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Here are some good ideas[1] if you want to know which businesses that can be started with little capital:Prepackaged school supplies based on the lists from the school districtsIncubator site providing office space, hosting, etc. for startups that are seed funded.In-grocery store prep service (buy vegetables first and drop them off to be chopped to your specs – pick them up on the way to checkout)A website that you can submit your design and work with vendors to manufacture your own clothes line.Online discount brokerage that has no closing hours and provides access to all exchanges throughout the world and handles currency and legal issues automatically.Stamphead, where stamps can be custom made in the likeness of an avatar the user creates.Aggregator site for affiliate marketers to collect daily reports. Would work on a tiered pricing structure giving you more info the higher up you go. Basic would be for people like me who just want to know if they made something and how much; advanced would pull down all available data.A gadget to cook beans/lentils/vegetables/rice for the same amount of time (quicker than what can be done in a slow cooker.)Herbal Flavored Gum. MMM…rosemary.Church issued credit card with automatic 10% for religious tithingBrainstorm Consulting – Teach companies how to brainstormCreate a website for non-profits and a list of their projects/needs and a similar list for foundations. Its a central meeting place for people to look for funding from the right people.Tutorial videos for technological skills (word processing, internet applications, email) for people who don’t know how to navigate technology but need it.Website where musicians can collaborate on tracksSigns in restaurants on menus that list all the ingredients.Network of chefs that rotate being personal chefsNuclear waste storage facilities completely removed from society.Hedge fund that makes bridge loans to homeowners facing foreclosure.Small-town / city – websites (through city and Chamber of Commerce) – most small town websites are terriblewhistle It.com: download audio of people whistling popular songs.A food service where you can email them your mom’s recipes and have them make it for you and deliver it during lunch time.Multiple camera angles on live television (especially for sports) or online viewingReplicate reality TV shows for foreign countries.SIM card swap serviceAn alternative medicine or homeopathic online service that diagnoses your ailment and gives you the proper corresponding alternative treatment; also provides you with doctors in your area that treat with alternative medicines.Create the double snuggie.A company that installs a monitor at a building entry and hosts virtual door attendants.Web-based language tutors leveraging Skype and Google Docs for curriculum.Meal delivery in hospitalsWeb package for a school system that allows parents to see their kids’ school schedules, grades, lunches, sports activitiesSanta Delivery Service – A dressed Santa will come to your home during the holidays and deliver your gifts as SantaPresentation Consulting – Teach companies how to improve their presentationsA website that boasts: Buy one product, have one mystery product of lesser value shipped to a friend for the cost of s&h. the user don’t know what they’ll get, but it will be fun as hell and keep the user in touch in an interesting way.Tribe Consulting – Help individuals (not companies) discover what Tribes they should lead and help them do itMusic Box manufacturer that will turn any inanimate objects mailed to then into music boxes. Users buy direct online, picking from available stock. Added charge for sending in the you own inanimate object.Customer feedback software for mobile phones sold to marketers.Web-based rent-a-CFO.Application to sell to universities that allows students to access their transcripts onlineExchange clothing (like PaperBackSwap. com) for men’s & women’s clothingMonthly subscription service for unlimited ebooks on your ebook reader device. Rhapsody/Netflix model for ebooks.Cell phone made of biodegradable materialsiPhone app that tells you odds for a poker handLet’s Get Coffee – for couples; pairing service similar to matchmaking service “It’s Just Lunch” to help couples find other couples with similar friendsA service that discounts newspapers after 8pm the day of print. I do not want to buy a NYTimes for $5 at the end of the day.Nonprofit that takes old CD electronics to developing countriesA unified affiliate marketing program for every store on the web.Contest company for small local businessesWebsite mock-ups for people seeking VC fundingGrocery delivery service for college campuses: Fresh direct for dorm rooms.Independent calorie testing company – A company that actually does testing on food for consumers to determine calorie contentEarplugs that go deeper into your ears but don’t hurt them.Online transcription service for conference calls – leveraging Amazon Mechanical Turk.Google for video & audio – Dragonspeak -> voice to text -> with searchable tagsA service that just vacuums and dust your apartment.A way to ensure that each person can only create one account on a website, without having to sacrifice anonymity.A small flap or cover (separate or built-in) for computers and technology with little lights that flash when they sleep (so you can sleep.)Crutches that stand upright by themselves. The base would have feet that fold up or spring out so you don’t have to lay them on the ground where they get in the way and people trip over them, etc.Interior Design on a Budget – for people who still want their homes to look good but can’t afford Restoration Hardware – client defines a set budget and consultants design using budget pieces from TJMaxx or Marshall’s or odds & ends shopsLandscaping business specializing only on LEED rooftop gardens.Company that records your voice message and other digital forms of media and sends it out into space as a message in a space bottle.Fireplace rehab – clean, paint, resurface, whatever to make it pretty. Big in places where same brick as outside the house is used for the fireplace and it’s not attractive (I’ve had two houses now like this.)“How to live on a small budget” website written by college students for college student for all sizable college towns sponsored by local merchant coupons.Someone/service that keeps inventory in a household (products, cleaning, etc.)A drink company that brings the Shirley Temple back – the best drink ever!Online Firewood Sales – Sell firewood online directly to consumers and businessOn-click online data storage and streaming of a clients entire media library. (never lose anything again and have anywhere anytime access) Charge by the GB and bandwidth. Provide conversion services from CD to MP3.Cupcake Shop (fresh and creative cupcakes)A mobile job fair in a tractor trailer that pulls up in the parking lots of offices of businesses performing massive layoffs. You then make money by renting out space in the trailer to companies in similar industries.Online Professional Wrestling Memorabilia Store – Sell pro wrestling memorabilia online directly to consumersSoftware to manage energy sales back to the grid generated by home systems.Analytics software for customer profiling, not just click analysis.Disposable mittens made from biodegradable materials in vending machines at supermarkets.OnlineConfessions. com. read other confessions and share the you own anonymously.Instant Flash builder, providing motion templates with easy upload graphic and text to fit template animation.Technology licensing agency.Consulting business creating a series of Squidoo lenses for an author on and around specific book titles. Pitch to publicists and literary agents, as well as directly to authors.Hire voice actors to read popular blogs to create podcasts where they don’t yet exist (“Best of the Blogosphere Podcast”)Personalized cereal boxes (to celebrate child’s achievements)Local business-sponsored community bike programAntique wedding jewelry shopping – another eco/reuse theme. Rather than buy something new that requires mining and blood diamonds, get an antique that comes with a story (about the long marriage of the previous couple, of course). We find it for you in the style you think you want.Surgery-specific recovery packs in hospital (if Sucrets and a can of cranberry juice is better than water & crackers, that pack exists)Personal lessons for using your cell phone featuresVenture capital consulting for startups.A company that makes hybrid fruits. Instead of drinking a lime pomegranate drink you can eat a lime granite.Photography studio that casts you as characters from moviesCentralized web portal for university technology transfer offices.A service that gives well-known business people a public email address, so that if an email get sent there you can read the email and then read the response. The idea being that it will give you insight into the mind of the person and what made them successful, and it will also teach you how to write good emails and learn email etiquette.A technology/service that Preachers/Rabbi’s/Clergy can use that creates a way to gather feedback during the service from the attendees.Online flower shop with one-hour deliveryA non-profit that provides credit consulting for youth and impoverished.Local fresh restaurant that opens off-peak hours to hold cooking classes to locals.Fractional Ownership of Technological Conference Room in Office ComplexRent-a-wife – a woman who cleans, cooks, does laundry, picks up dry cleaning, mail, etc but without the emotional relationship.Project management website for volunteer shift scheduling – church nurseries, hospital auxiliary members, protest marches, etc.Pencil sharpener type device to grind down dog toenailsAn iPhone app that allows you to search a database of unique and obscure beers and drinksGlow in the dark non-toxic washable paint that you can spray on your kids/pets when they are trick-or-treating so they don’t get hit by a car.A party planner for kids birthday parties, but with educational and fun activities.Instant Ad builder where sexy templates are linked with creative common photos.Spring Shadows – program where kids can shadow business people for a short two weeks internship to see what they want to major in once they go to college and what they might want to do once they graduateGPS / location based meetup based on pre-selected preferences (i.e. – Manchester United)A paperless hospital.Whack-a-Mole style game with political figuresAutomatic thank you notes, sent to contacts as a postcard generated from uploaded contact lists. On the card is a highly realistic signature that users digitally upload.National Restaurant Customer Service Association – traveling customer service training systemArtists’ Playground. This is a building that has rooms setup for ceramics, painting, silk screenings, theater, writing, open mic, dance, photography, etc. This building will also have social areas for people to mingle over coffee and healthy foods. People pay a monthly membership to have access and it is also rented out for events.Concierge services for an office building. Sell as a value add to the building managers or to the specific businesses as a benefit for their employees.A place where people could go to eat/study but would HAVE to do it at the same table with a stranger. It would encourage us to talk to/meet new and interesting people.Street Bar – bar inside a huge vehicle that drives you aroundFeng Shui website where you upload your floor plan and other details and get a recommendation on how to construct your space.Web-based rapid prototyping service that allows amateur product designers to test their designs and have our RP machines “print” their products in 3D.Training company that teaches people to be brave.Retractable spikes for boots to walk on the ice on sidewalksRecipe Subscription Business – Subscribe to the website and pay for premium recipesOnline eulogies (recorded by your friends before you die)Software that automates Search Engine Optimization without registering as black hat.iPhone app that is specifically for ski maps. Shows number of runs made and has lots of cool interactive features. Even shows which runs are groomed. Every ski resort would need their own app. Probably provide the app for free and the ski resort pays an arm and a leg for being featured in the app.Publisher of web-based children’s games.Horoscope porn site that provides the user with a personalized video stream based on preconceived notions assigned to your “sign”.A travel company that arranges people to stay at others houses.Slow cooker meals in a bag – drop it in and forget itA travel mug that’s not plastic or aluminum (ceramic) but with a rubber lid (tight-sealing), that has a good grip. Most travel mugs retain the smell of the material and give the liquid a strange taste after a while. Also, something that doesn’t allow Tupperware to smell.Employee monitoring service that provides detailed reports on employee’s online and offline activities. – include hardware scrubbing and “forensic” proof removal of data, footprints and histories.Tattoo parlor for petsStory Consulting – Help companies define and refine their story and teach them how to tell itA service that tells your neighbors to be quiet (nicely). They will draft a letter or note and send it to your neighbor so you can be the objective third party.Graffiti generator .com, where friends can type in text and, using different styles and palettes, create a tag-like version of their name, poems, etc.Internet and webcam set up for seniors to communicate with grandkidsTheme restaurant – Karaoke in front of a live bandGraph consultants – Company that specializes in making effective graphs from any data you give them.A conflict negotiation service (online or regular) where you submit a problem and we provide an answerSchool real estate rental company – Company that specializes in renting out underutilized school rooms during the summerTuning rooms for entry level home theatersBuild Your Own Perfume Shop.Front door cleaning service – get rid of all the stuff that gets left on your door every day before you get homeA company that sells framed book covers, so that you can hang the covers of your favorite books on the wall of your office.Standing in line for peopleBrainstorming. com – put in an idea and gives you 10 ideasChewing gum that is good for braces and dentures.Glass repair company – Company that specializes in repairing scratches or slightly broken glassTwitter third party app to help drivers find vacant parking spaces in crowded metro areas; sponsored by local merchants and advertising.Make your own batch of whiskey – takes 12 years, you pick flavors and types of oak barrels at the distillery, pickup in 12 yearsFranchise business consulting that helps large franchisees improve their businesses.Repossession agency for the most dangerous jobs in foreign lands.Digital tombstones that serve rich multimedia messages and memories of the deceased.Marketing and Advertising agency that excels at telling stories.A site like intrade that would allow investors to make bets on analytics type data, like the number of page views Wikipedia will get next January, or how many copies Malcolm Gladwell’s new book will sell.A service that pairs you with the right tools and helps you find a career by pairing you with the right resources (NOT an uninformed headhunter)Web portal to help large corporations source physical building contractors for specialized facilities.A company that digs your car out of the snow and removes snow from your driveway and starts car in the morning.A trend of the month club, where each month you get mailed something hip with a glossy booklet explaining how cool that item is about to become. Limited to 5,000 people. Gives you a cool story to tell yourself for the rest of the month, and something to talk about with your friends.Come to your house alterationsCustom adventures using Virtual Reality goggles and a windfan (i.e. – basejumping a waterfall or the Grand Canyon)Performance art as advertising – i.e. Cirqu de Soleil actors selling an item / brandService to teach doctors how to use technology to reach out to patientsCompany working with magazines to help them develop a social networking presencesA pill that gradually dies body hair.Technology scout for large corporations that looks for acquisitions.Create book trailers for small publishers/authors to place on YouTubeIP Scanner. Scans images straight to flickr or similar service. Great for archiving family pictures or kids drawings.Independent account fraud company – A company does regular checkups of your online accounts (like social networking sites) to check for fraudulent activityWeb-based devil’s advocate service for startups looking for someone to critique their ideas. Perhaps also provides referrals to cheap services for startups, like cheap graphics design, cheap business plan feedback, etc.A half refrigerator/freezer/microwave in the trunk of a car. That way, wherever you go in the car, you can always have food handy. How great for road trips.Pandora for spin bikesA non-profit that documents the stories told by elderly in nursing homes.A company that will apply your logo in glow-in-the dark removable paint around a town so it appears at night.Replication service for clothing items – make you a new pair just like the old pairImprove soft contacts so you can wear for 1 year at a time.An online counseling program via webcam (for therapists, inmates)Not-for-profit that works with people to help cure their acne.Service that takes your text messages from your phone to your computerFast food restaurants with healthy, organic and tasty foods. Similarly, healthy foods in cafeterias and grocery stores in low-socioeconomic areas.Software company that builds marketing dashboards sold to CMOs, brand officers, etc.A Build Your Own Robot Shop.A store that sells all inflatable products. So, for example, large inflatable cushions that kids can use to make play forts that compress flat afterwards. Good for Manhattan apartments where you buy a 30 dollar toy that takes up $800 of floor space.Offshore software development company based in China.A company that will sell a large digital frame for close to free and then provide a service for a fee that sends artwork to the digital frame every week.Desk mounted lighting systems for video chatThe Black Cow (least innovative product)A way to turn recipes on the web into peapod orders. A way to turn the customized diets that Weight Watchers or WebMD create into a peapod order.Online petition forms with compliant signature verification software.Customer service solution for general contractors that allows them to put in notes and they’re generated as an email or voicemail to customer to provide them with regular reports on how the work is going and what is holding things upNetworked home alarm systems so that neighbors can check on each other’s homes during break-ins.Scavenger Hunts, Inc. – We setup pre-defined scavenger hunts for small business team building (like a mini Amazing Race) or even for kid’s birthday parties (in a local park)Eyeglasses that you can adjust to the light. Not that they adjust on their own.Worst, etc.Clipping service for high school athletes parentsEZPass for humans on public transportation (key fob?)A company that creates baseball trading card style packets for restaurant chains.A cleaning service using only eco friendly productsCustom Hypnosis CD Business – Customers go to a studio and record their own voice reading self-hypnosis scriptsOnline Suspenders store – Company sells nothing but suspendersA non-profit that supports poor on using technology in classes taught by young freelance artists.Online Antique Appraisal Service – Send pictures of your antiques online to have them appraisedMicrobrewery with high-octane beers with boutique labels that cost as much as good winesTechnology consulting for venture firms.Harmonica It. com: download audio of people playing popular songs on the harmonica.A site that aggregates naturopathic medicines that have been shown to work to peer reviewed journals, but which for whatever reason aren’t prescribed by mainstream doctors. The site would then show the medicines proven effective for each condition, with a link to the journal article or clinical data or whatever.A hyperlocal iPhone app to find food, but for a series of use cases more specific than what Zagats and yelp are designed for. For example, list the best 3 food places within one mile of each highway exit that take less than 20 minutes. Or what wine tastings are within 10 miles of me within the next week.Anti-drug education program for inner city schools. In conjunction with a touring program that sends a message to parents across the country.Food consulting company that helps local restaurants with calorie measurement and other health issues – and publishes calorie, fat, vitamin data to third party apps on smart phones.Mushroom/compost farm that uses the Matter of Trust “lasagna” to eat cleaned up oil spillsCompany working with small local newspapers helping them develop a social network presenceGiant Magnadoodle for teachersComputer Remote Technician – Fixes your computer by remotely connecting to your PC or MacConference organizing company that handles all the logistics of creating and running conferences for other organizations.Retro-fitting older school buses with seats with seatbeltsOnline Town HallSpeed dating online.Adopt a prisoner “charity” that allows people to adopt an inmate and assure that they are visited and receive cards/letters on important dates.Prius Cab Fleet (www.greenfares. com)Buzz-monitoring of brands with software tools sold to enterprises and brand managers.The Purple Cow (most innovative product)Feng Shui experts that tells you the best way to put your stuff/furniture in your house.Expert roommate negotiator – Expert negotiator specializing in solving roommate disagreementsRent a scout that gives you a good idea of your child’s sports potentialBook packaging for great college writing and emerging writers.Online Palm Readings – Readings done via SkypeSelf-righting picture hangerWebsite for online job searching with IM capabilities so you can ask questions to other people searching or people who are advertising.An IMDB for people who have won awards. I want to a quick way to find the professors with the most citations in any given subject, the chefs in the area with the best Zagats reviews, the local high school football players with the most touchdowns, etc.A digital coloring book.SMS messaging for television tickers scrolling at the bottom of popular shows.A website that users can program in advance to take some action if some event happens. For example, a national chain of tire stores could send an email reminding its customers to get their snowtires on two days before the first predicted snowfall of the season in that user’s zipcode.Garanimals for adult menDating Consulting – individualized service on how to find, meet and date quality peopleBoat or Yacht share program.DeadCells Art: mail your old, hated cell phones to a factory that smashes it and returns it to the user as framed work of art.Girls Getaway – a company that sets up ‘girls week / weekend’ vacations (shopping, Broadway shows, hiking, wine tasting, etc.)Toilet seat covers for public toilets that don’t fall off the seats.For younger students, nap time is often hard to relax and in a bright room. There should be a company that creates comfortable nap time products for kids.A company that you can hire that will come to your town and make a 4 show realty series that you can show to your friends or play on local stations.A service that manages the risk of house swapping.Build the you own Barbie. com. Pay $100 to dress and color the you own doll.Twitter or SMS restaurant reservationsDigital family whiteboard on fridge (like an iTablet for the family)A website that connects people with Objects of Sociability. For example, you have a group of friends you want to do something with and it connects you with an open hibachi table or a deep sea fishing boat rental. You can also rent objects of sociability, so for example you could rent a bread maker or a super nice tea set with 30 looseleaf teas. It would also connect people to third places that would be suitable for, say, a first date.Dissolvable contacts that deliver medicine to the eye overnightA charity that works with major retailers to encourage buyers to bring in their old clothes for a discount. The company will then take the clothes from the retailer and send to a third world country or American red cross.TV Show Summary Service – Brief summaries of your favorite shows are e-mailed to you in case you missed them.Globalization consultancy.Creating a website community for freelance professors (professional educators with a specific expertise; different from tutors in the sense that they are not preparing students for a certain test or “actual class”) to connect with students who want to be educated via an alternative route. The ideal site would essentially be a hub for professor profiles/portfolios with a variety of mediums for conversation between the students and the professors for hire. comments/rating system (no anonymity including a record of that person’s comments rates so you can get a better picture of who is saying what). The idea is similar to something like pick a prof. that most college kids use now to pick their classes; however, the emphasis with pick a prof is typically to pick easy classes to graduate where as this would be an effort to allow professors and students to negotiate the value of the education without going through the university as a middle man. Monetization routes: site advertisements, membership fees for students and/or profs, selling books written by professors for commissionBuild Your Own Back Pack Shop.Group dating website.Online mentoring program for students of all ages (Pen pals that teach each other skills online.)Citizen journalism website.A website that teaches kids how to do quant trading. The site would come with a data feed, dozens of pre-packaged statistical tests that could be deployed or combined in various ways, and the ability to do backtesting to see if this strategy would have been effective on previous data.Designer bibs for patients during their teeth cleaning. This could be a cartoon design for kids or pattern for adults.RingTone this. com: upload audio and have computer-like versions automatically generated and sent to the you cell phone as a ringtone.A book about the history of the internet made from recycled phone books.A cutting-edge fashion company that tells the story of wearable computing.A virtual interior decorator that will make suggestions via digital photographs.A company like 800CEOREAD that does bulk book rentals. So for example you could rent 1,000 copies of Jeff Jarvis’s new book for a month instead of purchasing them.A company that sells high quality sawdust / sand in bulk to put on the floor of restaurants or bars. The company would then be responsible for removing the old material and replacing it when necessary.IP Camera security company – software solution – offsite remotely recordingMilitary Peak Performance Consulting – Consult the military on matters of peak performance (nutrition/stress management/etc.)An event planning organization that markets to stay-at-home moms with big ideas, eager to share there inventions and business ideas with other mothers.Educational youth iPhone gamesAn automatic dimming light bulb coated in that same stuff that is on glasses that the user wear on the you face.Frozen venison shipping store – Company that specializes in shipping the best venison across the countryA safe vice for your head when you have a headache (made from strong fabric maybe)Apprenticeship management company for high profile business personalities.Celebrity voice wake up callsObama TARP consulting for financial institutions.For Americans book series – Cricket for Americans, Bollywood for Americans, World History for Americans, French Film for Americans – would be topics that go outside our country to hopefully explain that the rest of the world isn’t just like us, nor should it beA consulting company that works with aerobic instructors to trademark their exercise routine.Webcam security set up to watch kids, dogs, older parentsA recipe holding cell online for all your recipes to edit, print, and send to friends.A small handheld set up to video chat only via satellite.A business model that implements a ‘couples fare’ if you are in a long distance relationship you register for a year deal where you get specialized pricing and discounts.iPhone app that has similar features to Nike plus (who doesn’t have an app yet). For runners. Pace, speed, etc. All reports back to a website that has monthly subscription costNetwork of restaurants where chefs rotateAgency that trains people for doctor’s offices. Doctor’s offices need etiquette training for their employees desperately.A service that transfers CDs -> LPs (not the other way around)Organic grapes and wine brokerage.If you’re doing home exchanges, a personal website devoted to you and your trip and all of its details. It will have an itinerary on a main page so you don’t have to access your email to see it and it has all the details there.Collectibles investment club.After Party Art where the user send in bottles collected from special gatherings to a factory that melts and shapes glass into guest gifts.Stickers for backpacks (stick well to nylon, etc.)Roomba type toy for dog to chase around house. Should move faster, be fairly indestructible and make appropriate noises.An online toy that introduces children to audio mixing through a Mr. Potato Head-like interface. Add a nose, add an instrument to the band!Children’s school that finds the right balance between Marine Corps boot camp methods, Montessori methods, and liberal arts education – located half the year in big cities worldwide and half the year in wilderness camp setting.Dashboard painting of car interiorsAmusement park based on video gamesPersonal chef for a day. A formalized service where you can call and get a chef with a specific specialty to come and cook a few days worth of food. In cities, this would be very popular and in demand.After party cleaning crewFence store – Sells nothing but designer fences with custom designsInvestment bank that helps non-profits tap the public market with stock listings.An invite only site that allows friends to create video playlists from Hulu, the userTube and third party programming.Online Astrology Readings – Readings done via SkypeGym review company – Company that reviews gyms and creates a guide like ZAGAT that we sellNetflix for freshwater aquariums. If you get bored of some of your old fish you can swap them out for new ones.Phone booth style device that you walk into and it steam presses your clothesA website that you can put in a characteristics of a person and it will come up with an avatar.A store for really obscure food products that change regularly. Every time a customer checks out, you ask them if there are any products that we don’t know about that we should know about, and if there is then the customer ten dollars off their bill and that product shows up in the store within a week or two. Maybe even more narrow than just foods in general, so for example a store for niche meats that come from animals you’ve never heard of.Religious Karaoke Bar – Bar where only religious songs allowed to be sung, and no alcoholSoda Pop flavored mochi balls.A sophisticated website that uses an eHarmony-like algorithm to match nannies and familiesSolid state lighting retrofits of vintage light fixtures (art deco, 40’s 50’s 60’s fixtures) get them at Goodwill and other second hand shops and turn them into $300 “green works or retro art”A dating service where only your friends can set you up on dates.Fractional Ownership of Season Tickets / Box SeatsAffordable home matting and framing kitConsumer fingerprinting business – Company provides trained experts to apply fingerprints tests for household eventsA relocation service for Americans looking to permanently move to a new location for global opportunities.Website where musicians can collaborate on lyricsRoomba for the backyard to pick up dog poop and enzyme it. Or it works with the Doggie Doolie and dumps the poop in it on a regular basis.Fish Burial Kit.Mail in your old mixtapes and get a playlist sent to you via email with a digital version of that mix.A not-for-profit that pairs experts with eager and willing pupils for an apprenticeship program.Actor research consulting service – Consulting service for actors to be setup with people who can mentor them in research for their rolesAutomatic documentary maker, where families upload clips, photos and music from their special occasions generating a smooth transition clip that captures the essence of the moment.Executive Power nap business – Locations where executives can take a 20 minute energizer nap during the dayGraphic novels based on video gamesProfessional protesters agency.Lip reading school – School that teaches students how to read lipsDigital Workflow Consulting – “We’ll get your precious memories (files) into the cloud”Brand of energy ice cream.Cereal Bar (cereal the way you like it with “extras” – open late)Sell web stats back to website owners who are computer illiterate (Google Analytics)Anti-inflammatory Hot SauceFirefox Plugin for Autosave ClipboardUndercover Teenager – hire a teenager to spy on your kidiPhone app that uses the GPS to measure amount of accumulated gains in altitude for climbers, hikers, skiers.Pay at the pump carbon offsetsRoof-mounted Active Denial System (pain ray) to keep deer out of your garden. Or possibly a sounder laser. The system would be mounted next to a camera with an image recognition system that could pick out a deer.Netflix for conversation pieces. Get bored of your coffee table book and send it back for another.Box removal after people move – green message of recyclingRelationship Hotline – Relationship experts answer phone calls 24/7Age verification company – A company that solely does research to verify someone’s age (little league baseball would be a big client)Campaign Suite -> Political Suite – set of web apps – all the good things Obama did in a “package”A seminar business that trains people to be tribe leaders.Mobile software for a picture-based wishlist. When you see something you like at a store, you can take a picture of it and record the info about it either so you can comparison shop or someone can shop for you. Might replace the print-out-20-sheets-of-info gift registries in places like Target.An Urban Dictionary for texting.Salon for blind peoplePicture book series for adults explaining technology topicsCloset organization for adult men – Garanimalize their clothing for them after the factVision board builder site that provides a virtual design for users to focus on their goals daily.A website that teaches you about what is involved in the day-to-day training for different sports, so you can make an intelligent decision about what sport to take up instead of just choosing based on what it looks like on TV. Has links to where you can go to take up a sport as a beginner, what the competition structure looks like, how far you typically have to travel, etc. Can drive traffic to consultants who help get kids recruited to college, to genetic testing firms, to sports physiology labs, etc.DeadTV Art: mail your old, hated TV sets to a factory that smashes it and returns it to the user as framed work of art.Organic vitamin pills and supplements for vegetarians sold through health stores.Teach companies how improv comedy can help them in their business (comfort zone, setup your partner)Herb garden planting – plan, plant and basically get your herb garden going.Legal Advice Hotline – Legal staff and call center charges consumers to call in for advice charge per hourA website for elementary and middle school kids that helps them build a portfolio of their best artwork, writing, sports accomplishments, etc.A data aggregator of global statistics on death, destruction, catastrophes, violations of human rights, freedom in media, etc. There is so much attention put on the middle east. It would be interesting to see and compare what is happening in your neighborJelly Belly for gumTeam in Training – supporting a family or village instead of a causeFilmmakers café-It would be great to have a place that people could go and use a TV and DVD player in a cool coffee house setting–similar to how the internet cafes are run…It could be a place where people could just leisurely watch something midday and also people can meet and discuss their work and others’ work while watching through it.Website that allows people to share clothes and shoes (fractional closet ownership)iPhone app that measures the speed of a pitch. Utilizing the built in accelerometer, you throw the phone as fast as you can. When you pick it back up it tells you the speed of your throw. This pairs well with the iPhone insurance idea.A site that gives you a step-by-step system to build a large web presence and a large group of followers, and makes it easy to track how many people are reading your blog, commenting, sending you emails, etc. so you can work on growing your number of followers and increasing your influence as efficiently as possible. Gives you detailed instructions each step of the way based on your performance.Affordable set top box with video chat (less functionality and cheaper than Media Center)Robotics sports entertainment company – Create robotic athletes that play sports using AI, charge for admissionA storytelling site that markets to families who want easy ways to archive the voices of their elders.Interactive billboard advertisements with codes for coupons sent to mobile phones (mobile-location-based-presence advertising).One stop shop custom photo labs – bring in digital pictures and you have access to Photoshop expert, printing matting, framing,A village in china that paints portraits of pop culture icons as various dog breeds. Look, Obama as a GreyHound!Fish babies. Specifically market a female and a male fish capable of mating to customers interested in seeing the miracle happen at home.A consumer hotline to advise people on electronic purchases and installation on a $ per minute basis.BestOf. com A website that only sells one specific type of each product…but you know its the best.Create YouTube channel with local restaurant reviews (or any kind of local business)Words sewn into a certain brand of clothes – need to find people with same clothes to spell out a word or phrase on the website to win a prize (like McDonald’s Monopoly but with clothes and a website)Public bathrooms that you pay to use. There is a room attendant for your bags.A bio-degradable “plastic” to be used in developing countries where outsiders come in and drink lots of things in plastic bottles because they can’t drink the water. These countries don’t have trash and thus no way to deal with the plastic trash Westerners bring with them.A free live music concert video site streaming a huge archive with limited commercial breaks.Portable safety landing zone for emergency extractions sold to government first responders.A mentoring program (website) that connects entrepreneurs with specific mentors they seek.Art co-op that sells memberships and the art travels among the homes of the members. Kind of a Netflix for art, maybe. Art to eventually be sold at auction via exclusive contract with Sotherby’s and profit made. Could be sold as a shared-art program or as an investment co-op. Or both.Senior friend – hire people to come by to spend 30 minutes just hanging with your older relative who live away from you.Website that generates naming and branding ideasTravel agency that shows you dying civilizationsA service that provides specific enthusiastic people who want to volunteer or have time to go engage with people at nursing homes and doing activities with them.Private investigation company for restaurant employees – Restaurant companies pay to have PI’s investigate whether employees are washing their hands in the bathroomA service that takes your records and digitizes them into MP3’s.Unique Bachelor & Bachelorette Parties – we work with you to design a fun, unique bachelor or bachelorette party experienceA singer/song-writer could offer to write, record, and perform personalized songs for people’s special occasions (anniversaries, birthdays, holidays, or just because). Many would love to have the ability to write, record, and perform an original song for a special someone, but they just don’t have what it takes musically. Then, there are those of us who do have the ability but don’t know how to market themselves.Outsourced sales staff for high-ticket items.Manufacturer of flying cars (courtesy of Tim Draper).Mobile app with hospital floor plans, maps, info about area restaurants, etc.Digital Painted Blinds. Every time a dorm student closes their blinds, the window becomes a poster of rotating images he/she uploads from a flash drive.A service of highly trained babysitters specialized in all ages that you could call upon last minute any night of the weekA service where people are employed to talk specifically to antisocial or shy people at a party (they’ll feel better about themselves and will be more likely to open up next time with their new confidence.)Live Tutor (online tutoring via webcam, chat)The “C-Games” – like the X-games, but real people and celebrities compete in wacky (think Japanese game show) events & all $$ goes to charityA website that aggregates tastemakers. Collect the people who do for other things what Fred Wilson does for music. Give them a really good platform. Give them points for picking up on trends before they go mainstream.A consulting company that creates tribes for your business.USB stick into a cell phonesite that allows people to upload stock video in HD for use by other filmmakers. Low cost per minute. 50/50 split between site and uploader when a purchase is made.Positive news TV channel (PNN) – TV news network that only plays positive TV programming, nothing negativeGrocery checkout efficiency consulting service – Company consults grocery stores on their checkout process to increase efficiency10% for the world – an association of businesses who add 10% to their prices and donate the 10% to charity.Traveling Poker school – weekend intensive poker school – get some prosAssist business people (authors, bloggers, executives) organize speeches and presentations – establish relationships with many venues & marketing channelsBaby fitness class company – Organize fitness classes for babies and their parentsiPhone app that is a recorder (like the ocarina app, but more familiar).Globalization import-export trading company.Really good collaboration software for creating mindmaps, with version control that’s as easy to use as Wikipedia’s history tab. Uses the same open data format as FreeMind.English speaking, US educated virtual assistantRestaurant where you cook alongside the chef , choose your ingredients and learn a new technique.A virtual world future’s exchange, so you can bet on how much a Linden dollar will be worth in US dollars six months from now.Script Assistants-A business that assists screenwriters as they are developing their script by aggregating credible/hard to find reference material that is diverse. Often a young (or firsttime) screenwriter has a great idea and may not know what to reference to develop their idea. This business would evaluate the theme/tone/genre of the script and assist in coming up with a solid and effective reference package pulling from books/TV/movies/screenplays.Web cam at school events so you can see your kid when you can’t make itUse Google Earth to determine green improvements to a given residenceA personal education coach-they pick blogs, books, resources for you to learn based on your personal interests and career goals. They gather all the materials and for you and make recommendations on the best way for learning the material.Non-profit that takes old technology items (computers, monitors, phones) to make them usable for non-profit use – clean off the software, ensure it has all the pieces, etc.)We come to your house to list only your good stuff on eBay or Amazon so you don’t have to bring it to a store. You could have a portable photo studio in the van so the product never leaves the premises until they have cash in hand after the sale. Charge a commission and have college students working for you.All electronic gadgets in one. Phone charger, mp3 player, computer, radio, watch) should all be able to be charged by the computer. (Travelsize/lightweight.)Gold reclamation service from old electronicsA guitar pedals that make exotic animal noises.Kids choose a week’s worth of lunches online with their parents and a van delivers them everyday at lunchtime.An organization devoted specifically to growing awareness over an obscure problem that no one really looks at (i.e. kids that grow up in negative environments and what that does to them as adults.)Fractional Ownership of Segway3D personalized bubblehead dolls ordered over the Internet based on submitted photos and scans.Art for the blind – Company that translates art into some type of Braille system where blind people can appreciate it.A place online that has all your usernames and passwords for all of your bank accounts, bill paying (cell phone, grocery, utilities)…keeps track of them.Vanity audiobook company for Lulu. com authorsWebsite that allows groups to document their lottery ticket purchases and each individual members’ shares to avoid problems when they win – there are a lot of winners beside the jackpot winner.Wikipedia search hotline for those who don’t have access to the internet and simply want quick answers to their inquiries sent to them as SMS texts.Build Your own Mardi Gras mask site.RSS Batches – supported with ads (so you can subscribe to a batch of the top 10 marketing blogs and in addition to the feeds you get an occasional relevant and anticipated ad)Philanthropic tourism – pay a huge fee with part of it going to help the people you visitGrocery store with only junk foodHeadphone buds that look like headphones but that are really powerful earplugs so people can’t tell the difference.A store completely filled with weird vending machines.Offer private curbside recycling service in towns where it isn’t offered. For a monthly fee, provide a container and service it once a week. All the contents are taken to the city’s collection site.Marketing Awards (like the Oscars, potentially online)A private site where corporations hire street artists to Tag over their build boards in an ongoing campaign to draw attention to the location of the sign. Then at its peak awareness, replace it. And start the process again.Online judge dispute resolution – Submit your disagreements onliine and for a fee a judge will email you back the verdict on who is rightSteroid scandal consultant – Firm specializes in advising baseball players that are in the midst of a steroid scandalCasino Airlines – Airline that has casino games on the plane as soon as the plane gets over the seaTrade Ya. com where users trade their personal goods for other user’s crap.A drive through fast food place that has healthy sandwiches.A laundromat/video arcade – Play video games with your kids as you wait for your clothes to be doneA crowd-sourced social filter. So you type in something you think would be funny to say or do to someone else, and the site tells you whether it’s actually a good idea or whether it would be wildly inappropriate.Instead of cryogenic freezing of bodies, store genetic signatures and compile multimedia memories.Change Consulting – Teach companies how to change – “stories of change” workshopsWebsite that is an aggregator of all the charities that celebrities support and how you can get involved.To-go food materials that are 100% recyclable such as a straw.Company that anonymizes current case studies from real companies facing real struggles that others can subscribe to to be aware of current day challenges, sold as subscriptions.Delivery service of food from local restaurants that don’t offer deliveryRestaurant of the month club – diners pay $100 – get $200 gift certificates for a rotating set of restaurantsPolice texting alerts-in case of an emergency you can text the police instead of calling them.An application that is a Thesaurus for your phone.Will you marry me. com, that allows viewers to share video of wedding proposals and live stream real ones.Neighborhood phone bookTravel agency for the rich and famous.Improv comedy coach comes to dinner parties or can order “Improv Party” DVD setMobile pet neuter vans.A software that will allow you to change the channel and adjust the lights in the room through you computer.Etiquette guides for various social mediaStart a multi-level marketing company that specializes in sex toy parties for men.Snow Man kit: a packaged snow man parts, encouraging fun times building a Snowman with family and friends. Biodegradable.A digital book reader that provides an OED, so that readers can look up words as they come across them.Web-based portal that allows people to invest in or fund movie scripts written by new writers and allows studios to scout for good stories.FamousNoses. com, pick the you favorite hollywood star and get a rubber version of their beak for the you own!Online Executive Betting System – Online trading system that allows you to trade CEOs like securities to make moneyCreate your own ice cream flavorsTech adventures. Expensive one and multiday adventures where we put the players in scenarios where they get to play with the latest cool technology…night vision…robots…James bond type stuff. Real life role playing game.A PhD site where students and teachers post thesis.Casual escrow service – Escrow service for more casual transactions, like between family or friendsA software program that functions like Pandora for television programs.A company that creates baseball trading card style packets for fashion designer lines.Government sign business consultant – Company finds infrastructure signs (roads, bridges, etc.) that are ineffective or misleading that by fixing will save the government moneyStacked Twitter iPhone App (like TweetDeck or PeopleBrowsr for the iPhone)A place where kids can go and dress up like their favorite childhood book. There will be a set and costumes to dress up. Perfect for birthday parties.A car sharing service that manages the risk and coordination of sharing a car in major cities.Video messaging service where people can tape their messages (birthdays, holidays, etc.) and they give us the content from their own camera and we edit them and send them to the people they love.A not-for-profit company that works with the NYC government to spray paint North/south/east/west signs on the street or on a sign outside the subway stations to help the directionally challenged.A YouTube VC firm, that will invest money in people who are willing to do stupid stuff on video in hopes that the video will go viral and they will make a profit.AngryClickers. com: a streaming site that allows users to cue live face punches in an online fight. This is sick.Job swap board for stay at home moms – I’ll do your laundry if you do my grocery shopping.A high school homework site that markets to the schools and teachers who want to know what their neighbors require and post best-of results.Bicycle that generates electricity to run the clubInterview practice company – A company that will do mock interviews with you and give you feedback on your performance.A website or iPhone app that finds places for large groups to hang out in real time. So for example if you’re wandering around union square with fifteen friends at night, find the nearest ten places that can accommodate that many people right now.In store sales (for the next 60 minutes – x is 50% off – sent via SMS to customers who have signed up)Design agency for Second Life and other digital worlds.Private Security Insurance – promise trained “Blackwater-esque” security in the event of civil disturbance.Green Architecture Best Practices CommunityNap stations throughout the city. To integrate it into our culture, we can introduce them through napping stations similar to coffee houses.Video creation for local businesses to put on YouTubeInstall digital screens on shopping carts and sell advertising to national packaged goods companies. This would replace the current printed adds on the shopping carts. Grocery store shares in ad revenue. The carts could be location aware and tell you about products you were close to.Company that creates widgets that advertise high-end jobs on the most influential and specialized bloggers in the areas of tech, medical, finance, etc.Pillow case store – Store that only sells customers a large variety of custom pillow casesTactical fire arms training for urban environments.Be a sports team mascot for dayA digital medicine cabinet where patients upload their latest prescriptions and receive automatic feeds that relevant Google alerts.Dedicated sms/twitter service responding to shopping people about “greenness”/ethics of products.Software to build social graphs based by comprehensively integrating all the different online and offline social networks.Footnotes[1] Software Secrets Book

Pigeonpea [Cajanus cajan (L.) Millspaugh], having originated in India, is one of the major grain legume (pulse) crops in the Indian subcontinent. The crop is known to be susceptible to a few virus and virus-like pathogens, belonging to different genera [1–4]. While characterizing the dsRNA from the sterility mosaic disease (SMD) [3] affected pigeonpea plants, we noticed that two sets of symptomless controls plants containing four dsRNA molecules while none were noticed in the remaining sets of healthy samples. Healthy control plant samples (Mg1-H1 and Mg1-H2) that contained the dsRNA were collected from one of the farmers’ fields in Chevella area (near Hyderabad, Telangana state). The pattern and the number of dsRNA segments did not match with dsRNAs associated with pigeonpea plants infected by PPSMV-P sub isolate-Chevella (separate manuscript under preparation). Sequence of the four dsRNAs showed neither homology nor alignment to any region of PPSMVs. However, these dsRNAs share close similarities with individual genomic segments of several plant cryptoviruses. Evidence is provided to show that three of the four dsRNAs constitute the genome of a new cryptovirus tentatively named “Arhar cryptic virus-1” (ArCV-1) with a genome size of 4.64 kb. Pigeonpea is referred to as “Arhar”in Hindi. The genomic sequences showed its phylogenetic relationship to members of the genus Delatapartitivirus. The fourth dsRNA (dsRNA-2A) contained cryptic virus coat protein- like sequences with no similarity to that of ArCV-1 genome. We discussed different possibilities of its occurrence with ArCV-1 genome and its relevance.Cryptic viruses usually contain 2 to 3 monocistronic genomic dsRNA segments which are encapsulated individually [5, 6], and referred to as bipartite and tripartite viruses. Occurrence of tripartite cryptoviruses is less frequent [7]. Plant cryptoviruses, in general are either pollen or seed transmitted, belong to the family Partitiviridae which include five genera viz. Alphapartitivirus; Betapartitivirus; Gammapartitivirus; Deltapartitivirus and Cryspovirus [8]. Members of the family Partitiviridae are known to infect mainly fungi, plants and protozoans. This is the first report detailing association of a cryptic virus with pigeonpea (Cajanus cajan(L.) Millspaugh) plants which belongs to the family Fabaceae, harboring one third of the reported cryptovirus infections.Mixed infections involving different cryptoviruses as well as host specific pathogenic viruses are common [5, 9, 10], effecting a single plant or more. Beet cryptic virus- 1 (BCV-1) and beet cryptic virus- 2 (BCV-2) were reported infecting the same host [9], as a mixed infection, similarly in the case of pepper where Pepper cryptic virus-1 (PepCV-1) and Pepper cryptic virus-2 (PepCV-2) were found as mixed infection [11].In the present study, ArCV-1 genomic segments were characterized by Sequence Independent Single Primer Amplification (SISPA) method. This comprehensive method was found to be precise with a high degree of specificity to investigate the evolution and genetic diversity in dsRNA viruses. The four dsRNAs eluted from the agarose gel were purified and have been used as templates for RT-PCR amplification employed in SISPA to generate full-length cDNAs.It is of interest to examine if ArCV-1 RNA dependent RNA polymerase (RdRp) structurally resembles the known RdRp of the dsRNA bacteriophage Փ-6, reovirus, or with other viruses like calciviruses and picornaviruses [12–16]. Their RdRp molecules, whose structural details have been described, are larger than RdRp of ArCV-1. RdRps of several important human and animal picornaviruses have been extensively studied that correlated functions with structural details, which contributed to the understanding of the mechanics of this enzyme activity leading to viral replication [12–16]. Similar efforts were made in the past decade to study the 3D structural characterization of RdRps of a very few plant viruses and detailed studies remained to be carried out for RdRps of double-stranded plant viruses including partitiviruses. ArCV-1 RdRp sequence analysis revealed the presence of several conserved amino acid sequence motifs common in other tripartite cryptoviruses. These motifs have been described to be important for biological functions in several RdRps [17]. We report here the results of elaborated computer-assisted analysis of ArCV-1 replicase which revealed the presence of conserved sequence motifs (A to G) present in the fingers and palm subdomains of the polymerase that are shared in most of the RdRps. Interestingly, ArCV-1 replicase has more structural resemblances with several members of ssRNA (+) mono-partite Picornaviruses (viral replication by primer-dependent initiation), than the de novo dsRNA bacteriophage Փ-6 and reovirus polymerases. Variations found in ArCV-1 motifs’ sequence that may be involved in polymerization mechanism and the conserved motifs unique to cryptoviruses have been described. This report illustrates several interesting features of the ArCV-1 3Dpol and its complete structure was determined.Isolation of dsRNAs from pigeonpea plants and their amplification by SISPASymptomless pigeonpea leaf samples, Mg-H1, Mg-H2 collected from MG-1and MG-2 fields contain four dsRNA species with an estimated size of ~1.71, ~1.49, ~1.43 and ~1.6 kb (Figs 1A and 2), whereas as symptomless leaf sample (Mg-H3) collected from another field (MG-3) were devoid of similar dsRNAs. The pure dsRNAs yields ranged 700 to 800 ng was obtained from 7g of fresh leaves with consistent dsRNA unique profile which is entirely different from the PPSMVs. Post SISPA, PCR amplification product profile was similar to the dsRNAs obtained from the initial dsRNA extraction from the symptomless leaves (Fig 1). These amplification products were cloned and characterized.thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 1. Resolution of dsRNA species isolated from symptom free pigeonpea plants and SISPA-PCR amplified dsRNAs fractionated on 1.5% agarose gel.(A) Lane-2, dsRNAs isolated from symptomless pigeonpea field collected plants; (B) Lane-2, amplified dsRNAs. The ArCV-1, dsRNA-2 and 3 were almost similar in size. Lane 1 (A, B) contains 1kb DNA marker (Fermentas, Thermo scientific, USA).Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.g001thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 2. Schematic representation of genome organization of Arhar cryptic virus-1(ArCV-1) and associated CP-like dsRNA-2A.(A) ArCV-1, dsRNAs were denoted (RNA1, RNA2 and RNA3) are represented by solid black lines. The larger RNA was identified putative RdRp encoding a single peptide of P1, marked as grey box. The two smaller RNAs encode two individual peptides of P2 and P3 and were identified as putative capsid proteins, marked as grey boxes. (B) Schematic representation ofa fourth dsRNA isolated along with the ArCV-1 genome with an unusually long 3’NTR, referred as RNA-2A (Black solid line) is predicted to encode a putative coat protein P4 marked as grey box.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.g002Genome organization of ArCV-1The cloned amplification products corresponding to the four dsRNAs were sequenced. BLAST analyses of the full-length sequences revealed their identity with the orthologs of several cryptoviruses. ArCV-1 genomic segments shared distinctive conserved 16 nt stretch (GATAATGATCCAAGGA) at the 5’- non-translated region (NTR), a feature commonly observed in multipartite cryptic viruses and constituted as the genome of ArCV-1 with a size of 4.64 kb (Fig 3). Whereas the dsRNA-2A, 5’-terminus sequence (AGAATTTGCCCTGTAT) did not share the conserved sequences and thus treated as a separate entity. The genome organization of ArCV-1 was thus established during the present study (Fig 2), together with dsRNA-2A depicting the single open reading frames (ORFs) of dsRNA-1 (RdRp), dsRNA-2 (CP-1) and dsRNA-3 (CP-2) with the predicted molecular weights of proteins encoded by the RNAs (Table 1). Unlike the 5’ end, the 3’-region is least conserved in several cryptic viruses. The majority of the members of the family Partitiviridae typically contain few pyrimidine bases conserved at the 3’-terminal and in tripartite viruses, conservation is seen mainly in two of the RNA segments. ArCV-1 RNAs encoding capsid proteins are conserved in the last three nt (TTC), like in Raphanus sativus cryptic virus-2 (RsCV-2), the three ArCV-1 RNAs end with TC. The majority of the Deltapartitiviruses contain RNA segments consistently ending with nucleotides TC except in Rosa multiflora cryptic virus (RmCV) and Cannabis cryptic virus (CanCV) (Betapartitivirus) interestingly, the 3’ termini, each with a poly (A) tract.thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 3. Alignment of the nucleotide sequences of 5’ non-coding regions of dsRNAs-1, 2 and 3 of ArCV-1, Fragaria chiloensis cryptic virus (FcCV), Rose cryptic virus-1 (RoCV-1) and Raphanus sativus cryptic virus-2 (RsCV-2).Conserved residues are marked as in grey box.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.g003thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageTable 1. Genome characteristics of aArCV-1, encoded proteins and associated dsRNA-2A.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.t001The dsRNA-1 (HG797710) is 1,714 nt long, containing a single open reading frame (ORF) between nt 1,606 and nt 179 with 178- and 108-nt- long 5’ and 3’ NTRs, respectively (Fig 2, Table 1). This ORF encodes for a 55.34 kDa protein (P1) containing 475 amino acids. P1 protein was identified as viral RdRp and contains conserved short amino acid sequence motifs (A to G) which are essential for viral replication [18–21], and known in several picornavirus RdRPs. The A–G motifs are conserved in the RdRps of several genera of plant and animal viruses containing monopartite, linear, ssRNA (+) genome [17, 18, 22], segmented, tripartite ssRNA (+) viruses [23], as well as multi-segmented linear ssRNA (-) viruses [24–27]. ArCV-1 RdRp similar to the other cryptoviruses contained these signature motifs in the central region of the polypeptide. Specific amino acid sequences mostly conserved formed these seven motifs: SSAAGYGYVGLK (motif G; 117–128) KVRNVW//DPL//SFYFIGQD (motif F; 181–219), FDWSGFD (motif A; 241–247), GIPSGSCFTNIIGSITN (motif B; 302–318), THGDD (motif C; 338–342), DKSD (motif D; 372–375), and TFL (motif E; 384–386) known to be functionally active in viral replication [17]. Computer analysis predicted the active residues in each of the conserved motives (S1 and S2A Figs), indicated in bold letters. Motif G is less conserved amongst the human and animal viruses belonging to the picornavirus ssRNA (+) super family as well as viruses containing dsRNA genome. Active residues in motif G are comparatively well conserved in the dsRNA cryptovirus RdRps (S1 Fig). Aided by RdRp sequence of Փ-6 and reovirus active residues in motif G were identified [12, 13]. Conserved proline (in this motif) of most of the picornaviruses is substituted in ArCV-1 RdRp by glycine in the C-terminus. SSAAGYGY-G-K sequence is highly conserved in cryptic viruses. In addition to the above mentioned seven conserved motifs, several conserved sequences in ArCV-1 RdRp were present flanking the central region towards 5-’ and 3’-terminal regions. Motifs GWARS (53–57), STPD (162–165), TRTQL (168–172) are present in N-terminus and RDE (397–399), CLRML (402–406), LRA (421–423), DAG (429–431) YLY (436–438), WDP (460–462) in the C-terminus devoid of predicted active residues. Motifs GWARS and TRTQL (motifs-1and 2, S1 Fig) were conserved in most of the bi- and tripartite cryptoviruses. However, specific functions of these exclusive motifs are yet to be determined.Motif C represented by GDD sequence (Fig 4D, S2A Fig) is almost universal and has been described as the center of catalytic activity of the RdRp in all RdRp classes [18, 28]. The 3D structural analysis revealed that the GDD sequence present in the loop formed by antiparallel β–sheets 6 and 7 is located in the bottom part of the central cavity of the molecule in the palm region (Fig 4B, S2A Fig). Several investigators conducted mutational studies of the catalytically important residues of the polymerase of different viruses [29–32], and determined that the first aspartate is an essential residue of this highly conserved motif for the enzyme activity and its coordination in the binding of a magnesium ion that would be part of the rNTP binding site [12, 33]. It was found that the first Asp (D341 in ArCV-1) is a strict requirement and any change leads to loss of the enzyme activity. Some flexibility was suggested with regard to glycine and the second aspartate and subtle changes in the position of these two residues are tolerated with exceptions [34, 35]. Similar observations were made when the conserved aspartate of brome mosaic virus (+ssRNA) was replaced in the 2a gene with glutamate (D470E), as the DR4 mutant did not replicate in the barley protoplasts [36]. The polymerase structural domains and the regulatory mechanism of the enzyme with respective different viruses are well documented in a recent review [17]. Possible functions of the residues of the A to G motifs described for identical RdRps was conserved with respect to the ArCV-1 3Dpol structure and was discussed in structural analysis of ArCV-1 RdRp section. ArCV-1 RdRp has a high degree of homology with RdRps of tripartite viruses belonging to the genus Deltapartitivirus, compared to the RdRps of bipartite viruses. The RdRp has 72.4% amino acid sequence identity with FcCV P1, 71.2% with RmCV P1 and percent sequence identity of 71.0 to 12.1% with the P1 proteins of other tripartite viruses (S1 Fig, Table 2) as compared to 35–39% sequence identity found with RdRps of bipartite viruses. The 3’-terminus contained the sequence GCACCCGTCTC.thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 4. Molecular graphics of ArCV-1 RdRp 3D structure at 7.9Å resolution.Molecular graphic of ArCV-1, RdRp 3D structure of 7.9Å resolution with central cavity flanked by N-terminus and C-terminus structures and the folding topology resemble the RdRps of picornaviruses: (A) Cartoon representation of the molecule in a conventional orientation of the closed right hand depicted with sub-domains, fingers (shades of blue- cyan), thumb (shades of red) and palm. (B) Cartoon representations of ArCV-1 3Dpol Motifs A, B, C, D, E and F are represented in blue, purple, light pink, cyan, brown and green, respectively. (C) Surface representation of the ArCV-1 3D polymerase showing a donut shaped structure with a central catalytic cavity and the top channel. Few of the representative motifs of ArCV-1 3D with residues known to interact with the RNA and the incoming NTP substrate were identified close to the catalytic center (GDD cyan). (D) ArCV-1 3Dpol, turned 90° to the front showing view from the top, with three subdomains, Fingers, Thumb and Palm farming putative RNA binding cleft. The N-terminal (1–55 residues) over layered on the enzyme surface to show the fingers region tether to the thumb region (shown as yellow coil with residues Arg-24, I-29, S-36 binding to the thumb subdomain). (E) Structural details of the altered conformation of motif A and B side view of the RdRp surface representation exposing the three channelsArCV-1 polymerase that are described for the picornoviruses. Positioning of channels as template channel (top) NTPs channel (rear) and product exiting channel (front) were denoted. The subdomain motifs A, B, and C in part, as cartoon was over layered to exact position. Figures were developed with PyMol.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.g004thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageTable 2. Percent sequence identity of dsRNA genomic segments of ArCV -1 with tripartite and bipartite cryptic viruses.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.t002The dsRNA-2 (HG967639) is 1,491nt long, with a single ORF (nt 207 to 1253) of a 38.5 kDa protein with 348 amino acids (P2). This RNA contains 206- and 237-nt as the 5’and 3’NTRs, respectively (Fig 2, Table 1) and the 3’ terminus contained the sequence “GCA CCCATATTC”. The P2 was identified as the capsid protein-1, having 40.6% amino acid sequence identity with corresponding protein of RsCV-1- (EU024677) (Table 2).The dsRNA-3 (HG967638) is 1,433 nt long, containing a single ORF between nt 171 and 1,205 with 170- and 227nt long 5’ and 3’ NTRs, respectively (Fig 2, Table 1) and the 3’ terminus contained the sequence “GCACCACGTTTC”. This ORF encodes a 38.51 kDa protein (P3) of 344 amino acids, identified as the second capsid protein. The P3 protein has 42.7% sequence identity with coat protein (RNA-3) of RsCV-1 (EU024677) (Table 2).The dsRNA-2A (HG917912), extracted along with the ArCV-1 genome was found in lower concentration in comparison to the genomic dsRNAs (Fig 1A). The RNA is 1,608 nt long, contains a single ORF that encodes a 44.5 kDa protein (P4) of 389 amino acids. The ORF starts at nt. 85 and terminates at 1,254 nt (Fig 2B, Table 1) with 84- and 353-nts as the 5’ and unusually long 3’- NTRs, respectively. Its 5’ terminus has AGAATTTGCCCTGTAT sequence and did not share sequence identity neither with the corresponding region of ArCV-1 genomic segments (Fig 3), nor with the C-terminus (TTC) of the capsid proteins.Amino acid sequence identity analysis of P4 revealed similarity with the putative genomic capsid proteins of several cryptic viruses (Table 3), as well as few eukaryotic genes of plant genomes. P4 shares 36.7% sequence identity s with coat protein of RsCV-3 (FJ461350), 35.8% with Citrullus lanatus cryptic virus (CiLCV; KC429583), 34.4% with PepCV-1 (JN117277) and 26.0% PepCV-2 (JN117279) (Table 3). Interestingly, the P4 protein has high degree of sequence identity with cryptic virus coat proteins of bipartite viruses (Table 3). Efforts were made to confirm the presence of a second RdRp assuming there may be in existence and dsRNA-2A belongs to another cryptic virus, but were not successful. In each attempt similar four dsRNAs were isolated. It is hard to explain, in absence of a related RdRp gene, and to speculate that the dsRNA-2A coat protein like sequence belong to a yet unknown second cryptovirus infecting pigeonpea.thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageTable 3. Percent sequence identity of amino acids of P4 protein (dsRNA-2A; HG917912) shared with cryptic virus coat proteins.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.t003Apart from this, the four RNA showed significant sequence homology with plant genes. Interestingly in BlastX, RNA-1 showed 64% sequence identity with unknown protein of Picea sitchensis or Sitka spruce (ADE76327), an evergreen conifer tree. RNA-2 showed 34% sequence identity with unknown protein (XP008459635) of Cucumis melo. Further, RNA-3 showed highest sequence identity with plant genes as 61% with uncharacterized proteins KOM46435 and XP017431256 of Vigna angularis. Similarly, dsRNA-2A shared 34% sequence identity with ATP-binding cassette (ABC) transporter F family member-1 (KHF98106) of Gossypium arboretum (tree cotton) found conserved in eukaryotes and prokaryotes. These eukaryotic lineages are not the known host plants for cryptic viruses.Structural analysis of the ArCV-1 RdRpThe peptide sequence of ArCV-1 RdRp (P1) was used for developing the 3Dpol structure. Structural templates were identified from the PDB and the 3D model created using Zhang lab server [37, 38]. PyMol was used to develop the three dimensional figures. Crystal structure of RdRp has been described depicting the “closed right hand” configuration [17, 39]. RdRps belonging to various genera of viruses containing amino acid sequence motifs are most conserved and occur in a typical order [20, 28, 40], reflecting structural conservation in most cases. Besides size, origin, and some structural variations, all classes of RdRp including several known picornaviruses share two main features by (1) having the 3D structure divided into three major subdomains described as palm, thumb and fingers [17, 39, 41, 42], and (2) the interconnected subdomains that encloses the conserved catalytic region (Fig 4, S2 Fig) within a largely hollow center referred to as the catalytic center [43]. The 55 kDa ArCV-1 3Dpol is a spherical molecule resolved at 7.9Å resolution showed the basic features described above comprising of characteristic 16 α-helices, 7 β-sheets interconnecting with coiled structures (Fig 4A) forming a structure of a cupped right hand. Unlike in picornaviruses the partitivirus genome is devoid of 5’ terminus genome-linked protein (Vpg) or particularly the polymerase with polyadenylated C’- terminal with the exception of CanCV (JN196536) and RsCV-1RNA-3 (DQ181927).The N-terminal residues (1–55) in fingers subdomain (Fig 4A, Blue) of the RdRp molecule extend over to the thumb subdomain binding them together (Fig 4A and 4D) encircling the molecule, ensuing central cavity. This architecture makes the molecule spherical or globular (Fig 4A and 4C), and not U shape normally seen in DNA polymerases and DNA-dependent RNA polymerases [44]. ArCV-1 3D pol has a big cleft opening in to central cavity groove in the front and one at the top left channel referred to as template channel leads to the centre of the cavity supposed to be allowing the access of the RNA template and NTP substrates to the central cavity [17, 33, 45, 46] and that central cavity opens out to the side on the right (rear channel) (Fig 4E).The N-terminal portion of the fingers subdomain is mainly composed of random coiled web (residues 1–55) on the top portion molecule sometimes referred as index finger with the N-terminal (Met 1) protruding out. N-terminal coiled structure runs across, connecting the fingers and the thumb subdomains, folds back on itself into a loop providing the conserved residues GWARS (53–57) seem to be unique to the bi- and tripartite cryptoviruses (S2A Fig) located at the top of the molecule.The fingers subdomain (residues 56–232 and 252–309) consist of eight α-helices (α2 to α7 and α9, α10) and four β-strands (β1—β2 and β4-β5). Broad structural variations are seen in fingers subdomain followed by the thumb in general than the palm in the viruses belonging to picornaviruses. Whereas these variations in fingers subdomain being less significant in cryptic viruses. The fingers region contains motif F and motif G, the latter being geometrically closer to the catalytic center of the palm subdomain.Motif F: A stretch of 42 amino acid residues mostly conserved in cryptoviruses spanning few residues before β-sheet 2 to α-6 and connecting coil containing conserved active residues as motif F. Like the N-terminal long loop, a portion of motif F extends from the fingers to interact with the thumb. The size of the loop, however, varies in different RdRps [21]. Motif F contains predominantly hydrophobic residues, along with at least six active residues (R183, W186, L203, Y214, G217 and D219) supposed to be interacting with the RNA in the ArCV-1 3D complex (S1 Fig) along with the conserved Lys181. Basic residues Lys181 and Arg183 of ArCV-1 3D RdRp have been identified associated with motif F of other RdRps; FMDV (K172). Lys172 residue along with two arginine residues (R168 and R179) predicted to interact with the incoming NTP substrate in FMDV RdRp [33]. Motif F of cryptoviruses unlike the other classes of RdRps is well conserved.Motif G: The motif consists of residues from amino acid positions 117 to 128 (SSAAGYGYVGLK). This motif is highly conserved in cryptoviral RdRps. Amino acid residues of motif G form part of a long coiled loop connecting α-helices 4 and 5 in the fingers subdomain (Fig 4B) of the 3D pol. Motif G is not well conserved in general and does not exist in Human immunodeficiency virus-1 (HIV-1) RT, but observed across several genera of plant, animal and human picornaviruses, as noted from RdRp sequences [47]. Glycine and lysine seem to be a strict requirement of motif G as they are conserved (Gly121 and Lys128 in ArCV-1) in all classes of RdRp. The conserved proline residue noticed in some picornavirus [Avian infectious bronchitis virus (AIBV), Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), RHDV] and dsRNA virus [Infectious bursal disease virus (IBDV), Infectious pancreatic necrosis virus(IPNV), bacteriophage Փ-6] RdRps, is not present in the highly conserved motif G of tripartite cryptoviruses (FcCV, RmCV, RoCV-1, and RsCV-2) including ArCV-1. Proline is substituted by glycine (Gly123), suggesting some flexibility at this position. It has been described that the residues of motif G contact the nucleic acid at its 5’ template overhang and form part of the channel for the template strand observed in the structures of reovirus and bacteriophage Փ-6 RdRps [12, 13], and the motif also predicted to be involved in positioning of the 5’template strand [21].The palm subdomain(residues 233–251 and 310–398) consists of three β-sheets (β-3, β-6, β-7) sandwiched between helices α-11 and α-12 and a long loop from α-12 connecting α-13 in the thumb, α-7 being at the junction of palm and thumb (Fig 4A). The palm domain shows the maximum conservation in the conserved motifs A, B, C, D and E (S1 Fig, Fig 4B), and has been shown to play different roles in the catalytic activity of the enzyme, found in most of the known RdRps [20, 28]. Palm sub-domain contained the structural architecture to suit to the functions, has remarkable alignment with the known RdRps. ArCV-1 RdRp sequence identity with cryptoviruses is high, like the structural alignment. Interestingly, with less sequence homology (Table 4) a close structural alignment was noticed between ArCV-1 and Sapovirus (SV) and Norwalk virus (NV) (+ssRNA) RdRps as evident from the panels shown in Fig 5E–5H. Residues playing key roles in active site interactions, distributed across the motifs A to E, in different classes of RdRps have been identified in ArCV-1 dsRNA RdRp as well as the RdRps of other cryptoviruses have been discussed as below.thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 5. Comparison of cryptoviral and picornaviral polymerase structures.(A) Cartoon representation showing ArCV-1 3Dpol; (B) Fragaria chiloensis cryptic virus (FcCV) (C) Sapporo virus (SV; PDB ID, 2wk4A) and (D) Norwalk virus (NV; PDB ID 1SH0). The molecules are shown in conventional right hand orientation with spectrum colors. The N-terminus colored blue and red for C-terminus for all the molecules. The three subdomains the fingers (shades blue and green) palm and thumb domains are in yellow and red. Carboxyl-terminal ends of the ArCV-1, FcCV, and SV were seen protruded out and interestingly in NV C-terminus was found to be located in the catalytic region of the central cavity. (E) ArCV-1, 3Dpol (blue) in a cartoon representation was superimposed by FcCV (green) polymerase. (F) in a backbone representation ArCV-1 3Dpol (blue), with every 40th residues numbered was superimposed by FcCV (grey) polymerase and (G) by Sapporo virus (green) polymerase (PDB ID, 2wk4A) and (H) by Norwalk virus (brown) polymerase (PDB ID, 2b43D) A high degree of alignment was noticed ArCV-1, 3Dpol with FcCV and SV followed by NV especially in the palm region. Structural comparison analysis provided for SV and NV an RMSD value of 2.10 Å and 2.15 Å, respectively.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.g005thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageTable 4. Structural sequence identity ranking of ArCV-1 RdRp to the top five identified structural analogs in PDB.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.t004Motif A consists of conserved residues present between the β-sheet 3 running into α-helix 8. The RdRp contained active residues D242, F246 and D247 are conserved in motif A of several picornaviruses. Motifs A and C located in the 3Dpol structurally adjacent to each other contain aspartate residues (D341), involved in a two-metal (Mg2+ and/or Mn2+) mechanism of catalysis [48–50]. Motifs A, B, and F are key for nucleotide triphosphate (NTP) binding, motifs A and C are also involved in binding of metal ions [51].Motif B: It consists of conserved residues as a single block (S1 Fig) containing Ser305, Cys308, Thr310, Ile312 and Asn311 (α-helix 11; Fig 4) and are predicted to play a major role in the catalytic process conserved in picornaviruses as well (Fig 6). In the N-terminal of the motif, the coil forms a loop commonly referred to as B-loop (Ser, Gly, Ser and Cys), connecting to the long α-helix11. Motif B provides important orienting interactions with the incoming deoxyribonucleoside rNTP [52], and the B-loop has been described structurally flexible and moves its conformations [53], the key residue glycine (G306 in ArCV-1) was described to act like a “hinge” for the movement. Substitutions of this glycine residue in 3Dpol basically abolished RNA synthesis in vitro [35]. The high sequence and structural conservation of the B-loop among viral RdRps have been reported [17]. Rearrangement residues (SGSCFTNIIGSI) of ArCV-1 motif B which are conserved in cryptoviruses (S1 Fig and Fig 6) did not affect the structural sequence identity of the motif with picornaviruses. While describing the function of B-loop in FMDV the authors speculated that the rearrangements in the template channel and the B-loop occur in a concerted manner and that these changes collectively serve to regulate RNA replication, processivity and fidelity, and the active residues in motif B, each has a definite role to play [17].thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 6. Conserved residues the RdRp B motif of ds RNA cryptovirus and ss (+) RNA picornaviruses.The RdRp multifunctional B motif, comprising of conserved residues, forms structurally important entity containing B-loop (see Fig 5F) as demonstrated in picornaviruses. Shown here is a similar motif in cryptovirus RdRps with the conserved gly306 residue that acts as axis for the conformational changes, highlighted in green block. The positions of the catalytic Asn (in α-helix11, predominantly substituted by gly314 in dsRNA viruses) and of the Ser and Thr residues of picornaviruses interacting with the incoming NTP substrate also are common to cryptovirus RdRps (details in Text). ArCV-1 (HG797710), FcCV (DQ093961), RmCV (EU024675), RoCV-1 (EU413666), RsCV-2 (DQ218036), Phi6 (PDB 4A8W), Reovirus (RV; PDB 1MUK), SV (PDB 2WK4), NV (PDB 2B43), RHDV (PDB 1KHW), FMDV (PDB 1WNE). Middle = middle finger of the fingers subdomain; Loop = structures in palm subdomain.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.g006Motif B takes part in template binding, incoming nucleotide recognition [39] and determining whether RNA or DNA will be synthesized by selecting rNTPs and dNTPs and correct positioning of the sugar in the ribose-binding pocket [54], asparagine residue thought to be playing a vital role in its functioning. Briefly, Asn residue in this motif (Asn307 in FMDV) has been confirmed to interact with incoming rNTP (play a role in differentiating between dNTPs and rNTPs and thus determines whether RNA or DNA is synthesized) during RNA elongation in a number of picornavirus elongation complexes [39, 49, 54–56]. Similar positioning of Asn residue (N297) is present in the long helix α-H [39], 6.6Å away from the active aspartate of the catalytic center. However, the highly conserved Asn residue which is critical for enzymatic activity was not evident in motif B of the ArCV-1 RdRp (Fig 6). This is a conspicuous difference between the RdRps of picornaviruses and cryptoviruses. The Asn residue appears to be substituted by glycine (G314), present at the same position in the α-11 at a distance of 6.9Å away from the active aspartate of the catalytic center and is conserved in most of the dsRNA cryptovirus RdRps and also exist in phage Փ-6 motif B (Gly403). Crotty et al. [49] demonstrated that an absolutely conserved residue (Asn) of motif B within the nucleotide binding pocket of the poliovirus polymerase can be substituted for a different amino acid, yielding replication-competent virus. Glycine that replaced the Asn in cryptoviruses does not have amide group [49]. Employing one of the PV mutants (PV-297G) developed; authors explained that the molecular modeling suggests that a glycine at position 297 leaves sufficiently a large pocket for an additional water molecule [49]. Therefore, glycine may substitute for asparagine 297 by allowing a water molecule to become the hydrogen bonding partner for the NTP 2′OH. These results attest to the extreme evolutionary flexibility of the viral RdRp, in terms of both structure and cation usage [49], and could explain, devoid of Asn (Fig 6), in motif B of ArCV-1 and other cryptoviruses the polymerase is able to function.Motif C is comprised of β-sheets-6 and 7 in an anti-parallel manner connected by a loop towards the central cavity and this loop bares the highly conserved Gly340-Asp341-Asp342 observed in most of the RdRps, now observed in plant cryptic virus RdRps. Like the B-loop the beta bend GDD motif is structurally similar in all classes of RdRps, described as the center of catalytic activity of the enzyme in coordinating the metal ion. The first aspartate (Asp341in ArCV-1) is known to facilitate the rNTP transfer and by binding to the metal ion at the catalytic site. It has been shown that first N-terminus aspartate is absolutely required for enzyme function [29, 32, 35, 57, 58]. The presence of the second aspartate almost is universal in GDD motif with a couple of exceptions [replaced by asparagine (ADN) in IBDV and glutamate (GDE) in case of KF of E. coli DNA polymerase I] signifying flexibility at this position. But any substitutions made in the second aspartate were not tolerated as reported in poliovirus RdRp [35, 58]. Phage Փ–6 and IBDV (dsRNA) RdRps contain the catalytic motif as SDD and ADN respectively that makes the requirement for the glycine residue of GDD motif also somewhat flexible in vitro. However, substitutions at glycine position in Encephalomyocarditis virus(EMCV) 3Dpol make the RdRp completely inactive in vitro [35], as in the case of potatovirus X (PVX) RdRp in vivo were not tolerated [57], suggesting that the requirements for this position may be more strict [59], with some RdRps to function. Aspartate in Motif A interacts in catalytic reaction as they, together with the first aspartate of GDD, coordinate the rNTP transfer [60]. ArCV-1 3Dpol contains a couple of highly conserved aspartate “active residues” in motif A; D242A and D247A present at a distance of 12.4Å to each other (S3 Fig) and the D242A, 7.0Å from D341 are similar to those referred above [60], could be interacting with Asp341 by coordinating catalytically essential metals as noticed in many RdRps [23, 39, 61]. Many of the active residues of the conserved motifs (S1 and S2 Figs) present in the central conserved region of the RdRp were located in the central cavity or close by. This region forms a domain that is partially resistant to protease digestions [59].Motif D has been identified in ArCV-1, 3Dpol in a long loop between α-helix12 and α-helix13 (Fig 4A and 4B). A positively charged residue lysine373 along with serene is conserved in this motif (DKSD) across all cryptoviruses (S1 Fig). Lysine seems to be sustained in the sequences of motif D of all RdRps [20]. Motif D is inconsistent and not so well conserved in members of the family Picornaviridae and the cryptoviruses analyzed in the present study. Structural variability was noticed in the reported RdRps as well, but the majority showed motif D in a coil. The N-terminal Asp372 in the motif of the 3Dpol was conserved in many primer-dependent RdRps of calciviruses and picornaviruses, but not in the rest of cryptoviruses and in de novo RdRps (dsRNA-phage Փ-6, IBDV and reovirus). Active residues in motif D have been postulated to play critical roles in catalysis [62]. Motif D for long considered “scaffolding” for the palm, keeping the structural integrity [39, 59]. Movement of motif D facilitates active site closure and is sensitive to incorrect NTP binding and mismatches at the RNA terminus. These studies link the conformation of motif D to the efficiency and fidelity of nucleotide addition in elongation [63]. Closing and reopening of the active site happens as a result of association of motif D with motif A, which undergo coordinated conformational changes [64].Motif E was located at the junction of the palm and thumb, and was described as not integral to the conserved core structure [39], contains characteristic motif XFL where leucine was predicted as active residue. The conserved sequence at amino acids position 384 to 388 (TFLSR) was detected in the ArCV-1 3Dpol at the mentioned location α-helix turn α-helix as part of the long moving loop structure, adjacent to motif D close to the catalytic center (Fig 4B and 4C). There was variation in the amino acid T384 position in the cryptovirus primary sequence (S1 Fig) as well as in picornaviruses. The hydrophobic L386 residue of this motif was found in the analysis to be active residue; described essentially for catalytic activity. This motif described interacts with the elements of β-sheets of the palm core structure and the elements in the thumb subdomain in both poliovirus RdRp and RT [39, 65]. Substitution in the vicinity of active leucine residue is not tolerated in polio virus and an insertion of a leucine after R376 of PV (R388 in ArCv-1) was shown to abolish replication in vivo and in vitro [30], and a double mutant K375A/R376A of the same virus was found to abolish viral replication in vivo [66], suggesting that the critical role these amino acids play in RdRp functioning.Thumb subdomain (residue 399 to residue 475) constitutes mainly of helical structures. Out of four α-helices, α-13 and α-14 helices adjacent to each other runs antiparallel while α-16 helix runs across and ends with protruding Gln 475 residue (Fig 4A). The α-13—α-14, and α-15—α-16 are interconnected resulting in larger loops at the crest of the thumb subdomain and bending over forward to tie into the N-terminal region of the finger tips, creating a complete annulus around the catalytic site on the palm (Fig 4A). The small size of the domain contributes to the formation of a large central cleft [45], which is located at the front of the molecule (Fig 4A, 4C and 4D) and leads to the active site shown in the surface representation of the molecule. ArCV-1, 3Dpol visually seems to be having similar central cavity architecture and dimensions with FMDV, PV and HRV-16. The C-terminus region is conserved amongst the cryptoviruses studied showing conserved residues in motifs that are not observed in the single stranded picornaviruses or in the dsRNA viruses. CLRML (402–06) motif as part of α-13, facing the central cavity, the rest YPEY (408–11), DAG (429–31), and WPD (461–63) motifs are present in the remaining 3 helices.The overall topology of ArCV-1 3Dpol smaller thumb subdomain structure resembles more with ssRNA (+) RdRp analogs of NV, SV, HRV, RHDV, PV and FMDV than the de novo dsRNA viruses. The main structural differences between RdRps of ArCV-1 and the closely related FcCV and RoCV-1, RmCV, RsCV-2 (Deltapartitivirus) are in the thumb region (Fig 5A–5D). The thumb subdomain of these cryptoviruses contained four α-helices (α-α-α-α) on the contrary FcCV thumb had an extra helix making total thumb α-helices to five (β-β-α-α-α-α-α). The second difference noticed was the palm and thumb interface region. ArCV-1 3D pol, consists of long coiled loops connecting α-12 (palm) and α-13 (thumb) with no β-sheets in this region. While the RdRps of other cryptoviruses consists of two antiparallel β-sheets followed by motif E as was observed in NV and SV (Fig 5A–5D) making the thumb configuration as β-β-α-α-α-α.The thumb C-terminus observed in ArCV-1 3Dpol was protruded out away from central cavity like other cryptoviruses except in case of RsCV-1 (Alphapartitivirus) where the C-terminus helix (α-16) extends in to a compact tuft of coil packs against the front face of the molecule and the end pointing towards the central cavity, a similar structure (S3 Fig) seen in rice tungro virus [RTSV; ssRNA (+)] a plant picornavirus and NV (S3D Fig, Fig 5D). The C-terminus of RmCV (EU024675-77) genomic segments has a unique poly “A” tract. Flaviviruses, IBDV, Bacteriophage Փ-6and Reo (dsRNA) viruses with larger polymerase molecule contain elaborate thumb architecture with 7 to 15 α-helices and 2–4β-sheets. The phage Phi6 pol C-terminal extends back ploughing parts of it through the molecule and ends in the middle finger region of the other side [12]. Interestingly, substitutions or deletion of specific residues in the thumb subdomain of Brome mosaic virus (BMV 2a) and poliovirus 3Dpol abolish RNA replication [30, 67].Structural comparison with other RdRpsThe analysis showed ArCV-1 RdRp structural alignment with the viruses belonging to Picornaviridae and Deltapartitiviridae (Fig 5 and S3 Fig). The range of identity varied with the ranking viruses which had high identity with the RdRp of ArCV-1. Different genera of picornaviruses showed high template modeling (TM) score with ArCV-1 3D pol; NV (0.898) [68], SV (0.898) [69],Rabbit hemorrhagic disease virus (RHDV) (0.895) [70], all members of the family Caliciviridae (Fig 5G and 5H) and with moderate TM score with alignments of viruses members of the family Picornaviridae; FMDV (0.834) [33], EMCV (0.830) [71] and Poliovirus (PV) (0.823) [39]. These economically important viruses are human and animal pathogens and the structural details are available in PDB archives which doesn’t have the information on the RdRps of cryptic viruses to compare. For the first time using computer analysis we are reporting structural details of RdRps of few plant cryptic viruses with high structural similarity to ArCV-1 3Dpol (Fig 5E and 5F). Plant cryptovirus and human picornovirus RdRps that showed structural identity were superimposed on ArCV-1 3D RdRp in a cartoon and backbone representations. ArCV-1, 3Dpol (blue) was superimposed by FcCV (green) RdRp (Fig 5E) in a cartoon representation showed exact structural alignment. Similar high-level alignment was noticed when ArCV-1 3Dpol (blue), superimposed by FcCV (grey) RdRp (Fig 5F), by SV (green) RdRp (PDB ID, 2wk4A) (Fig 5G) and by NV (brown) RdRp (PDB ID, 2b43D) (Fig 5H) in a backbone representation. Every 40th residue from N-terminal to C-terminal was numbered in a stereo back the bone representation of the RdRps. A high degree of alignment was noticed ArCV-1, 3Dpol with FcCV and SV followed by NV especially in the palm region containing the A to E motifs with conserved residues. Structural comparison analysis provided for SV and NV an RMSD value of 2.10Å and 2.15Å, respectively. Our findings validate the analysis predictions and reveal a possible evolutionary linkage between cryptoviruses (dsRNA viruses) and the picornaviruses (+ssRNA viruses).Phylogenetic analysis.BLAST analysis involving several tripartite cryptoviruses revealed that the ArCV-1 genome shared a low level of identity with the orthologs, mentioned in Table 2. Besides 5’ terminus 16 nt similarity, a high degree of sequence homology between the ArCV-1 5’NTR sequences of the three genome segments was noticed while lacking decipherable identity in the 3’proximal un-translated regions. The genomic segments of several Partitiviridae members usually have sequence homology in the terminus ends [72, 73]. ArCV-1 RdRp (dsRNA-1) and dsRNA-2A sequences were used in the phylogenetic analysis with corresponding sequences of members of the genera Alphapartitivirus, Betapartitivirus, Gammapartitivirus, Deltapatitivirus and Cryspovirus (Fig 7) mentioned in the S1 Table. The analysis revealed that the viruses used in the study assembled in five groups, as recognized previously [8]. ArCV-1, dsRNA-1 clustered with tripartite RoCV-1, FCV and FcCV and with members of the genus Deltapartitivirus (Fig 7A). Phylogeny of dsRNA-2A revealed close relationship with members of bipartite Deltapartitivirus; PepCV-1, PepCV-2, RsCV-3 and CiLCV (Fig 7B). The findings further support the hypothesis that dsRNA-2A does not belong to the tripartite ArCV-1.thumbnail Download:PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 7. Phylogenetic analysis of ArCV-1 dsRNA1 (RdRp) and dsRNA-2A.(A) Phylogenetically though all members originate from common ancestor as they diverge from a single clade formed five distinct groups corresponding to their genera (Group 1- Alphapartitivirus; Group 2- Betapartitivirus; Group 3- Gammapartitivirus; Group 4- Deltapartitivirus and Group 5- Cryspovirus). Members of group 1 and 2 clustered separately but originate from common point however origin of members of group 3 were different. Group 4 and 5 originate from common sub-clade, which is different from other groups. ArCV-1 RNA-1 clusters with tripartite members of the genus Deltapartitivirus and is closely related to FcCV and RsCV-2. *Beet cryptic virus-2 was an assigned member of Deltapartitivirus but it was noticed to fit in with Alphapartitivirus. (B) Phylogenetic analysis of dsRNA-2A (P4 protein), displays its proximity with bipartite PepCV-2 and RsCV-3. The bar represents base substitution per site. Sequences of different partitiviruses from NCBI database were used in the analysis (S1 Table). RdRps of hPBV (AB186898) and oPBV (JQ776552) and CP sequence (hPBV: AB186897 and Q776551) were used to provide an out group root in the analysis.Molecular characterization of a novel cryptic virus infecting pigeonpea plantsA new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5’ non-coding region (5’-GATAATGATCCAAGGA-3’). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.https://doi.org/10.1371/journal.pone.0181829.g007DiscussionIn this study, the complete sequence of four dsRNAs isolated from asymptomatic pigeonpea plants was determined. Three dsRNA segments formed the genome of a new cryptovirus and named Arhar cryptic virus-1 (ArCV-1). The fourth dsRNA (dsRNA-2A; P4) was not related to the ArCV-1 genome and has characteristics like a viral coat protein. ArCV-1 dsRNA-1 dsRNA-2 and dsRNA-3 have been identified as encoding RdRp, CP-1 and CP-2, respectively (Fig 2A). Concentration of dsRNAs varied, dsRNA-1 (RdRp) was the most predominant transcript followed by the two capsid proteins (Fig 1). The ArCV-1 genome segments shared high degree of sequence identity in the 5’NTR including the marker 5’terminus 16 nt conserved sequences. Similar conservation in the tripartite viruses (Fig 3) was noticed; FcCV, RoCV-1, RsCV-2 [7, 74, 75], and were clustered together phylogenetically to form the genus Deltapartitivirus, a member of the family Partitiviridae (Fig 7A). The 3' NTR (50 nt) was rich in GC content and the three dsRNAs of ArCV-1 terminated with TC (Fig 2, S1 Fig). Furthermore, these findings were supported by a phylogenetic analysis which revealed that ArCV-1 formed a unique phylogenetic cluster with FcCV, RmCV and RoCV-1 within the genus Deltapartitivirus, infecting different genera of the family Rosaceae.The status and role of dsRNA-2A which encodes 44.5kDa protein (P4), which was found in least concentration, is not known. Association of an additional dsRNA segment (s), discrepancies with cryptic virus infections was not uncommon. In an earlier finding, the association of an extra component in many Vicia faba cultivars infected with Vicia crypticvirus (VCV) was reported [72, 76]. While characterizing the cryptovirus infection of radish (Raphanus sativus-root cv.Yidianghong) the authors suggested that more than one partitivirus was co-infecting radish leaves [73, 75]. The second virus in the complex has two smaller segments (RasR4 and RasR5) contained conserved 5’ termini sequence with RasR3 (RdRp) deem to be with unknown functions not having sequence identity with the known capsid proteins. In the present study, the four dsRNA shared sequence identity with members of Deltapartitivirus. The dsRNA-2A, (P4) contains sequence that resembles viral coat protein showed an unusually long 3'-terminal 353nt NTR region. The 5’-terminus of this dsRNA is different, does not share sequence identity with the 5’-terminus conserved genome segments of ArCV-1. However, P4 showed sequence identity with bipartite and tripartite cryptovirus coat proteins with pronounced resemblance to the bipartite cryptoviruses (Table 3). Phylogenetic analysis indicated that the P4 was closely related to RsCV-3 segment-2; coat protein (FJ461350). This indicates that the origin of P4 is related to Partitiviridae, but we have no evidence that P4 may interact with ArCV-1 in its replicative cycle or it is a derivative of rearranged sequences of the ArCV-1 coat proteins-highly unlikely or it could be part of the genome of evolving yet another cryptic virus.Primary sequence analysis of ArCV-1 RdRp revealed, besides several conserved sequences amongst the cryptoviruses; at least seven conserved signature motifs in a particular order which were well characterized [20, 28, 59]. These motifs were aligned with the RdRps of picornaviruses ssRNA (+) that led us to examine the structural similarity, if any, of ArCv-1 3Dpol with known RdRps in further detail. However, detailed studies of RdRps encoded by partitiviruses have not been reported. Amongst the double-stranded viruses studied; representatives of Cystoviridae [12], Reoviridae [13], and Birnaviridae [14, 77], in terms of the structure, mechanism of initiation of replication and transcription by their active RdRp was explained with the 3D structural analysis.This report illustrates several interesting features of the ArCV-1 RdRp and a cryptovirus 3Dpol and full-length structure was determined for the first time. It was revealed that the 3Dpol shared structural details more with calciviruses, members of the family Picornaviridae. Computational analysis was used to develop and characterize ArCV-1 3D pol structure at 7.9 Å resolution. We studied several RdRp analogs in the PDB which showed homologous structural identity with ArCV-1 RdRp mostly of animal and human picornaviruses. Unlike the genomic RNA of picornaviruses, the ArCV-1 dsRNA-1 that encodes RdRp (55kDa) protein does not show 5’ cap or VPg or 3’ polyadenylation, but a stretch of NTRs present on both the termini with no internal structures. The 3Dpol structures of Norwalk virus, Poliovirus and Sapporo virus which were previously determined and the respective sequences used, produced identical 3D structures in our computer analysis, confirming authenticity of ArCV-1 structural analysis.The overall topology of the ArCV-1 RdRp structure resembles the general description of the three-dimensional structures of the primer dependent initiating single stranded RNA (+) RdRps [33, 39, 45, 68–71, 78–80], depicted as a closed right hand (Fig 4A), differentiated as fingers, palm and thumb sub domain [52]. 3Dpol structure of ArCV-1 and other cryptoviruses analyzed are found to be different from the dsRNA viruses; bacteriophage Փ-6, Reovirus and IBDV [12–14, 77]. In general, the topology of the cryptoviruses; BCV-2, FcCV, RmCV, RoCV-1, RsCV-2, RsCV-3 (Deltapartitivirus) and RsCV-1 (Alphapartitivirus) RdRps, had close identity with ArCV-1 with minor differences. The ArCV-1 RdRp sharing 72.4% sequence identity with FcCV and only 14.8% sequence identity with RsCV-1 (Fig 4). Despite low amino acid identity (15%), interestingly the ArCV-1 RdRp shares more structural identity with the members of the family Caliciviridae; NV and SV, (Fig 5, S3 Fig). The structural identity of NV at 2.17 Å resolution (1SH0, 2B43) and SV at 2.3 Å resolution (2CKW) 3Dpol with the ArCV-1 RdRp, indicate a closer evolutionary link between the very divergent groups of viruses, and the polymerase has no structural alignment with other dsRNA, bacteriophage Փ-6, reovirus and IBDV which have larger RdRp molecules with complex dense subdomain structures.The analysis of RdRp sequence and structure revealed the occurrence of motifs A to G which play an important role in NTP binding and catalysis [70], described for Calicivirus and Picornavirus RdRps has structural similarity (Fig 4B) as described earlier. The similarity was confirmed by the visual observation of their superimposed structures (Fig 5E and 5F). The unique feature noticed in ArCV-1 RdRp is an N-terminal portion of the fingers reaches to the thumb bridging the fingers and thumb sub domains [43], was observed with all cryptoviruses studied. This feature was noticed in ssRNA (+) and dsRNA RdRps visualized in less cluttered IBDV RdRp. Fingers subdomain contains motifs F and G. Motif F of cryptoviruses unlike the other classes of RdRps is well conserved and extends from few residues before β-sheet 2 to α-helix 6 and the connecting coils. Computer analysis predicted that Motif F contains several active residues supposed to be interacting with the RNA in the ArCV-1 3D complex were detected (Fig 5B, green colour, S1 Fig). Basic residues Lys181 and Arg183 of this motif were also conserved in other RdRps; for example, in FMDV motif F, Lysine residue along with two arginine residues (K172, R168 and R179) predicted to interact with the incoming NTP substrate [33]. Motif G forms part of a long coiled loop connecting α-helices 4 and 5 in the fingers subdomain (Fig 4B, red). Motif G is highly conserved in the cryptoviral RdRps. The conserved Gly121 and Lys128 (in ArCV-1) of this motif in all classes of RdRps seem to be a strict requirement of motif G.Mutational analyses in PV and FMDV also have demonstrated that some substitutions in residues located far from the active site, in particular at the RdRp N-terminus, have significant effects on catalysis and fidelity [17]. Several highly conserved stretches of sequence motifs of ArCV-1 in the N-terminus to the central region, have been identified uniquely to cryptic virus RdRps. Motifs 1 and 2 are observed in the RdRp sequence away from the palm subdomain could play a role in catalysis (S1 Fig).The palm sub domain contains A to E motifs in a series. β-sheet 3 to α-8 consists of residues 241–251 were identified as Motif A (Fig 4B, blue color) contains conserved Asp242 and Asp247. Like SV and NV, RHDV 3Dpol also has a high degree of structural homology with ArCV-1 (Fig 5 and S3 Fig) and the aspartate residues share identical positions in motif A and C (AspA250 and AspC354 in RHDV). It has been described that metal ions that are likely to be involved in the nucleotide transfer reaction, interact with aspartate residues at positions 250, 354, and 355 in RHDV [70]. Similarly, in ArCV-1 3Dpol the corresponding two aspartate residues AspA242 and AspA247 which are separated by a distance of 12.4Å and AspA242 being closer (7.0 Å) to the catalytic aspartate AspC341, could be performing the same functions. Ser, Gly, Thr and NS/T are identified as highly conserved active residues in motif B (Fig 4B, purple), each with specific function in many picornaviruses. Asn residue (Asn307 in FMDV) of motif B plays a role in differentiating between dNTPs and rNTPs and thus determines whether RNA or DNA is synthesized [39, 55]. Later it was determined that the active Asp residue of motif A, and Asn of motif B (Asp240, Asn307 in FMDV) play a critical role in rNTP selection as evident from the FMDV replication process [33]. Asparagine residue is highly conserved in the long helix, about 6.6Å away from the active aspartate of the catalytic center in picornaviruses. On the contrary, the Asn residue was substituted by Glycine (314) present almost at the same position (Fig 6), in the α-helix (α-11 in ArCV-1), conserved in most of the dsRNA cryptovirus RdRps. In poliovirus mutational studies, Asn (297) was substituted by glycine (PV-297G) resulted in replication-competent virus, which probably explains how cryptoviruses are able to multiply with altered functional residue (Gly314) involved in rNTP selection (S3A Fig) [49]. Rearrangement (SGSCFTNIIGSI) of few residues of the ArCV-1 motif B which are conserved in cryptoviruses did not affect the structural similarity of the motif with picornaviruses. The architecture of the palm subdomain of ArCV-1 3Dpol with three β-Sheets (β-3, β-6 and β-7) sandwiched between two α-helices (α-7 and α-12) is highly conserved feature in most of the RdRps as motif C (Fig 4B, pale pink). The anti-parallel β-Sheets 6 and 7 connected by a short loop containing GDD (Asp 341 and Asp 342) sequence described as catalytic center. Structural properties and catalytical aspects of palm and thumb (residue 399 to residue 475) sub-domains are discussed in greater detail in the results section.Understanding structural details of known viral RdRps and associated mechanism described for the initiation of RNA synthesis and chain elongation reveal, that these viruses use different strategies to initiate replication. It has been described that viral replication can be initiated by two principally differing mechanisms; the primer-dependent [42], and primer independent or de novo initiations [81–83]. However, Influenza virus employs a combination of the two mechanisms with the choice being determined by the type of RNA to be synthesized [84]. The de novo synthesis involving the interactions of required components, the initiation essentially a one-nucleotide primer provides the 3’-hydroxyl for the addition of the next nucleotide [42]. The de novo RdRps have specific structural elaborations that function to stabilize the initiation complex. Such initiation platforms have been found in the crystal structures of viral RdRps of Cystoviridae, Reoviridae and Birnaviridae [12, 13, 77]. In the second mechanism, the primer-dependent initiation requires the use of either an oligonucleotide or a protein primer to provide the hydroxyl nucleophile. Members of the Picornaviridae family use exclusively the protein-primed mechanism of initiation [17, 81].Viruses belonging to different groups containing ssRNA (+), dsRNA as genomes adopt de novo initiation mechanism. For example, members of the genus Flavivirus: hepatitis C virus (HCV) [85], and dengue virus (DENV-2) [86], as well as Enterobacteria phage Q beta (Qb) [87], all have ssRNA (+) genome. The second group to adopt de novo initiation are the dsRNA viruses namely, cystoviral bacteriophage Փ-6 [12], Infectious bursal disease virus(IBDV) (family Birnavirdae), [14, 77], and reovirus (family Reoviridae) [13].By contrast, very little is known about knowledge of the mechanisms underlying dsRNA cryptovirus (Partitiviridae) replication as the polymerases of this group have never been studied. ArCV-1 has segmented linear dsRNA genome, each genomic segment is monocistronic: dsRNA-1 that encodes RdRp does not have either 5’ termini or 3’-end structures like template-directed nucleic acid polymerases and does not resemble the vastly intricate 3Dpol structure of known dsRNA bigger molecules that adopt de novo, to speculate the polymerase preferred mechanism.Unlike the de novo RdRps, primer dependent RdRps of the viruses of Caliciviridae and Picornaviridae have significantly smaller thumb subdomains, wider template tunnels and large central cavity with exposed active sites [33, 45] as observed in ArCV-1 3D pol. FMDV RdRp was shown to have the wide enough central cleft to accommodate the bulky priming peptide during the initiation of RNA (complexed with RNA, divalent cation and protein primer VPg), synthesis [46]. Similar structural details of the polymerase mentioned above share a number of unique features by ArCV-1 and other cryptoviruses (Fig 4A, 4C and 4E). If the structural architecture reflects the functions of a polymerase as often supposed [17], the simple thumb organization, arrangement of the motifs (A to G) in a structural order (Fig 4B), large central cavity and the entry and exit channels like in picornaviruses and having remarkable structural conservation with SV, NV and RHDV 3Dpol, lead to a strong possibility that ArCV-1 RdRp could adopt primer dependent initiation mechanism. This important aspect of research of cryptovirus RdRps is yet to be explained. The remarkable structural similarities between ArCV-1 and the other tripartite cryptoviruses (dsRNA) and the positive-stranded RNA viruses of the picornavirus family, in particular, caliciviral RdRps, offer evidence of probable functional and evolutionary relationships between these two virus groups.Several highly conserved stretches of sequence motifs have been identified that seem unique to cryptic virus RdRp; Motifs 1–2, observed in the RdRp sequence away from the palm subdomain are inimitable and observed in both bi- and tripartite cryptoviruses (S1 Fig), whose exclusive or overlapping functions are yet to be determined. We hope in the near future, biochemical analysis experiments would reveal the function (s) and their relevance in RNA synthesis. Substantial data that has been generated on ArCV-1 3Dpol is being authenticated by X-ray crystallography studies and biochemical analysis.Material and methodsSample collectionLeaf samples of pigeonpea were collected from fields near the Chevella area of Hyderabad; (Telangana state, India). A popular local pigeonpea cultivar Erra Kandulu, is being traditionally cultivated in this area which is susceptible to sterility mosaic disease (SMD), fungal blight, and wilt diseases. Leaves from four healthy looking plants, with no disease symptoms, were collected randomly from three fields MG-1, MG-2 and MG-3 (approximately three km apart). Collected leaf samples were designated as Mg-H1, Mg-H2 and Mg-H3 respectively, along with the SMD samples. Leaf samples were placed in separate ziplock bags placed in ice chest with dry ice and transported to the lab. For sample collection, no specific permissions were required from these locations, as these were collected from a private farm land. Also, this study did not involve endangered or protected plant species.Extraction and purification of dsRNALeaf samples, Mg-H1 Mg-2 and Mg-H3 were used for dsRNA extraction [88]. Briefly, 7g leaf tissue were crushed to fine powder in liquid nitrogen and homogenized in 20 ml of 2× STE extraction buffer (0.1 M Tris-HCl, pH 7.0; 0.2 M NaCl; 2 mM EDTA), containing 1.5% β-mercaptoethanol, 1.5% (w/v) polyvinylpyrrolidone (PVP), 2% (w/v) SDS, and 16 mg of bentonite powder and incubated at 37°C for 15 min. The clarified contents were extracted with equal volume of Tris (pH 8; phenol pH 4.5 ± 0.2) saturated phenol: chloroform: Isoamyl-alcohol (25:24:1) mixture followed by vigorously shaking at room temperature for 20 min. The contents were centrifuged at 11,000 rpm (Heraeus Multifuge 35R+, Thermo Scientific, USA) for 10 min. One fifth volume of ethanol was added to the supernatant and mixed well, followed by the addition of 1g of cellulose CF-11 (Whatman, USA) and the slurry was incubated at room temperature for 30 min with continuous shaking. The mixture was centrifuged at 5,000 rpm for 5 min. and the cellulose pellet with dsRNA was resuspended in 40 ml wash buffer (1× STE with 16% ethanol) and washed twice for 5 min with mild agitation. The cellulose slurry was applied to a 20 ml syringe blocked with glass wool and washed with 20 ml of washing buffer. The bound dsRNA was eluted stepwise (two times) with 15 ml elution buffer (1× STE without ethanol). The eluted dsRNA was further purified by adding 1/5th volume of ethanol and 0.8 g CF-11 cellulose and the suspension was vigorously shaken for 30 min at room temperature. The suspension was transferred to a new 20 ml syringe and the dsRNA was eluted again twice with 2.5 ml elution buffer. The extracted dsRNA was precipitated by adding 1/10th volume of 3 M sodium acetate pH 5.2, and 2.5 volumes of absolute ethanol and incubated overnight at -20°C. The contents were centrifuged at 14,000 rpm for 20 min at 4°C and the resulting dsRNA pellet was washed with 80% (v/v) ethanol, air dried and resuspended in 500 μl MilliQ water. MgCl2 was added to the extracted dsRNA to a final concentration of 300 mM and was digested with 500 ng RNase A and 2U DNase I (RNase-free) at 37°C for 45 min followed by phenol/chloroform extraction and precipitating with ethanol. The precipitates were suspended in 500 μl of MilliQ water. Purified dsRNA were separated by electrophoresis on a 1.5% agarose gel in 1x TAE buffer and stained with ethidium bromide and documented by gel doc system.Anchor-primer ligation of dsRNAThe obtained dsRNA was resolved on 1.5%agarose gel and the dsRNA segments were excised. DsRNA molecules that were close to each other were excised together as a mixed population. These dsRNAs were further purified by gel elution kit (Macherey Nagel, Germany). 200–300 ng of dsRNA was used for self- priming anchor primer ligation in a 60 μl reaction with T4 RNA ligase (Fermentas, Thermo Scientific, USA). Self-priming oligo (5′p-GACCTCTGAGGATTCTAAAC/iSp9/TCCAGTTTAGAATCC-OH 3′), having C9 internal spacer region (iSp9) was used for ligation at 3′end -OH group of dsRNA [89]. The ligation buffer [90] was modified to contain 50 mM HEPES/NaOH buffer pH 8.0, 15 mM MgCl2, 0.01% BSA, 0.75 mM ATP (Fermentas, Thermo Scientific, USA), 1.5 mM DTT (Fermentas, Thermo Scientific, USA), 8% DMSO (Sigma, USA), 15% polyethylene glycol 6000 (PEG6000) (Himedia, Mumbai, India) and 10 U T4 RNA ligase (Fermentas, Thermo Scientific, USA). Ligation was performed at 37°C for 12–16 hrs.Full-length cDNA synthesis and PCR amplificationLigated dsRNA was purified using gel extraction columns (Macherey-Nagel, Germany) in 15 μl volume. About 50–70 ng of ligated dsRNA were denatured in the presence of 1.5% DMSO and 2M betaine at 98°C for 2 min (optimal) and immediately chilled on ice. The denatured dsRNA was used for the first strand cDNA synthesis by reverse transcription (RT) reaction. The RT reaction mixture consisted of 1X RT buffer, 1 μl dNTPs (10 mM each), 1 μl (200 units) of RTase (Primescript, TakaraBio, Japan) and the final volume was adjusted to 25 μl with nuclease free water. The RT reaction mixture was incubated at 42°C for 1 hr and then 70°C for 15 min to deactivate the enzyme. The cDNA obtained was amplified by a primer (5′- GAGGGATCCAGTTTAGAATCCTCAGAGGTC-3′) complementary to anchor sequence [89]. Briefly, PCR reaction contained 25 μl reaction mixture [1.5 μl of first strand cDNA, 1.0 μl of 2.5 mM dNTPs mix, 1X GC melt buffer, 0.5 μl of 10 pmole primer and 0.2 μl (1unit) GC LA Taq (TakaraBio, Japan)]. Amplification cycle was: 94°C for 1min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s and extension at 72°C for 2.30 min. Final extension was carried for 72°C for 5 min. RT-PCR amplicons were resolved on 1% agarose gel containing ethidium bromide and visualized in gel doc.Cloning and sequencingAmplicons corresponding to the dsRNA bands were excised from gel, purified using GenElute Gel Extraction kit (Sigma–Aldrich, USA) and cloned into pGEMT-easy vector (Promega, Madison, Wisconsin, USA). Recombinant plasmids were purified using GenElute Plasmid Miniprep kit (Sigma–Aldrich, USA) and the target cDNAs were sequenced in an automated DNA sequencer (ABIPRISM®3130xl Genetic Analyzer) using ABI prism Big Dye™ Terminator v3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems, USA), with 2X coverage. Nucleotide sequences were translated using Expasy server (ExPASy - Translate tool), and the obtained amino acid sequence was used to determine homology by Blastp (Basic Local Alignment Search Tool) analysis.Computer analysis of the ArCV-1 3D structure of RdRpWe studied the three dimensional crystal structure of RdRp using computer aided analysis. The amino acid sequence of ArCV-1 (Table 1), RdRp (P1), was used to develop 3D structure with characteristic folding. I-TASSER (I-TASSER server for protein structure and function prediction), an online server was used to study the RdRp [37, 38]. This integrated platform generated three-dimensional atomic models from multiple threading alignments and iterative structural assembly simulations. Data provided on topology similarity (TM) with the proteins structurally close to the target protein (s) in the PDB is helpful contrivance to assess the relationships of the polymerase. The outputs of the I-TASSER data of possible predicted models and the molecular graphics are of high quality that contained full-length secondary and tertiary structure predictions. Developed structures were subjected to simulations by an open-source viewer, the PyMol (PyMOL | pymol.org) and Jmol (Jmol: an open-source Java viewer for chemical structures in 3D) an interactive graphics program [91, 92], for illustrating the three-dimensional (3D) chemical structures of the crystal. These programs were used to alter the scheme of the images to characterize the proteins. Using this program, individual and conserved residues in the motifs were identified which facilitated understanding over all nature of the RdRp. Structural adjustment was made for this model of ArCV-1 3D pol of about 90° to study from top surface and side ways to visualize the orientation of channels. Amino acid sequences of 3Dpol structures of Norwalk virus (1SH0), poliovirus (4R0E) and Sapporo virus (2wk4A) from PDB and RTSV sequence (NCBI NP734463) was used in the analysis and to develop 3Dpol structures.Construction of phylogenetic treesMultiple alignments and sequence identity matrix of RdRp and dsRNA capsid protein-like sequences were carried out by ClustalW [93]. To deduce the evolutionary relationship, the phylogenetic tree was prepared by MEGA 6.0 program [94], by using neighbor-joining method [95], and evolutionary distances were computed using the maximum composite likelihood method with 1000 bootstrap value [96]. The bar represents base substitution per site. Sequence identity matrix and phylogeny were studied by using NCBI GenBank database (GenBank Overview).

We at QAFox, have answered the below 500 Software Testing Interview Questions and Answers (Read answers for the below questions here)1) Why do you want to pursue your career in the field of software testing?2) Please explain your nature, habits, or likes that you think you are the right candidate for the testing?3) Why should we test?4) What do you mean by testing?5) What are the features you check before you purchase the mobile?6) What do you mean by quality?7) What do you mean by performance?8) Can you write possible test cases for a pencil?9) What do you test in One Time Password (OTP) creation application?10) What are the essentials of software testing?11) What is required for the tester to know before testing software applications?12) What are desktop applications?13) How to test desktop applications?14) What are web-based applications?15) How to test web-based applications?16) What are anomalies in software?17) What are the types of defects in software applications?18) How many different ways you can test software?19) What is expected from the testing team on testing a software application?20) What is Unit testing?21) What is sanity testing?22) What is smoke testing?23) What is exploratory testing?24) What is integration testing?25) What is system testing?26) What is Interface testing?27) What is Regression testing?28) What is Alpha testing?29) What is Beta testing?30) What is performance testing?31) What is Load testing?32) What is Stress testing?33) What is Security testing?34) What is Static testing?35) What is dynamic testing?36) What is Compatibility testing?37) What is Reliability testing?38) What is Compliance testing?39) What is Localization testing?40) What is test case?41) What columns are present in test case template?42) What columns are important in testcase?43) What test case management tool have you used?44) What is use case?45) Explain main elements of use case.46) What is test scenario?47) What is a positive test case?48) What is a negative test case?49) What is Behavior Driven Testing?50) What is Acceptance Testing?51) What is Vulnerability testing?52) What is black box testing?53) What is white box testing?54) What is grey box testing?55) What are the different levels of testing?56) Explain integration testing of modules in real life example ?57) What is Top Down Approach?58) What is Stub in Top Down Approach of integration testing?59) What is Bottom Up Approach?60) What is Driver in Bottom Up Approach of integration testing?61) What is GUI or UI testing?62) What is functional testing?63) What is non-functional testing?64) What is requirement traceability matrix?65) What is the defect traceability matrix?66) What are the techniques used in test design?67) Give example of Equivalence Class Partition.68) Give example of Boundary value analysis69) Give example of Decision Table70) Can you explain Defect Life Cycle?71) What is Defect severity?72) What is Defect priority?73) Difference between quality assurance and quality control.74) What is difference between inspection and audit?75) What is Quality assessment measurement?76) What is test management measurement?77) What is test maturity model?78) How is level of software development organizations decided?79) What is Process Capability Measurement?80) What is difference between Corrective actions and preventive actions?81) What is Software Testing Life Cycle?82) What is Requirement Specification Document?83) What documents will you refer to understand about features in the software?84) What is a virus?85) How antivirus program works?86) Give few examples of sensitive information can be shared across network?87) What is IDE and their use ?88) List name of IDE made of Java.89) Give some examples of use of Java applets used in your application90) What is required to run Java applet in a browser?91) Can you explain methods used in applet life cycle?92) What is JRE?93) What is JVM?94) Why do we need JDK?95) How JRE, JVM and JDK related?96) List types of class loaders in Java97) What is a difference between JRE and JVM?98) What is difference between interpreter and compiler?99) What exe stands for?100) Give some examples of executables in Windows101) Give name of four Java framework102) Can you explain Hibernate framework?103) A type of testing in order to expose defects in the interfaces and in the interaction between integrated components is?104) Identify type of testing wherein we subject the target of the test, to varying workloads to measure and evaluate the performance behaviors and the ability of the target and the test to continue to function properly under these different workloads?105) What do you ask for in order to purchase computer?106) What is a Difference between Regression and Retesting?107) What is a difference between Debugging and Unit testing?108) What are Unit testing techniques?109) How do you decide which test cases to consider for Regression Testing?110) What is a Difference between Integration Testing and System Testing?111) What are the various contents of a Test Plan?112) Do you know Exploratory Testing?113) What is Prototyping? What are the various types?114) What are SDLC phases?115) List some of the SDLC models used in software development?116) What kinds of reviews are conducted during SDLC phase?117) What is the role of Metrics in Testing?118) What are key challenges while testing a software application?119) Explain your views about Quality.120) What is the advantage of using any bug tracking tool?121)Explain about website Usability testing.122) What are defect attributes?123) What is pesticide paradox?124) Explain V model.125) What is the difference between Web application and Client Server Application?126) What is Localization testing?127) What is difference between Black box testing and White box testing?128) What is the difference between Authentication and Authorization give an example.129) Other than functionality what else you should test in Web Application?130) How you know that testing is enough?131) How will you test the reports?132) What is Database? Which database you are aware of?133) Can you name some of Relational database?134) Can you name some of nosql database?135) Can you name some of cloud database?136) Can you name some of distributed database?137) Can you name some of object-oriented database?138) Can you name some of graph database?139) What is Database Testing?140) What do you mean by Cookies?141) How do you test Cookies?142) What are Stored Procedures?143) How many types of Joins are in sql?144) What are DML and DDL commands give example.145) What is Normalization?146) What is Primary key and Foreign key?147) What is RDBMS?148) What is difference between delete & truncate?149) What design documents you receive from PM?150) What are documents are created in Design phase?151) What challenges you have faced in testing so far?152) Realizing you won’t be able to test everything – how do you decide what to test first?153) What is SQA?154) What are the contents of defect report?155) What is Big Bang type of testing?156) What is Traceability Matrix & Coverage Matrix?157) What is UAT and Alpha and Beta Testing?158) List OOPS features?159) What is Hot fix?160) Where Exit and Entry Criteria are written in Test Plan?161) What are different scenarios for verifying Email address?162) What is the query for update table?163) If there is mistake in company Logo, what is Priority and Severity Justify your answer?164) What is A/B testing?165) What is Test driven development?166) Explain error guessing with an example.167) In bug life cycle, who opens the bug?168) What is Compatibility, Multiplatform and Configuration testing?169) What are Session and Cookies and what is difference between them?170) What is Mutation testing?171) Difference between Ad-hoc & Exploratory testing172) Difference between HTTP and HTTPS, Explain how the data is secured in HTTPS?173) Have you heard of Agile Methodology? Explain?174) What is scrum?175) What is sprint?176) What is epic?177) What is product backlog?178) What is burn down report?179) Have you used any Configuration Management tool?180) Do we always need to do regression after retesting?181) What is Web server, which web servers you know?182) What is Inter-system testing? Have you done that?183) What is a build? What is the release?184) What is production server?185)What is Recovery Testing?186) What are the properties of a good Requirements Document?187) What is Accessibility Testing?188) How can you make your software ready for accessibility?189)What is Jenkins?190) What is an Insurance?191) What are some of the types of insurance?192) How many types of banking have you heard of?193) What is retail banking?194) What is NetBanking?195) Name some digital payment platform196) What entities are present in health care domain?197) What are the seven layers of OSI model in network?198) What do you mean by protocol?199) What are various types of e-commerce sites?200) What are system integration test types?201) Name few of System integration testing tools.202) What is Defect cascading?203) Give some Test Management Tools you are aware of.204) Where are Jenkins files stored?205) Have you heard about GIT?206) Have you worked on SQL query? What you have done?207) Explain REST API.208) What are tools used in software industry for API testing?209) How to upload data in Jenkins?210) Have you heard about GIT?211) Have you worked on SQL query? What you have done?212) What is Primary Key please explain.213) If there are same multiple entry in the table, how to find Unique element in the table?214) How to get 3rd Highest Salary from employee table?215) How to find any particular date of joining of employee from Employees table, date is in number format then how to change it to date format?216) What is end to end testing?217) Arrange the following testing types in sequence in which we test any applicationFunctional Smoke Integration System Sanity Retesting Regression218) List Name of HTTP methods for RESTful services219) What are status codes in HTTP?220) Have you used SOAPUI?221) Have you used Postman?222) Have you used REST Assured?223) What is Installation Testing?224) Have you come across any severe bugs in your application and did you contribute so that it gets resolved?225)What is Application Server? Give example.226) If any bug is reproducible at client-side (in production environment) not in the test environment what are the different possibilities that this scenario occurs?227) If any bug is reproducible only on Testing server and not on Development server, how you make sure that the developer fixes that bug?228) What is Data Migration Testing? What are the different approaches followed in Data Migration?229) What are broken links?230)What is ETL testing / Data warehousing testing?231) What is Closer Report?232) write test cases on online software product for free?233) write test cases on company web portal (say ACC cement)234) write test cases on gaming software235) write test cases on image editing software236) write test cases on online file format conversion237) what is captcha? Where did you see captcha?238) explain functionality of captcha.239) Why all websites visited want to have your consent to use cookies?240) Give example of Statement coverage.241) Give example of Decision coverage.242) Give example of Branch coverage.243) What is TestNG?244) What is Test Driven Testing?245) What is Behavior Driven Testing?246) What is Latent Defect?247) What is fault-masking?248) Explain what the difference between latent and masked defects is?249) Explain what LCSAJ is?250) What is Fuzz testing and when it is used?251) what is test planning?252) What are commonly used testing types across industries?253) what are the steps to follow when you discover defect?254) How would you resolve conflict amongst team members?255) What is Charles Proxy testing?256) What is meaning of Code Walk Through?257)What things to consider while project monitoring?258) List common pit holes or mistakes that creates issues in project delivery?259) What the basic components of defect report format are?260) What is risk-based testing?261) When is used Decision table testing?262) What is a Proxy server?263) what is test environment?264) what is test execution?265) what is the difference between test scenarios, test cases, and test script?266) What all things you should consider before selecting automation tools for the AUT?267) How will you conduct Risk Analysis?268) How would you allocate responsibilities to your team members?269) What Is Docker?270) What is ETL testing / Data warehousing testing?271) What steps would you verify in case your desktop takes too long to start up?272) What do you mean by Defect Triage?273) What is a test harness? Why do we need a test harness?274) Why software has bugs?275) What is Cause Effect Graph?276) What are the different strategies for roll out to end users?277) What are error guessing and error seeding?278) Do you feel comfortable standing up to developers who disagree with your results?279) Are you willing to cut corners to save time?280) What is validation in software testing?281) What is verification in software testing?282) What is API testing?283) What is web service testing?284) What is Postman?285) What is REST Assure?286) What is Cucumber?287) What is Capybara?288) What is Salesforce?289) What is TestNG?290) What is TeamCity?291) What is Gradle?292) What is Framework?293) Have you heard about Teradata?294) Have you heard about Hadoop?295) Have you heard about Hansoft agile planning tool?296) What is JIRA Issue and project tracking tool?297) What do you think our company could do better?298) What experience do you have with developing corner cases?299) Explain any of bug tracking tool300) What is DevTrack cloud based development?301) Did you use TestRail Test case management tool?302) What is configuration management?303) What are popular configuration management tools you have heard of?304) What is scalability testing?305) What is concurrency testing?306) What is endurance testing?307) Have you appeared for ISTQB foundation test?308) When should you stop software testing activities?309) Can you explain in short 7 principles of software testing?310) What is Defect Clustering ?311) What is Defect Density?312) What is Age of Defect?313) What is Build?314) What is release?315) What is a difference between Build and release?316) Can you list some Continuous integration tools?317) Can you list some Continuous Deployment tools?318) What tools for version control have you used?319) What API testing tools you are aware of?320) What Test management tools did you use?321) What is CRUD stands for?322) What important web browsers do you know?323) What Operating systems you are aware of?324) What is CRM software?325) List few of CRM software used for customer relationship management.326) What Is Cyclomatic Complexity in software testing?327) Mention the categories of debugging?328) How do you go about learning a new product?329) How you coped with having to learn any new skills?330) What is performance testing?331) What is Load testing?332) What is Stress Testing?333) What is Spike Testing?334) What is Volume Testing?335) How well do you work with others?336) What motivates you?337) Have you worked as independent contributor?338) Explain PDCA Cycle (Plan-Do-Check-Act).339) Give one example of High priority and high severity340) Give one example of High priority and low severity341) Give one example of Low priority and high severity342) Give one example of Low priority and low severity343) Describe a situation where you successfully collaborated with developers to deploy a program or application344) Describe a time when you acted proactively to increase system performance.345) How do you keep up-to-date with industry developments? Are there specific blogs or forums you read?346) What process are you using for testing currently? Can you describe how you might improve it?347) What is your favorite testing tool? Why? If some technical constraint meant you were unable to use it, what would you do instead?348) How to handle a situation when you don’t have time for complete testing?349) Do you measure how effective (or not) your testing is? What metrics do you use?350) How well do you work under pressure and with deadlines?351) What different software testing methods are used by a software company?352) Name some of the test cases that you can automate.353) Name some roll out strategies for end-users.354) What is Application Binary Interface (ABI)?355) What is a interruption testing?356) What is CMM – Capability Maturity Model for a software?357) When is a case where automating a test case is not viable solution?358) How well do you deal with Ambiguity?359) What is Emulator?360) What is Simulator?361) What is CAST?362) What is Race Condition?363) What is Binary Portability Testing?364) What are the challenges that you face as a software tester in the current/previous company?365) What is a difference between functional and non functional testing?366) List REST constraints that describe basis of RESTful style?367) What 201 HTTP response codes describes?368) What is a status code for internal server error?369) Have you come across 404 response code?370) What is a difference between PUT and POST in case of REST API?371) What is expected for a manual tester to master automation testing?372) What could go wrong during execution of automation script to test application?373) What is a unix command to read contents of file?374) What is a Unix command to run programs with security privileges?375) What is a unix command to copy file from remote machine?376) How multiple inheritance feature can be used in Java?377) What is an interface in Java?378) What are various permissions to access file?379) What is a Unix command to change the access mode of a file?380) What is silk test?381) What is an error seeding?382) What is a data driven testing?383) What is desired by a project head as a candidate while taking an interview?384) List classes and interfaces from Java collection framework.385) what is a difference between SVN and GIT386) What is Git?387) Can you explain how a page of any web portal would display that sell software online allowing free downloads388) What is Artificial Intelligence?389) Have you heard of (IoT)Internet of Things?390) Explain how can IoT can be used in advantage?391) In order to check the condition frequently, What do you use in a program?392) what various loop types are you aware of, say in Java?393) What are the steps you take when things does not work?394) What is your approach when automation script does not work?395) How would you approach your seniors and request them to help you resolve the issue?396) Have you done test environment set up yourself?397) How do you configure Tomcat server?397) How do you configure Tomcat server?399) What is a sentiment analysis?400) How would you list features from the requirements?401) What is a difference between multi tasking and multi threading402) What are differences between collection types eg enumeration and iterator?403) Why do we use Joins?404) What is a penetration testing?405) What can one track with history of changes using version control software?406) How would you test application related to Payments domain?407) What do you know about iOS and mobile testing?408) Explain what do you mean by UML diagram?409) How would you configure any open source software into your system?410) Have you heard of Oracle PL / SQL,411) What are stored procedures?412) Give a situation where Triggers are used413) What is the difference between XML and JSON?414) What do you mean by protocol testing?415) How would you test free online video making software?416) Write test cases for PDF to PNG converter software417) Write test cases on WhatsApp application418) Write test cases on Google Drive419) What is a difference between Arrays and collection in Java?420) Write test cases on Sharefile / Fileshare software421) What is an advantage of using collection over arrays?422) What are differences between class and interface?423) How would you plot a pie chart between two parameters SalesAmt vs Years using spreadsheet say MS Excel?424) Have you heard of Headless Testing?425) Why API testing is so important over Graphical user interface testing?426) Give name of API testing tools other than SOAPUI and Postman427) What are few important queries to enquire before automating a web service?428) Explain web service terminologies429) What are elements of SOAP message?430) What is an importance of Cookies ?431) What are main requirement for web service to be RESTful?432) What is phishing?433) What is test driven testing?434) What is task scheduler in Windows?435) How gaming software is tested?436) What gaming platforms are used to test gaming software?437) What are test techniques for testing gaming software?438) What is visual testing?439) What are the requirements of ETL testing?440) Can you think of some areas where very huge data exchange take place?441) Give examples of some of the Big Data databases442) Give some example of unstructured data?443) What is log4j use in Java program?444) Name few cloud service providers445) What are the components of AWS?446) Do you know any Java web services framework?447) Give an example for simple join.448) where will you use left join449) where will you use right join450) Give an example for full join451) What Java Script is used for?452) Name some of the JS libraries used in client side programs453) What are Apache Tomcat components454) What is Catalina in Tomcat?455) what can be used to handle multiple elements, in absence of Collection classes and interfaces?456) What is List?457) What is Set?458) Difference between List and Set459) What is Array List?460) What is Linked List?461) What is similarities and difference between class and interface462) what is a command to print out comments information in Linux?463) How would you view file kept in machine having Linux operating system?464) I want to read a file one page at a time in Linux, what command should I use?465) I want to find out what were the first 10 lines of text file, what command should I use in Linux?466) Tell me a command to read last 10 lines of say log file in Linux467) I want to create an empty text file, what command you think I should use in Linux?468) I want to copy all files and directories to the destination directory, tell me Linux command469) What is a command to display ip address of your system?470) I tried using ifconfig command to find out my ip address, but it did not display, how can I still get IP address using ifconfig.471) What is ping command in Linux?472) How would you resolve the issue, if your chrome browser stops working?473) what is nslookup command in windows used for?474) What is Regex and where can you use them?475) Have you used JMeter before and what did you test using JMeter?476) Why do you use Thread Group in JMeter?477) Why we use timer in JMeter?478) How would you achieve page refresh by press of a single key?479) What is a difference between Alt+Tab and Ctrl+Tab?480) What are features?481) What is a functionality of software or any product?482) What is root cause analysis?483) What preventive measures should be taken to design defect free software?484) What is Data warehousing Testing?485) What is an importance of BI?486) What are the benefits of data warehouse as a solution to store and gather data across various department?487) What is ETL process488) What is a data structure?489) What operations can be performed on various data structures?490) What are basic types of data structure?491) What would you test zoom before an interview?492) What are requirements for testing Electronics medical reports or equipments related to healthcare industries?493) What file sharing software you know that are available in market?494) What role does UX and UI play in an application?495) What online education sites you know that are available in market?496) What online video streaming platforms in India you have used or aware of?497) What are common video formats and when to use them?498) What is the use of Grep command in linux ?499) How will you justify your position as Quality Engineer?500) What would you feel when your product is appreciated by clients?We at QAFox, have answered the below 500 Software Testing Interview Questions and Answers (Read answers for the below questions here)