Some residents of Corpus Christi, Texas, resumed turning on the tap after an industrial chemical leak at an asphalt plant sparked fears of widespread water contamination.
Officials said Saturday that there has been no evidence of tainted water, although testing of water samples was still being done in Houston and could be made available Sunday. As a precaution, pallets of bottled water were trucked in to alleviate the situation following a ban that was put into place Wednesday night.
The EPA said there were four "unconfirmed reports" of symptoms possibly related to prohibited water use. Corpus Christi Mayor Dan McQueen called the reports "rumors."
City water towers have also been flushed out to ensure the water is safe, officials said.
A ban remains in place in some portions of the Gulf Coast city of nearly 320,000, and officials are left grappling with closings of schools and day cares, and moving dozens of animals in a local shelter to San Antonio out of concern.
"We know the citizens are suffering. We’re suffering too," Corpus Christi Councilman Greg Smith said at a news conference Saturday.
Here's what you need to know about the ban.
What is the chemical that may have contaminated the water?
An incident report described the leak as a "backflow incident from a chemical tank impacting the public water system." The chemical is a combination of Indulin AA-86 and hydrochloric acid.
Indulin is an asphalt emulsifying agent that's corrosive and can burn the eyes, skin and respiratory tract if a person comes into contact with concentrated amounts. The amber liquid is considered a hazardous material by the Occupational Safety and Health Administration and could cause damage to internal organs.
"You don't expect to see it in water," said Terry Clawson, a spokesman with the Texas Commission on Environmental Quality.
Up to 24 gallons of it may have seeped into a pipeline carrying water, allowing it to move to other areas of the city, Kim Womack, a spokeswoman for the city, said Thursday.
How did the contamination start?
City workers determined there was a leak in the asphalt plant's chemical tank and on Tuesday determined there was a backflow problem.
The city told the Texas Commission on Environmental Quality (TCEQ) about it Wednesday, said Assistant City Manager Mark Van Vleck, and hours later, the state banned the use of public drinking water. Mayor Dan McQueen has said local officials only learned of the leak Wednesday. TCEQ officials did not respond to a request for comment Saturday.
Van Vleck cautioned that the investigation is ongoing, "so we don't know what happened." He added: "We do not know (there was contamination) because we can't prove it."
How long have officials know about the leak?
Van Vleck said the first "dirty water report" came Dec. 1 from an asphalt plant in the city that's leased to Ergon Asphalt and Emulsions. City workers flushed the pipe. A second report came from the same building Dec. 7, he told The Associated Press, and the main was flushed again.
But Monday, Valero workers told the public works department there was a "white, sudsy liquid" in the administration building; Van Vleck didn't specify where the liquid was coming from.
How widespread is the ban?
Initially, the use of tap water was banned throughout all of Corpus Christi when officials said they learned Wednesday of a leak that contaminated a water distribution system.
The city has since been divided into three zones allowing water to be used depending on location.
For instance, those living in Zone 1 have been allowed to use tap water for drinking, cooking, bathing and all regular uses.
Those in Zone 2 have limited water use, while those in Zone 3 are banned from using the water completely as a precaution. That involves about 113,000 people still under the restrictions.
What does the company say happened?
Bill Miller, a spokesman for Ergon Asphalt and Emulsions, said the company is also looking for answers from the Texas Commission on Environmental Quality to know whether any water was actually contaminated.
"Inaction is bad for people in our position. The longer we sit here, the worse it looks," Miller said, according to the AP.
Has anyone been sickened?
Federal and state agencies say there have been four unconfirmed reports of people suffering symptoms that would be associated from using contaminated water. Details of those symptoms were not provided by the agencies.
Corpus Christi Mayor Dan McQueen also said at Saturday's news conference he heard about people falling ill.
City Councilman Michael Hunter told the Corpus Christi Caller-Times that it was unlikely the leaked chemical was concentrated enough to do harm, but that every precaution must be taken.
What is the response from residents?
More than a half-dozen lawsuits have already been filed against the oil refiner Valero, of which leases the asphalt terminal to Ergon. The Corpus Christi Caller-Times reported that several businesses and a class-action lawsuit representing all of the city's residents allege the companies recklessly exposed business owners and residents to toxic chemicals.
Van Vleck said the city made the Ergon plant install new valves because of the leak, but has not restored water service to the plant.
Editor's note: A previous version of this story stated that Ergon was a subsidiary of Valero. Valero simply leases the a portion of the plant to Ergon.


Blum-Novotest presents a universal radio probe for tool setting and monitoring in 3 axes
Blum-Novotest, the leading manufacturer of innovative and high-quality measuring and testing technology, presents the new 3D tool setting probe TC64-20 at the EUROMOLD in Frankfurt (Hall 8 Stand C20). The company is supplementing its tool setting probe series with a high-precision, wireless system which features BRC Radio Technology, for tool setting and tool breakage detection in three axes.
Winfried Weiland, Marketing Manager at Blum-Novotest, explains: “The TC64-20 is both a universal and cost-effective system for the automatic measurement and monitoring of tool length, tool radius and tool breakage in CNC machining centres. It is used on machines in mould and die production and medical technology, as well as in many other industrial areas. If several systems, for example a tool setting probe and a workpiece probe, are to be operated in a machine, this is possible thanks to the BRC Radio Technology integrated within the probe. The TC64-20 is also an excellent solution if wired devices cannot be attached, for example on machines with pallet changers.”
The compact tool setting probe excels through extremely fast and robust data transmission. Instead of the channel hopping method or channel assignment usually found on the market, this probe transmits the collected data to the receiver using Blum’s BRC technology. The advantage of this technology is based on each single bit of the radio signal running across the entire width of the frequency band, thus making transmission particularly resistant to interference. Then the data is transmitted to the radio receiver RC66 quickly and securely. Just one receiver can be used to control up to six measuring systems from the extensive Blum radio family. This includes all tool setting probes and workpiece touch probes, as well as the newly introduced DIGILOG measuring systems, the bore gauge BG60, the roughness gauge TC63-RG or the workpiece temperature measuring device TG81.
A further highlight of the TC64-20 is the integrated shark360 technology. The patented measuring mechanism offers a considerably higher measuring accuracy than comparable probes because of the modified face gear. During the measuring process the face gear causes a defined deflection direction at constant deflection forces. The torsion force which occurs in the measuring mechanism is absorbed by the gear and therefore has no effect on the measuring result. The switch point repeatability is a high-precision 0.4 µm 2σ.
Another advantage is the generation of the switch signal for determination of the axis positions. It is not generated by a mechanical contact, but opto-electronically by shading with a miniature light barrier inside the tool setting probe. As a result, it operates absolutely wear-free and guarantees maximum reliability over many years, even under the most adverse conditions. Further advantages of the TC64-20 are the high probing speed as well as the very low power consumption. Tools from a diameter of 1 mm (depending on tool geometry and material) can be measured.
The mechanically robust design of the compact tool setting probe is perfectly adjusted to the harsh environment of a machine tool as per device protection class IP68. The self-centring stylus allows a change to be made without re-aligning the probe using a dial gauge. In addition, an optional predetermined breaking point protects the probe system in the event of an operating error. “The TC64-20 is an interesting wireless 3D alternative to our already established and often copied linear-guided tool setting probes Z-Nano and Z-Pico. As with both these systems, we also offer a full range of accessories for the TC64-20, for example an integrated air nozzle for cleaning the tool and the measuring surface”, summarises Winfried Weiland.


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The Chicago Cubs ended their first road trip on a high note, holding on Sunday against the Colorado Rockies to finish .500 on the six-game trip.
From a productive offense to Seiya Suzuki staying hot to begin his career, here are three takeaways from the trip to Pittsburgh and Denver.
1. The doubles machine keeps rolling.
One of the most encouraging early trends for the Cubs offense is its ability to drive the ball.
The Cubs kept rolling in Denver. Their 17 doubles in the four-game series was their most in a series since hitting 19 in a three-game set against the St. Louis Cardinals on July 12-13, 1931.
“We don’t make it easy on the opposing pitcher,” second baseman Nick Madrigal said. “We work counts. We hit mistakes in the middle of the plate. This lineup really does scrap together some good at-bats every single day.”
Third baseman Patrick Wisdom hit another double Sunday, giving him five over a three-game stretch, while shortstop Nico Hoerner had a two-double game and Madrigal tallied his second of the season.
The Cubs’ 24 doubles through Sunday are tied with the Philadelphia Phillies for the most in the majors. While more balls in play also has yielded the most double-play balls in the majors, the Cubs are getting good production when getting elevation and taking advantage of mistakes.
“Line to line it feels like we’re taking good at-bats,” manager David Ross said. “Just complete usage of the strike zone and to all fields means really good approaches and staying within themselves. I’ve been really pleased with that. Guys are attentive and turn it over to the next guy and understand the offense runs as a flow and they’re willing to turn it over to the guy behind them if they’re not getting their pitches.”
2. Playing-time balance at catcher should pay off in the summer.
For the first time, catchers Willson Contreras and Yan Gomes were both in the lineup for Sunday’s series finale against the Rockies. Contreras was the designated hitter, also a first this year. Ross wanted them to start with Rockies left-hander Austin Gomber on the mound.
The Cubs made it a priority heading into the offseason to make sure Contreras didn’t repeat the heavy workload he took on behind the plate in 2021. Gomes’ experience and offensive profile made him the ideal fit as a free-agent signing.
Ross said it has been easy finding a playing-time balance for the catchers. Contreras has been the primary catcher, but Ross has found opportunities to mix in Gomes, who has started at catcher in three of the first nine games.
“Yan dove in, found his groove, got to know guys, how we do things, very communicative,” Ross said Sunday. “They both can really hit, so it’s nice to have those core tandem catching guys. It’s always nice to have two good ones.”
In addition to making sure players are getting days off early in the season because of the shortened spring training, Gomes’ presence should pay dividends for Contreras during the summer. The Cubs thought Contreras wore down by the end of last season.
Ross believes he already is seeing an impact. Contreras has been hitting the ball hard, resulting in two multihit games on the road trip and a home run Sunday.
When the grind of summer hits, a fresher Contreras could make a big difference offensively.
“His work, his engagement throughout a game with the pitching staff and the at-bats has really stood out so far to me and the way he’s running the bases,” Ross said. “I think he’ll be able to give us that little bit extra intensity now and not feel like he’s got to conserve so much. That should pay off in the long run.”
3. Seiya Suzuki already is commanding respect.
Suzuki’s nine years in Nippon Professional Baseball doesn’t erase his major-league rookie status. His performance, however, is forcing teams to treat the Japanese star like the threat he has become in the batter’s box through his first nine games.
The Rockies intentionally walked Suzuki to load the bases with one out in the fifth Sunday. It was the second time Rockies manager Bud Black chose that route during the four-game series, making Suzuki the only player in the majors with more than one intentional walk.
The decision didn’t pan out as the Rockies envisioned Sunday. Gomes delivered an RBI single to extend the Cubs’ lead to 5-0.
“The bases get loaded, so it creates a bigger chance for the team and that’s the most important thing,” Suzuki said through interpreter Toy Matsushita.
Suzuki quickly showed why the Rockies respect his bat. In his next plate appearance, he slugged a 1-1 fastball for an opposite-field home run.
The 382-foot homer extended Suzuki’s hitting streak to eight games. It’s the second-longest to begin a career for a Japanese-born player, trailing only Akinori Iwamura’s nine consecutive in 2007 with the Tampa Bay Rays.
“In terms of how I feel about my results, I don’t really feel anything — I just want to stay conscious and just hoping to get those results every day,” Suzuki said.

domain: news


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• Model No. LED450


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I have a cheese problem. Right now in my fridge I have the following cheeses: goat, blue (seen above), leek and morel jack, asiago, and parmesan. No, there is absolutely no reason to have that many. I live and eat by myself for the most part. I blame it on my high school job of working at the Hy-Vee Cheese Island. It hooked me and gave me expensive taste.
Today for lunch I had some curried pumpkin soup (thanks mom!) with toasted french bread and goat cheese. Then later for a snack I had some blue cheese on crusty bread. Seriously. I have a problem.
The weather is so nice lately. The sun makes me want to close my eyes and listen, not sit down and type. Sorry that these posts have been a little boring. Look at my bread and cheese and be soothed.

domain: food


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The Indian Navy will soon invite tenders for building six conventional submarines after a high-level committee of the Defence Ministry submitted its report identifying shipyards capable of executing the about Rs 60,000 crore project.
“The Committee has visited all the shipyards in the country and has submitted its report recently. It is under consideration of the Defence Ministry,” defence sources said.
Though they remained tightlipped about the name of shipyards that have been shortlisted, the sources said a Request for Proposal (RFP) will be issued soon once the report has been studied in detail.
Besides the state-run shipyards, private players like Larsen & Toubro (L&T) and Pipavav Defence & Offshore Engineering Company, in which Anil Ambani-led ADAG has announced its decision to pick up a controlling stake, are eyeing the mega deal.
Defence sources had earlier said both L&T and Pipavav are strong contenders, besides the state-run Mazagon Dockyard Limited (MDL).
MDL is already making six Scorpene submarines. Asked if MDL would be the natural winner, sources said, the eco-system has to be developed and hinted that the order may go to a particular shipyard which could then outsource a part of the project to others. Continue reading
NEW DELHI : Indian Railway Catering and Tourism Corporation (IRCTC) has floated tenders for setting up its premium Executive Lounge at 16 more stations that provides airport lounge-like facilities to passengers at an affordable rate.
The new stations where the Executive Lounge will come up are Jaipur, Vijayawada, Kathgodam, Sealdah, Howrah, Dhanbad, Patna, Ajmer, Darbhanga, Bhubaneshwar, Vishakhapatnam, Bilaspur, Secunderabad, Ahmedabad, Puri and Nagpur. IRCTC, a ‘mini-Ratna’ PSU of Indian Railways, had started the facility at New Delhi station in November 2012. Earlier this month, it signed MoUs with the railway administration to set up the swanky lounge at Agra Cantt, Lucknow and Gorapkhpur stations, added the release.
“We are enthused by the growing response to the Executive Lounge that is considered a showpiece in our portfolio of passenger amenities,” IRCTC Chairman and Managing Director Dr. A K Manocha said.
“The decision to set up lounges at 16 more stations is part of our efforts to provide top-rate facilities to travelling passengers and make their journey, especially waiting at stations, a pleasant one,” he added. Continue reading


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May Day Bank Holiday Offer - 15% Off - Use Code MAYDAY
Offer applied at checkout. Ends Midnight Tuesday.
Ultra-Violet / Infra-Red
Wide range of uses from Blacklight blue for glow in the dark effects, to fly killer tubes, varnish curing and photo chemical processes to “Germicidal for purification. The colour/finish of the tube is often a good starting point.

domain: chemistry


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I barely slept last night because I was so nervous about my medical appointment this morning, but it ended up going really well. I met with my rheumatologist for the first time in three years (I know! Yikes!) and she agreed to put me on a new med that is now available for my condition. So I’ll hopefully be starting that soon, assuming my labs and x-rays look okay. She also gave me a cortisone injection in my problem knee (ouch!) which should provide a few months of relief in the meantime. I’ve had good results with these previously.
The new drug sounds a little scary, but nothing is without the potential for side effects. I’m just grateful there is no cancer risk. Plus, one of the big side effects is weight loss. Um…count me in, please!
I’m icing my knee, per doctor’s orders, for what will hopefully be the last time for quite a while.
I’m so fucking relieved. I can’t even begin to describe it.

domain: medical


text:
Shoot 'Em Up
You are not logged in. Only registered users can vote without verification. Please login or register, or type the words below:
Shoot as many birds as possible on each level and try not to let any get away.

domain: game


text:
September 04, 2014 07:30:37
The flooded rice field at the Department of Agriculture and Food in Kununurra.
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This service may include material from Agence France-Presse (AFP), APTN, Reuters, AAP, CNN and the BBC World Service which is copyright and cannot be reproduced.
AEDT = Australian Eastern Daylight Savings Time which is 11 hours ahead of UTC (Greenwich Mean Time)


Colorado High School Volleyball - Manitou Springs over EllicottOctober 8, 2019: Calhan, CO 80808
In Tuesday's league contest, the Manitou Springs Mustangs (Manitou Springs, CO) volleyball squad knocked off the host Ellicott Thunderhawks (Calhan, CO), by a margin of 3-1.
Next up, the Mustangs battle the Buena Vista Demons (Buena Vista, CO) in a 3A Tri-Peaks contest, on Thursday, October 10. Manitou Springs will attempt to better on its 5-8 season record. The Demons come into the contest with a 6-5 record after their 3-0 league win over Ellicott (Calhan, CO).
With the loss, Ellicott drops to 3-15 on the campaign. The Thunderhawks travel to Trinidad (Trinidad, CO) to battle the Miners in a 3A Tri-Peaks contest on Thursday, October 10. The Miners come into the contest with a record of 4-7. Trinidad lost 3-0 in their recent league contest against Florence (Florence, CO)


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Table of Contents
Why was Petra so important?
Petra began as a main stopping point for Nabataean and foreign traders. These nomadic merchants carried textiles, incense, spices, ivory, and other precious goods grown or manufactured in Arabia, Asia, and Africa. As the trade market grew, so did Petra.
Why is Petra Jordan one of the wonders of the world?
The ancient city of Petra in Jordan became one of the 7 New Wonders of the World when it was chosen in 2007 by a vote of 100 million people. The city’s carved rose-red sandstone rock facades, tombs, and temples became known around the world with its appearance in Indiana Jones and The Last Crusade in 1989.
Why is Petra a tourist attraction?
Known as the Rose Red City due to the colour of the kingdom’s rock carvings, Petra is Jordan’s most visited attraction. The ancient Nabataean capital remained unknown to the western world until 1812 when a Swiss explorer discovered numerous spectacular sites in the area.
Is Little Petra worth visiting?
Siq Al Barid (Cold Canyon) is colloquially known as Little Petra and is well worth a visit. It was thought to have served as an agricultural centre, trading suburb and resupply post for camel caravans visiting Petra.
What is Petra Jordan famous for?
Petra (Arabic: البتراء, Al-Batrāʾ; Ancient Greek: Πέτρα), originally known to the Nabataeans as Raqmu, is a historical and archaeological city in southern Jordan. The city is famous for its rock-cut architecture and water conduit system.
What are facts about Jordan?
with a population of over 9 million people.
Is the city of Petra in the Bible?
The City of Petra is in the Bible. The city is located near Mount Hor which is the location in scripture, where the brother of Moses — Aaron — passed away,

domain: geography


text:
|Sweet Fire (1991) [Novel]|
by Jo Goodman
Rating: No votes (Rate!)
Reviews: None (show them) Review!
(From the publisher):
She longed to have him
Told all her life that she was plain, Lydia Chadwick knew no man would come courting because of her looks. So it was with some suspicion that the shy, sweet San Francisco heiress woke one morning with a tall, dark, dangerously handsome husband she couldn't recall marrying. Lydia had lost her memory, and was desperate to discover if there was truly a love to remember. For as she looked at Nathan Hunter's lean, muscular frame, she longed to abandon herself to the sensual stranger, and believe -- if only for a little while -- that the possessive passion in his smoky gray eyes was really for her.
He ached to have her
Business and pleasure weren't supposed to mix, but in this case Nathan Hunter was willing to make an exception. After all, it was in his best interests to keep his new bride's mind off the secrets of her past. Making sure she didn't remember her hatred for him turned out to be the easy part, as he initiated the innocent Lydia into womanhood. Not so easy was keeping sight of his own goals as Lydia's sweet surrender wove a seductive spell around Nathan's heart, arousing emotions he had thought forever buried...
Original title: Sweet Fire
Genre: Fiction→ Romance→ Historical
Fiction→ Historical→ North America→ Nineteenth Century

domain: literature


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Retaliation is the second album by American comedian Dane Cook. It was released on July 26, 2005 by Comedy Central Records. The CDs contain a concert done by Cook at The Comedy Connection in Boston, Massachusetts. The DVD contains Dane's appearances on Shorties Watchin' Shorties and Crank Yankers, ...
Dane Jeffrey Cook (born March 18, 1972) is an American stand-up comedian and film actor. He has released five comedy albums: Harmful If Swallowed; Retaliation; Vicious Circle; Rough Around The Edges: Live From Madison Square Garden; and Isolated Incident. In 2006, Retaliation became the highest charting comedy ...
Aug 22, 2008 ... Watch the video «Dane Cook BAMF Skit» uploaded by Jonathan Vera on Dailymotion.
Bamf Lyrics: I've made some upgrades in my life, sometimes you have to upgrade your life. There's things that you need to do, you need to invest in yourself, in your soul [laugh] / I wanted to.
Dane Cook's cement truck with padded cement drum for his friends to roll around in whilst eating watermelon Jolly Rancher candies. Dane custom made the 40" rims on the CT2004 in his basement out of aluminum foil and ornaments. Makes Dane feel like a BAMF while driving it.
Jan 12, 2008 ... Not to be confused with the very few people who can actually relate to and understand what Dane Cook is saying in his comedy routine. 75% of people who have listened to a dane cook cd, dvd, or have been to a Dane Cook event. ... BAMF: Do you even know what he said? Dane cook fan: YEAH!! Uh, no ...

domain: entertainment


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domain: hobby


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Seattle Mariners vs. San Diego Padres
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1250 1st Ave
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domain: sports


Thousands of former and current plumbers, pipefitters, and steamfitters may have been exposed to toxic asbestos exposure in construction, shipbuilding, and Maintenance Crews. Regardless of where a plumber was working, the chances are they have directly or indirectly worked around asbestos products in the last 50 years.
In fact, workers are still at risk of asbestos exposure as many buildings and workplaces throughout the country still contain the toxic material. Plumbers and pipefitters regularly worked with asbestos thermal insulation and asbestos pipe repair materials.
This includes pipes, boilers, ducts and tanks containing asbestos insulation as well as joint compounds, cement, pipe block, valves, gaskets, pipe coating and welding rods.
Nearly any plumber or pipefitter that worked into the 1980s could be at risk of developing severe health issues, including asbestosis, lung cancer, and mesothelioma.
When plumbers and pipefitters are in enclosed spaces as they cut, saw and assemble essential plumbing components that contain asbestos, toxic fibers are released into the air and easily inhaled and ingested. The accumulation of asbestos fibers in the lungs can cause inflammation and scarring that can lead to lung cancer, mesothelioma cancer and other asbestos-related diseases.
Asbestos was used in pipes, boilers, ducts and tanks because it is an effective heat and fire resistant material. Since many of the areas plumbers work are short on air circulation, the Workplace Ventilation Risks and asbestos fibers made the occupation extremely dangerous. Unfortunately, many employers failed to warn their workers of the health risks, even after it was well-known that asbestos causes cancer.
Asbestos fibers can spread up to 50-75 feet from the work being performed. The toxic fibers can be airborne and cause plumbing asbestos exposure, and also carried home on clothes and on the person, exposing families to the toxins.
The Occupational Safety and Health Administration (OSHA) warns of other plumbing and pipefitting hazards, including:
Plumbers and pipe fitters used asbestos-containing materials in both the pipe walls and the pipe wrapping. Asbestos seemed like the best material for pipes and wrapping because it is lightweight, cheap, durable, and fire resistant.
Asbestos also lowers electrical conductivity in steel pipes making them safer around current and static charges in ships and in manufacturing facilities. Potential asbestos products include the following:
Lung cancer and mesothelioma have a long latency period, and plumbers and pipefitters may not be diagnosed for up to 40 years after exposure. Symptoms of lung diseases like asbestosis, pulmonary fibrosis, lung cancer and mesothelioma can include:
Pipefitters, steamfitters and plumbers who have been exposed to an unsafe work environment may have a viable personal injury claim against a former employer and a list of manufacturers with Asbestos settlement funds. Any victim of plumbing asbestos exposure should seek professional medical attention and contact a mesothelioma cancer lawyer to investigate the root cause.
Each asbestos illness is unique and may be the result of either acute exposure or long-term exposure to toxic materials. The results are all devastating with victims falling ill, often many years after initial exposure.
Any family member who has been diagnosed with mesothelioma, lung cancer, pulmonary fibrosis or asbestosis may have a valuable claim against a former employer.
Many experience asbestos exposure through the workplace. Workplaces may have been filled with toxic materials, and employers may have failed to warn of the serious health risks of the job. Filing a suit helps to raise the awareness of job safety.
Asbestos is a naturally occurring mineral that was first utilized in the early 1900s for its insulating abilities, fireproof properties, and versatility. It has been estimated that over 30 million tons of asbestos was used in the American economy in industrial yards, homes, schools, shipyards, and other workplaces.
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Newly emerging influenza A viruses (IAV) pose a major threat to human health by causing seasonal epidemics and/or pandemics, the latter often facilitated by the lack of pre-existing immunity in the general population. Early recognition of candidate pandemic influenza viruses (CPIV) is of crucial importance for restricting virus transmission and developing appropriate therapeutic and prophylactic strategies including effective vaccines. Often, the pandemic potential of newly emerging IAV is only fully recognized once the virus starts to spread efficiently causing serious disease in humans. Here, we used a novel phylogenetic algorithm based on the informational spectrum method (ISM) to identify potential CPIV by predicting mutations in the viral hemagglutinin (HA) gene that are likely to (differentially) affect critical interactions between the HA protein and target cells from bird and human origin, respectively. Predictions were subsequently validated by generating pseudotyped retrovirus particles and genetically engineered IAV containing these mutations and characterizing potential effects on virus entry and replication in cells expressing human and avian IAV receptors, respectively. Our data suggest that the ISM-based algorithm is suitable to identify CPIV among IAV strains that are circulating in animal hosts and thus may be a new tool for assessing pandemic risks associated with specific strains.
Influenza A viruses (IAV) have their natural reservoir in aquatic birds. However, they may acquire mutations that alter viral host tropism, leading to efficient replication in and/or transmission to other species including humans. The major potential of IAV to cause seasonal epidemics or pandemics is linked to their genomic variability. A high number of mutations results from the error-prone RNA-dependent RNA polymerase during viral RNA replication (antigenic drift). Furthermore, reassortment of genome segments may occur upon co-infection of the same cell by two or more different virus strains, often resulting in viruses with reassorted genomes and profoundly changed antigenic and biological propertie1,2,3. These genetic changes may cause immune escape from pre-existing neutralizing antibodies, resistance to antiviral drugs as well as changes in host tropism and/or replication efficiency in specific hosts. In addition, “candidate pandemic influenza viruses” (CPIV) may emerge from avian reservoirs and evolve into viruses that are efficiently transmitted among humans. Such pandemic strains often carry mutations in the viral hemagglutinin (HA), the virus glycoprotein that mediates binding to the cellular receptor. In some cases, amino acid (aa) substitutions in the HA cause a change in receptor usage from an avian-type receptor (α2,3-linked sialic acid, α2,3-SA) to a human-type receptor (α2,6 linked sialic acid, α2,6-SA), resulting in variants that are efficiently transmitted in humans4,5. Furthermore, aa substitutions in the HA cleavage site and/or other viral proteins may enhance the pathogenicity of newly evolved virus strains in specific hosts6,7,8,9,10,11.
Pandemic strains of IAV that encounter populations with limited pre-existing immunity may cause significant mortality and economic damage, especially if there is a delay between the identification and characterization of a new IAV strain and the production and licensing of a matching vaccine. The previous century has seen several major pandemics, including the 1918 Spanish flu (caused by an IAV H1N1 strain resulting in more than 50 million deaths), the 1957 Asian flu (H2N2, 1.5 million deaths), the 1968 Hong Kong flu (H3N2, 1 million deaths) and the 2009 Mexican flu (H1N1, 18,000 deaths)10,12,13. Other IAVs, such as the highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype, are transmitted less efficiently in humans but feature extremely high case-fatality ratios of close to 60%. These viruses were first detected in humans in 1997 in Hong Kong, where six fatalities occurred due to infection with avian H5N1-type IAV14,15. After several years with sporadic outbreaks, a second and main wave of infections with H5N1 viruses started in 2003 in some Asian countries, which then spread to Europe, the Middle East and Africa16,17. In 2006, first human infections occurred in Egypt, with sporadic infections being reported in the following years18. From 2003 until March 3rd 2015, a total of 784 cases of human H5N1 infections were reported worldwide (292 in Egypt), leading to 429 deaths (99 in Egypt)19. Since 2006, clade 2.2 H5N1 viruses have evolved in Egypt by antigenic drift, resulting in distinct endemic subclades with altered virulence, pathogenicity, transmission, receptor-binding preference and drug resistance profile20.
Specific bioinformatic algorithms may provide tools to monitor genetic changes in circulating IAV strains, including H5N1 HPAIV and identify IAV strains posing an increased pandemic risk, especially in geographic regions where HPAIV are endemic and frequently transmitted to humans. In this context, we recently described a novel phylogenetic algorithm based on the informational spectrum method (ISM)21,22,23.
In the ISM approach, sequences (protein or nucleotide) are transformed into signals by assigning a numerical value to each element (amino acid or nucleotide). These values correspond to the electron - ion interaction potential (EIIP), which determines electronic properties of amino acids and nucleotides. The signal obtained is then decomposed into a periodical function by Fourier transformation, resulting in a series of frequencies (F) and their amplitudes (A). The obtained frequencies correspond to the distribution of structural motifs with defined physico-chemical characteristics that are responsible for the biological function of the sequence. When comparing proteins that share the same biological or biochemical function(s) this technique allows the detection of code/frequency pairs that are specific for their common biological properties. The method is insensitive to the location of the motifs and, thus, does not require previous alignment of the sequences. The ISM was successfully applied to predict the biological function of novel proteins, in structure/function analyses of different protein and DNA sequences and in de novo design of biologically active peptides ( http://www.vin.bg.ac.rs/180/anims/ISMEIIP_Approach.html).
The calculation of the informational spectra (IS) of IAV HA aa sequences allowed us to identify minimal changes (single or combined aa substitutions) that likely affect interactions of the viral HA protein with the cellular receptor24. We previously used this algorithm to monitor in silico the human tropism of highly pathogenic H5N1 viruses isolated in Egypt21.
In the present study, we extended the ISM analysis to H5-HA sequences of circulating viruses to identify aa substitutions predicted to enhance the tropism to human hosts, thereby possibly affecting the respective viruses’ pandemic potential. ISM analysis of H5N1 HA1 sequences of HPAIV circulating between 2006 and 2014 in Egypt suggested that the mutations K153D, S223N and G272S may significantly increase the affinity of these viruses towards the human receptor. This is based on the observed increase in amplitude at the frequency (F) 0.236, representing the interaction with human cells, which is accompanied by a reduction in amplitude at F0.076, representing the interaction with avian cells. To validate our conclusions derived from these in silico studies, we generated replication-incompetent HA-pseudotyped retroviral particles (PRP) expressing H5-HA proteins with the respective codon substitutions (single or combined) and studied the entry of these PRPs into cells overexpressing human HA receptors in vitro. To further corroborate this set of data, recombinant IAV carrying equivalent substitutions in their HA genes were generated and their replication was analyzed in cell culture. As a safety measure, the multibasic HA-cleavage site present in the pathogenic H5-HA was replaced by a monobasic cleavage site in both the wildtype construct (Wt-LP IAV) and the mutants, resulting in viruses that required trypsin for fusion activity. Viruses carrying the specific HA substitutions were found to replicate more efficiently in mammalian cells overexpressing receptors for human HA, when compared to the parental Wt-LP IAV.
Interestingly, our analyses of H5N1 HPAIV isolates collected in Egypt over the past few years revealed a strong increase in prevalence of the predicted mutations in the HA coding sequences. Also, the predictions and hypotheses derived from our in silico analysis combined with our subsequent experimental studies to validate these predictions are in line with the recent natural evolution of IAV in Egypt. Thus, a fairly consistent picture arises with respect to mutations that may play a role in the adaption of avian IAV to the human receptor. The available evidence leads us to suggest that Egyptian H5N1 HPAIV are evolving towards increased human tropism, a prerequisite for increased pandemic potential of these viruses. Our study illustrates the power of bioinformatic approaches in making early predictions on possible adaptation of specific strains to new hosts and the emergence of CPIV.
Materials and Methods
Cells and viruses
Madin-Darby canine kidney cells (MDCK-II), human lung adenocarcinoma epithelial cells (A549), MDCK-SIAT1 cells (MDCK cells engineered to overexpress α2,6 sialic acid transferase), human rhabdomyosarcoma (TE671) cells and human embryonic kidney cells (293T) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen) supplemented with 100 I.U./ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS). Quail fibroblasts (QT6) were cultured in Ham’s F10 medium (Gibco, Invitrogen) supplemented with 100 I.U./ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 10% tryptose phosphate broth, 5% chicken serum and 10% bovine calf serum. MDCK-, 293T-, A549-, TE671- and QT6 cells were either obtained from the cell culture collection in Giessen or from LGC Promochem GmbH. MDCK-SIAT-1 cells were kindly provided by Mikhail Matrosovich, Institute of Virology, Philipps University, Marburg, Germany25. All cell monolayers were incubated at 37 °C in the presence of 5% CO2. The HPAIV isolate A/Thailand/KAN-1/2004 (KAN-1, H5N1) was kindly provided by P. Puthavathana, Thailand. Cells were infected at the indicated multiplicity of infection (MOI).
Characterization of avian and human HA receptor expression
To determine the abundance of specific sialic acid (SA) species (SAα2,3Gal and SAα2,6Gal) on the surface of MDCK-SIAT1, QT6 and A549 cells, cell monolayers were fixed with 3% paraformaldehyde for 1 h at room temperature (RT) followed by 3 washing steps with PBS. Cells were incubated for 1 h at RT with fluorescein-labelled Sambucus nigra agglutinin (SNA, Biozol, Germany, 1:100 dilution) to detect α2,6-SA or with biotinylated Maackia amurensis lectin II (MAAII, Biozol, Germany, 1:100 dilution) to detect α2,3-SA. Binding of biotinylated MAAII was visualized using streptavidin-Cy3 (Sigma-Aldrich, Germany) at a 1:500 dilution. Cell monolayers were washed with PBS and permeabilized with 0.2% Triton X-100 for 20 min. After another washing step with PBS, nuclei were stained with DAPI (4′, 6′-diamidino-2-phenylindole; 1:400 dilution) (Roth, Germany) for 10 min at RT. Finally, cells were washed with PBS and water, embedded in Fluoromount-G (eBioscience, Germany) and analyzed by confocal laser-scanning microscopy (Leica TCS SP5).
Moreover, the presence of α2,3- and α2,6-linked SA on A549, QT6 and MDCK-SIAT1 cells was measured by FACS using different concentrations of biotinylated lectins MAAII (α2,3-SA specific) and SNA/EBL (α2,6-SA specific), respectively and PE-conjugated streptavidin. Briefly, 106 cells were incubated with 10–100 ng of the biotinylated lectins for 30 min at 4 °C. After washing in PBS, cells were stained with PE-conjugated streptavidin (1:500) for 30 min at 4 °C, washed again and PE-positive cells were quantified by flow cytometry (FACS Calibur, Becton Dickinson).
In silico prediction of HA mutations with pandemic potential
For in silico prediction of mutations in H5-HA that might affect IAV cell tropism we analyzed the HA protein of KAN-1 by ISM, a virtual spectroscopy method for structure/function analysis of proteins26,27. ISM is based on the assumption that the protein-protein interaction encompasses two basic steps: (i) recognition and targeting between interacting proteins (long-range interactions at distances >5Ả) and (ii) chemical binding (short-range interactions at distances <5Ả)27. The long-range interaction properties of biological molecules are determined by the electron-ion interaction potential (EIIP) representing the main energy term of valence electrons22. The EIIP for organic molecules can be calculated by the following simple equation derived from the “general model pseudopotential28”:
where Z* is the average quasivalence number (AQVN) determined by:
where Zi is the valence number of the i-th atomic component, ni is the number of atoms of the i-th component, m is the number of atomic components in the molecule and N is the total number of atoms. Applying the given equations (1) and (2) to 20 amino acids, the following EIIP values are obtained (in Ry): L 0.0000, I 0.0000, N 0.0036, G 0.0050, V 0.0057, E 0.0058, P 0.0198, H 0.0242, K 0.0371, A 0.0373, Y 0.0516, W 0.0548, Q 0.0761, M 0,0823, S 0.0829, C 0.0829, T 0.0941, F 0.0946, R 0.0959 and D 0.1263. The ISM technique is based on a model of the primary structure of a protein using a sequence of numbers, by assigning to each amino acid the corresponding EIIP value. The obtained numerical sequence, representing the primary structure of a protein, is then subjected to a discrete Fourier transformation, which is defined as follows:
where x(m) is the m-the member of a given numerical series, N is the total number of points in this series and X(n) are discrete Fourier transformation coefficients. These coefficients describe the amplitude, phase and frequency of sinusoids, which comprised the original signal. The absolute value of a complex discrete Fourier transformation defines the amplitude spectrum and the phase spectrum. The complete information about the original sequence is contained in both spectral functions. However, in the case of protein analysis, relevant information is presented in an energy density spectrum23, which is defined as follows:
In this way, sequences are analyzed as discrete signals. It is assumed that their points are equidistant with the distance d = 1. The maximal frequency in a spectrum (as defined above) is F = 1/2d = 0.5. The frequency range is independent of the total number of points in the sequence. The total number of points in a sequence influences only the resolution of the spectrum. The resolution of the N-point sequence is 1/n. The n-th point in the spectral function corresponds to a frequency f(n) = nf = n/N. Thus, the initial information defined by the sequence of amino acids can now be presented in the form of an informational spectrum (IS), representing a series of frequencies and their amplitudes.
The IS frequencies correspond to the distribution of structural motifs with defined physicochemical properties determining a biological function of a protein. When comparing proteins that share the same biological or biochemical function, the ISM technique allows detection of code/frequency pairs that (i) are specific for their common biological properties or (ii) correlate with their specific interaction(s). These common informational characteristics of sequences are determined by the cross-spectrum or the consensus informational spectrum (CIS). A CIS of N spectra is obtained by the following equation:
where Π(i,j) is the j-th element of the i-th power spectrum and C(j) is the j-th element of CIS. Thus, CIS is the Fourier transform of the correlation function for the spectrum. Therefore, any spectral component (frequency) that is not present in all the compared informational spectra is eliminated. Peak frequencies in CIS represent the common information encoded in the primary structure of the analyzed sequences. This information corresponds to the mutual long-range interaction between analyzed proteins or their interaction with the common interactor.
ISM analysis allows: (i) to predict the biological function of a protein; (ii) to compare the biological activity within a group of proteins with the same function; (iii) to predict mutations that may increase or decrease the biological activity of a protein; (iv) to design an artificial protein sequence with a desired biological function.
The web tool for the calculation of informational spectra of proteins is available at: http://www.vin.bg.ac.rs/180/istree/ and sequences of the different HA versions can be found in the supplementary information.
Cloning and mutagenesis of HA
The H5-HA gene of KAN-1 was amplified by reverse RT-PCR from viral RNA isolated from virus-containing supernatants of infected MDCK cells. The HA sequence was amplified using primers Xma-HA-U (5′-TATTCCCGGGAGCAAAAGCAGGGG-3′) and Xma-HA-L (5′-ATATCCCGGGAGTAGAAACAAGGGTGTTTT-3′) and cloned via XmaI into a pHCMV-based vector as previously described29, resulting in M288-H5-HA. Single amino acid changes were introduced into M288-H5-HA by site-directed mutagenesis (quick change kit from Agilent Technologies). Double and triple mutations were introduced consecutively and all mutations were confirmed by sequencing.
Generation of H5-HA-pseudotyped retroviral particles (PRP)
To generate replication-incompetent HA-PRP for single-round infection assays, we cloned (in addition to our HA constructs) the NA and M genes of KAN-1 into the M288 vector, resulting in M288-H5-NA and M288-H5-M. Primers for NA were Xma-NA-U (5′-TATTCCCGGGAGCAAAAGCAGGAGT-3′) and Xma-NA-L (5′-ATATCCCGGGAGTAGAAACAAGGAGTTTTT-3′ ) and for M Xma-M-U (5 ′-TATTCCCGGGAGCAAAAGCAGGTAG-3 ′ ) and Xma-M-L (5′-ATATCCCGGGAGTAGAAACAAGGTAGTTTTT-3 ′ ). Oncoretroviral PRP were essentially prepared as described previously29. Briefly, the three plasmids M288-H5-HA, M288-H5-NA and M288-H5-M (1 μg each) were cotransfected with the MLV GagPol vector (pSV-Mo-MLVgagpol30 M579 (12.5 μg)) and the GFP transfer vector (pMP71-eGFP-pre31 M56a (7.5 μg)) into 293T cells (6 × 106 cells per 10 cm diameter dish) by calcium phosphate transfection. At 6 h posttransfection (p.t.), the transfection medium was replaced with DMEM containing 10% FCS, 2% L-glutamine, 100 I.U./ml penicillin, 100 μg/ml streptomycin. PRP-containing supernatants were harvested at 48 and 72 h p.t. After filtration (0.45 μm filter), PRPs were concentrated by low speed centrifugation (16 h, 6,000 rpm, 4 °C), resuspended in PBS and aliquots stored at –80 °C until further use.
Electron microscopy of H5-HA PRP
Two days p.t., cells were fixed with 2.5% glutaraldehyde in 0.05 M Hepes (pH 7.2). Then, cells were scraped off, centrifuged, post-fixed with OsO4 (1%), block-stained with uranyl acetate (2%), dehydrated stepwise in graded alcohol, immersed in propylenoxide and embedded in Epon (Serva, Heidelberg) with polymerisation at 60 °C for 48 h. Ultra-thin sections (60–80 nm) were cut using an ultramicrotome (Ultracut S or UCT; Leica, Germany) and stained with 2% uranyl acetate and lead citrate. Transmission electron microscopy was performed using an EM 902 (Zeiss) operated at 80 kV. Images were digitized using a slow-scan charge-coupled-device camera (Pro Scan; Scheuring, Germany).
Determination of receptor specificity of mutant H5-HA PRP
Mutant H5-HA PRP were titered on A549 cells, which express sialic acid receptors for human as well as avian IAV. Briefly, 5 × 104 cells were seeded in the wells of a 24-well plate. The next day, cells were incubated in duplicate with dilutions (0.1–20 μl) of the appropriate mutant PRP stock. After three days, transduction rates were determined by quantifying GFP-positive cells by flow cytometry (FACS Calibur, Becton Dickinson). PRP titers were calculated as:
To compare potential differences in receptor selectivity of the different HA mutants, equal amounts of the respective PRPs (MOI = 0.2 as determined on A549 cells) were added to QT6 cells, which predominantly express the α2,3-SA receptor bound by avian HA and MDCK-SIAT1 cells, which also express the avian receptor, but overexpress the α2,6-SA receptor bound by human HA25. Transduction rates of the two cell lines with the different mutant HA-PRPs were determined by quantifying GFP-positive cells by flow cytometry. Differences in receptor usage were determined by comparing relative numbers of GFP-positive cells determined on both cell lines (MDCK-SIAT1/QT6) using wildtype and mutant PRP, respectively.
3D structure model of the HA1 protein of IAV A/Thailand/1(KAN-1)/2004(H5N1)
The structural model of the H5 HA molecule of KAN-1 was constructed using SWISS-MODEL32 and POLYVIEW-3D33 and is based on homology modeling using the crystal structure of the H5-HA protein of A/Vietnam/1203/2004 (PDB ID: 2FK0.1) as a template34. H5 numbering was based on the mature protein (Gene bank accession: AAS65615.2) and the residues involved in receptor binding were defined according to Duvvuri et al.35.
Generation of H5-HA IAV with a monobasic cleavage site
The reverse genetic system encoding the 8 viral segments of HPAIV strain A/Thailand/KAN-1/2004 (KAN-1, H5N1) was kindly provided by Stephan Ludwig, Muenster, Germany. To clone the H5-HA gene encoding the monobasic cleavage site, a cDNA to be used as template in subsequent PCRs was generated using the Uni-12 primer36. The multibasic cleavage site PQRERRRKKR/GLF between HA1 and HA2 was mutated to a monobasic cleavage site, PQRETR/GLF, using specific primers to amplify HA1: Ba-LPAI-F1 (5′-TATAGGTCTCAGGGAGCAAAAGCAGGGGTTCAATC-3′) and Ba-LPAI-R1 (5′-TATTGGTCTCTGTCTCTCTTTGAGGGCTATTTCTG-3′) and to amplify HA2: Ba-LPAI-F2 (5′-TATCGGTCTCAAGACGAGAGGATTATTTGGAGCTAT-3′) and Ba-LPAI-R2 (5′-ATATGGTCTCGTATTAGTAGAAACAAGGGTGTTTTT-3′). Briefly, 2 μl cDNA were mixed with 25 μl 2x “Phusion Master Mix” (Thermo Scientific, USA), 2 μl of each primer (40 pmoles each) and the total volume was adjusted to 50 μl using nuclease-free water (Ambion, USA). The PCR reactions were then subjected to a pre-denaturation step at 98 °C for 30 sec, followed by 35 amplification cycles (98 °C/10 sec, 58 °C/30 sec and 72 °C/1.5 min) and a final extension at 72 °C/5 min. The PCR products were purified using the “GeneJET Gel Extraction Kit” (Thermo Scientific, USA) and digested with BsaI-HF (NEB, Germany). The digested and purified PCR products were ligated with AarI-digested pMPccdB DNA as previously described37 and transformed into E. coli DH5α (New England Biolabs, USA).
A part of the HA1 sequence of monobasic KAN-1/HA cloned into pMPccdB was replaced with the corresponding sequences from the appropriate M288-H5-HA mutant constructs (Mut-I: S223N and G272S; Mut-II: K153D and S223N; Mut-III: K153D, S223N and G272S) using Mva1269I and EcoRI (Thermo Scientific, USA). The dual-promotor plasmids with mutated monobasic HA were transformed into competent E. coli XL-1 blue, screened, sequenced and then used for transfection (“NucleoBond® Xtra Maxi”; Macherey-Nagel, Germany).
Generation of IAV stocks and determination of replication kinetics
To generate the KAN-1 (Wt-LP) virus and its mutant counterparts, 8 μg of plasmid DNA (1 μg of each plasmid) encoding the eight viral segments were transfected into a co-culture of 293T/MDCK-II cells (ratio 3:1, 10 cm2) as previously described with minor modifications37,38,39. Briefly, the transfection mixture consisting of 180 μl “Opti-MEM” (Gibco, Invitrogen, USA), 8 μg of plasmid DNA (1 μg of each plasmid) and 16 μl “Trans-IT2020” (Mirus, USA) was incubated for 45 min at RT. The transfection mixture was then diluted to 1 ml using Opti-MEM and transferred to an 80–90% confluent cell monolayer to allow transfection. The cells were then incubated for 8 h at 37 °C in the presence of 5% CO2. The transfection medium was replaced with 1 ml of infection medium (“Opti-MEM” containing 100 I.U./ml penicillin, 100 μg/ml streptomycin and 0.2% BSA (PAA, Germany)) and cell cultures were incubated for another 12 h. Then, an additional 1 ml of infection medium containing 2 μg/ml TPCK-treated trypsin was added (Sigma-Aldrich, USA). The cell culture supernatant was harvested 48 h after addition of the TPCK-treated trypsin and cell debris was removed by centrifugation at 2500 rpm for 5 min at 4 °C. An aliquot of 500 μl of each supernatant was used to inoculate fresh MDCK-II cells (25 cm2) and cultures were incubated for 72 h in the presence of TPCK-treated trypsin (1 μg/ml). Titers of recombinant viruses were determined as FFU/ml in MDCK-II cells40.
To determine multi-step growth curves, confluent monolayers of A549, QT6 and MDCK-SIAT1 cells, respectively, were each infected in triplicate with KAN-1 (Wt-LP) and its mutant derivatives, respectively, at an MOI of 0.001 FFU/cell. After 1 h of incubation at RT, the medium was replaced with infection medium (minimal essential medium with 100 I.U./ml penicillin, 100 μg/ml streptomycin, 0.2% BSA and 1 μg/ml TPCK-treated trypsin) and the cells were incubated at 37 °C. Supernatants were collected at 1, 24, 36 and 48 h postinfection and stored at −70 °C. Virus titers were determined by focus assay40.
All experiments with infectious virus were performed according to German regulations for the propagation of influenza viruses. All experiments involving highly pathogenic avian influenza A viruses were performed in a biosafety level 3 (BSL3) containment laboratory approved for such use by the local authorities (RP, Giessen, Germany).
IS frequency analysis
According to the ISM concept, interactions between biological molecules encompass two steps: (i) specific recognition by interacting molecules (long-range interactions with distances between 5 and 1000 Å) and (ii) chemical binding (short-range interactions with distances <5 Å). Without the first step the number of collisions between interacting molecules will be small and without the second step the interacting molecules will fail to bind to each other. In conclusion, each of these two steps represents a “necessary but not sufficient” condition for efficient interaction between biological molecules measured by their “productive collision”.
As was demonstrated previously for an efficient interaction, the protein domains, which are essential for recognition and targeting (recognition and targeting site, RTS) and the receptor binding domain (RBD), should be located close to each other41. Specific long-range interactions between proteins are represented by the common frequency components in their informational spectrum (IS) and the strength of the interaction is represented by the amplitude values on this frequency. This common frequency is determined by a peak in the cross-spectrum of proteins (CS). The presence of a common frequency component in the IS of a group of proteins suggests that these proteins have common interactor characteristics.
Mutations that decrease the amplitude value of the IS frequency (responsible for long-range interaction between proteins) decrease the efficacy of protein interactions by decreasing the efficacy of their recognition and targeting resulting in a decreased number of productive collisions. Conversely, mutations that increase this amplitude increase the efficacy of protein-protein interactions. Therefore, mutations that affect the amplitude on a characteristic frequency, although located outside the chemical RBD, also affect the efficacy of protein-protein interactions.
We previously showed that HA1 from H5N1 HPAIV that predominantly interact with the avian receptor are characterized by the IS frequency F(0.076), whereas HA1 from viruses that preferentially infect humans are characterized by the frequency F(0.236)41. Thus, the amplitude values of F(0.076) and F(0.236) can be used as indicators of the respective viral tropism (bird versus human). A ratio of A(0.236)/A(0.076) <1 suggests that the virus preferentially infects birds. Conversely, A(0.236)/A(0.076) >1 suggests that the virus is more likely to infect humans. Therefore, mutations in the H5N1 HA1 that decrease the amplitude F(0.076) and/or increase the amplitude F(0.236) are predicted to improve the recognition of the human receptor, leading to a virus that, most likely, infects humans more efficiently. In other words, increased A(0.236)/A(0.076) ratios indicate that viruses expressing the respective HA1 protein are more likely to infect humans.
In line with this, a recent analysis of mutations in H5N1 viruses circulating in Egypt between 2006 and 2012 revealed a total of 10 aa substitutions (P74S, H110R, A127T, F143Y, K153D, S188K, S223(N/I), S234P, G272S, N275S) that lead to a significant increase of the A(0.236)/A(0.076) ratio21,41. Importantly, the prevalence of a specific G272S replacement (that also occurred in combination with S223N) increased over time. Interestingly, these aa substitutions are highly specific for Egyptian H5N1 viruses and not conserved or very rare in the HA proteins of viruses isolated in other parts of Africa or in Europe and America. To obtain further evidence for a potentially increased pandemic risk associated with H5N1 HPAIV carrying these mutations, we characterized the respective mutations in the structural context of an HA of an IAV strain from another geographical region. To this end, we introduced 10 mutations (alone and/or in combination) into the HA coding sequence of the prototype Asian strain A/Thailand/KAN-1/2004 (KAN-1, H5N1) and determined the A(0.236)/A(0.076) amplitude ratios (Supplementary Table S2). Among all the mutations tested, K153D, S223(N,I) and G272S were found to increase the amplitude at the frequency F(0.236) most profoundly, the latter representing the frequency determined for human-adapted IAV strains (Fig. 1). The amplitude increase at F(0.236) was linked to an amplitude reduction at F(0.076), indicating an improved interaction with the avian receptor. The A(0.236)/A(0.076) ratio was found to be increased from 0.77 to 1.26, suggesting an increased pandemic potential for viruses carrying these mutations (see above). As indicated above, the prevalence of the G272S mutation in H5N1-HA sequences isolated from virus strains circulating in Egypt has strongly increased since 2009, particularly in viruses isolated from humans (from 5.6% in 2009 to 95.7% in 2011, see Supplementary Table S1). Furthermore, the (only) two HA sequences determined in 2012 for Egyptian isolates of human HAPIV were shown to carry the double mutation G272S/S223N.
Generation of H5-HA wildtype and mutant pseudotyped retroviral particles (PRP)
To corroborate the predicted role of specific HA mutations in human receptor usage, we introduced the appropriate mutations into the KAN-1 HA sequence and studied potential effects on virus entry in two cell lines that either express avian or human receptors. We first generated retroviral particles pseudotyped with wildtype KAN-1/HA or mutant forms of HA carrying the mutations predicted by ISM to confer higher preference for α2,6-SA (Table 1). Pseudotyped retroviral particles (PRP) were generated from 293T cells transfected with a plasmid expressing wildtype and mutant H5-HA, respectively and a set of plasmids expressing the NA and M proteins of KAN-1, MLV GagPol and the GFP marker protein, respectively. PRP infectivity was confirmed by FACS analysis of GFP expression after transduction of A549 cells with supernatants of transfected cells (Fig. 2a).Titers of the different wildtype and mutant PRP stocks determined on A549 cells varied between 2.4 × 104/ml and 2.3 × 107/ml (Table 1). Thin-section electron microscopy of transfected 293T producer cells revealed the expected morphology of immature and mature PRPs as well as budding structures (Fig. 2b). The retroviral particles displayed a dense envelope layer suggesting efficient expression and incorporation of the IAV surface proteins.
Determination of the receptor specificity of mutated H5-HA PRP
To investigate if the HA mutations improve α2,6-SA receptor usage (as suggested by the ISM analysis, see above), we analyzed the infectivity of the corresponding mutant PRP on cells expressing different amounts of α2,3- and α2,6-SA (QT6, MDCK-SIAT1). QT6 is known to primarily express α2,3-SA (the avian IAV-type receptor), while MDCK-SIAT1 mainly expresses α2,6-SA (the human IAV-type receptor). Expression levels of the two receptors were determined by differential lectin-binding analysis (Fig. 3). Varying concentrations of biotinylated lectins, MAAII specific for α2,3-SA and SNA/EBL specific for α2,6-SA, were added to the cells and cell-bound lectins were quantified by FACS analysis using PE-labelled streptavidine. A549 cells, which were used to determine the titers of our PRP stocks, were included as control (Fig. 3).
As expected, both types of lectins were bound by A549 cells at high levels, indicating good expression of both receptor types (Fig. 3a,b). In contrast, QT6 cells only bound to MAAII lectin, whereas SNA/EBL binding was not detectable, not even at the highest lectin concentration used (100 ng) (Fig. 3b). MDCK-SIAT1 cells were shown to bind both types of lectins, indicating the presence of both receptors. However, the amount of α2,6-SA was significantly increased compared to A549 control cells (Fig. 3b). The differential expression of both receptors on these cell lines was confirmed by confocal laser microscopy (Fig. 3c). Based on these observations, we decided to use QT6 cells (expressing predominantly avian IAV receptors) and MDCK-SIAT1 cells (overexpressing human IAV receptors) in subsequent differential infectivity experiments.
Potential differences in receptor usage by individual mutant PRPs (compared to the wildtype PRP) were assessed by determining the ratio between GFP-positive MDCK-SIAT1 and GFP-positive QT6 cells following transduction with equal amounts of wildtype and mutant PRP, respectively. All mutants predicted by ISM to result in increased α2,6-SA usage showed better infectivity on MDCK-SIAT1 cells as reflected by increased MDCK-SIAT1/QT6 ratios compared to wildtype HA (Fig. 4). The double mutant S223N/G272S showed the highest MDCK-SIAT1/QT6 ratio, which was not further increased, but rather decreased, in the triple mutant K153D/S223N/G272S. Thus, the increased usage of human α2,6-SA receptors by the mutations in HA predicted by ISM was experimentally confirmed as reflected by better infectivity of the corresponding mutant PRP on cells overexpressing the human receptor.
Replication kinetics of IAV HA mutants in MDCK-II cells
To assess potential effects of HA mutations on viral replication, we used reverse genetics to produce and characterize a set of IAV mutants based on a genetically modified form of KAN-1 (designated Wt-LP) that (for biological safety reasons) lacked a multi-basic cleavage site in its HA protein (Fig. 5a). The mutant viruses derived from the parental Wt-LP virus carried the HA mutations that had been shown to cause the strongest effects in our PRP infectivity assays (see above) (M-I, M-II and M-III). Figure 5b,c indicate the location (in the globular head domain, HA1, of the molecule) of the three aa residues 153, 223 and 272 that we have analyzed.
Focus size and morphology were analyzed using MDCK-II cells (Fig. 6a) and the relative distribution of focus diameters was determined (Fig. 6b). The M-II virus (K153D, S223N) produced foci similar in size to those produced by the parental WT-LP virus, while foci produced by the M-I (S223N, G272S) and M-III (K153D, S223N, G272S) mutants were significantly larger than those of KAN-1 (Wt-LP). This indicates that the M-II and -III viruses have a propagation advantage in MDCK-II cells compared to the parental virus.
Replication of H5N1 IAV mutants in different cell types in vitro
The potential impact of the introduced HA mutations on viral replication was further investigated in (i) MDCK-SIAT1 cells, which overexpress α2,6-SA25, (ii) QT6 cells, which overexpress α2,3-SA and (iii) A549 cells, which express similar amounts of α2,3-SA and α2,6-SA (Fig. 3). Confluent cell monolayers were infected with parental or mutant viruses at an MOI of 0.001 FFU/cell and maintained in trypsin-containing cell culture medium. Virus titers in supernatants collected at 12, 24, 36 and 48 h p.i. were determined as FFU/ml on MDCK-II cells40. In MDCK-SIAT1 cells (Fig. 7a), the titersof all mutants were approximately 0.5 log10 FFU/ml higher than that of the KAN-1 (Wt-LP) at 24–36 h p.i. However, at 48 h p.i., the M-III virus showed a significant increase in titer of about 1.0 log10 FFU/ml compared to both the parental KAN-1 (Wt-LP) and the other two mutants (Fig. 7a). Conversely, in QT6 (Fig. 7b) and A549 (Fig. 7c) cells, the parental KAN-1 (Wt-LP) and M-III viruses replicated to similar titers with no apparent differences in infectious particle number. In contrast, M-I and -II virus titers were significantly reduced by 0.5–1.0 log10 FFU/ml compared to the parental KAN-1 (Wt-LP) virus at 24–48 h p.i (Fig. 7b,c). Taken together, these results suggest that the triple mutation in HA (K153D/S223N/G272S; Mut-III) enhances viral replication in cells that overexpress α2,6-SA (MDCK-SIAT1), while there was no such difference in A549 and QT6 cells, which express low(er) amounts of α2,6-SA (compared to MDCK-SIAT1). The double mutants, S223N/G272S and K153D/S223N (Mut-I, Mut-II), also replicated more efficiently in MDCK-SIAT1 cells. However, in this case, the increase was much less profound compared to the triple mutant. In A549 and QT6 cells, the double mutants replicated to lower titers compared to both the parental virus and the triple mutant, possibly indicating that the binding to α2,3-SA was reduced in these mutants.
H5N1-type HPAIV are associated with high mortality in humans (>60%) and therefore represent a serious threat to human health. WHO launched extensive surveillance and monitoring programs for IAV42 and the development of new tools for early detection of new strains and prevention of IAV pandemics is of high priority. To date, H5N1 viruses have not (yet) been transmitted efficiently among humans, but there is a risk of this host restriction being overcome by reassortment of genome segments between H5N1 and co-circulating human H1N1 or H3N2 IAV strains.
Recent studies suggest that H5N1 HPAIV isolated in Egypt over the past few years increasingly exhibit a receptor usage that is similar to that of seasonal human H1N1 viruses, possibly indicating more efficient human-to-human transmission and increased pandemic potential in the (near) future24,43. An antigenic cartography of viruses isolated in Egypt revealed 6 distinct antigenic groups44. In line with our data and conclusions presented above, 19 of the 27 mutations presumably involved in adaptation of H5N1 viruses to the human receptor are associated with an increase of the A(0.236)/A(0.076) ratio21. Others possibly increase the affinity of the HA to the human receptor by a better adaptation of the receptor binding domain (RBD).
In this study, we used the ISM bioinformatic tool to characterize a set of mutations in the H5-HA that likely increase the pandemic potential of a prototype HPAIV (KAN-1) strain. We found that the mutations, when introduced in silico into the wildtype KAN-1 HA sequence, led to an increase of the amplitude ratio A(0.236)/A(0.076) at the frequencies F at 0.236 and 0.076, the latter indicating critical interactions of HA with human and avian receptors, respectively (Fig. 1). The ratio increased from 0.77 to 1.26, with values above 1 suggesting increased human tropism24. By comparison, the amplitude ratio increased only from 0.72 to 0.96 (Supplementary Fig. S1) if substitutions known to increase IAV transmission by respiratory droplets in ferrets (N154D, Q222L, N220K and T315I) were analyzed in the context of the HA protein of A/Vietnam/1203/2004 (H5N1)45.
In our ISM studies, the HA aa substitutions K153D, S223(N,I) and G272S (alone or in different combinations) increased the amplitude that indicates human receptor usage most profoundly (data not shown). Importantly, S223N and, particularly, G272S have also been detected in H5N1 HPAIVs circulating in Egypt and the number of viruses carrying these mutations has significantly increased since 2008 (Supplementary Table S1), correlating with an increasing number of human H5N1 infections in this country. The fact that the number of human cases in Egypt did not increase more dramatically in recent years might reflect a need for further adaptive requirements, for example in the receptor binding domain. Interestingly, S223N substitutions were only detected in human samples and combinations of the two aa substitutions were detected in both human H5N1 HPAIV HA sequences reported in 2012, further supporting the significance of these aa substitutions in viral adaptation to human hosts and suggesting an increased pandemic potential for H5N1 HPAIV strains currently circulating in Egypt (Supplementary Table S1).
Substitutions at position 153 (150-Loop) and 223 (220-Loop) of the receptor-binding site in the globular head domain of H5-HA are expected to affect the structure of the receptor-binding site and, thus, may influence receptor specificity. Amino acid mutations K153R and S223N/G were shown to alter the antigenic properties and/or receptor specificity34,46,47,48. However, it is currently not clear if the recently reported adaptive substitution G272S in the H5-HA protein of Egyptian isolates has similar effects on the receptor specificity20. In a recent attempt to establish a model that predicts the evolution of the viral population, the analysis of 3,944 HA sequences from H3N2 IAV revealed that adaptive mutations, which influence biological and immunological properties of these viruses, occur with highest probability within the epitopes A–D49. The alterations at the three selected positions (153, 223, 272) investigated here are also located within epitopes A, B and C, suggesting that with high probability such variations can be expected to occur also in H5-type HA of HPAIV as a consequence of natural viral adaptation.
To provide more direct evidence for an involvement of specific HA aa residues in the adaptation to human receptor usage we made appropriate replacements (predicted by ISM to alter the amplitude ratio A(0.236)/A(0.076) towards human receptor usage) in the HA protein of the A/Thailand/KAN-1/2004 (KAN-1) strain. Single or combined replacements of K153D, S223N and G272S were introduced into the H5-HA of KAN-1. Potential effects of the introduced aa substitutions were studied by using (i) retroviral particles pseudotyped with either the wildytpe or the respective mutant HAs and (ii) recombinant viruses based on an attenuated KAN-1 virus (WT-LP).
Transduction of PRPs expressing GFP and a wildtype or mutant form of HA into MDCK-SIAT1 cells (overexpressing the human receptor type) and QT6 cells (with very low expression of the human receptor type) revealed an increased MDCK-SIAT1/QT6 ratio for all these mutants compared to wildtype (Fig. 4), strongly supporting our ISM-based predictions on more efficient human receptor usage for these mutants. Notably, ISM analyses of double and triple mutations consistently showed a decrease in amplitude at the frequency for avian receptor usage (0.076) accompanied by an increase at the frequency for human receptor usage (0.236). This was more pronounced for the triple mutation (Supplementary Table S2). Even though the triple mutations substantially decreased the amplitude of the avian frequency compared to wildtype (Supplementary Table S2), the results shown in Fig. 7B indicate no decrease of viral replication in QT6 cells. We think that combinations of mutations do not only affect long-range interaction properties of HA1 but may also affect its 3D structure. For this reason, the triple mutation (even though it increases the calculated efficacy of recognition and targeting between HA1 and the human receptor) might not necessarily disturb the chemical binding to the avian receptor.
The PRP study was complemented by the characterization of recombinant viruses derived from KAN-1 (Wt-LP) that express different mutant forms of HA with combined replacements at positions 153, 223 and 272 (K153D, S223N and G272S). The data show that mutants containing combined S223N and G272S replacements produce larger foci in mammalian MDCK cells (Fig. 6). Compared to the parental KAN-1 (Wt-LP) virus, viruses containing double or triple replacements replicated significantly more efficiently in MDCK-SIAT1 cells overexpressing α2,6-SA (as determined by FFU/ml). In contrast, differential growth kinetics of wt versus HA mutant viruses was not observed in cells expressing only the avian HA receptor type, indicating that these changes do not represent a propagation disadvantage in avian hosts.
Recent human infections with a low pathogenic avian influenza virus (LPAIV) of the H7N9-type have initiated a paradigm change50,51. Previously, studies into the pandemic potential for humans were largely focused on HPAIV of the H5N1 and H7N3/H7N7 subtypes. While H5N1 HPAIV are known to cause severe disease in humans and many birds, the H7-type is generally associated with mild disease in humans but causes fatal disease in a wide range of birds including economically relevant species (chicken, turkeys, etc.)52,53. However, recent work shows that also specific H7-type HPAIV strains have the potential to replicate in mammalian species40,54. These observations have led to more extensive research and monitoring efforts to better understand the specific pathogenic and epidemiological characteristics of these HPAIV. Additionally, the large group of LPAIV and the recently documented H7N9, H10N8 and H6N1 LPAIV cases in humans extend the list of subtypes potentially causing human disease and thus deserve further studies55,56. The increasing number of cases with IAV subtypes not considered previously to cause disease in humans highlights the need to develop new tools (including in silico methods) that are suitable to closely monitor the biological properties (tropism, pathogenic potential in specific species, etc.) of circulating IAV strains and identify mutations indicative of CPIV. These tools might help the health authorities, healthcare systems and vaccine manufacturers to plan ahead and respond earlier to newly emerging IAVs with pandemic potential. The bioinformatic method used in the present study to define mutations in H5N1 IAV that indicate viral adaptation to humans and thus increase the pandemic potential of these viruses may be a suitable tool to identify CPIV among circulating influenza viruses in different populations.
How to cite this article: Schmier, S. et al. In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses. Sci. Rep. 5, 11434; doi: 10.1038/srep11434 (2015).
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We thank Patricia Schult-Dietrich for excellent technical expertise and El-Sayed M Abdelwhab for helpful scientific discussions. We acknowledge Mikhail Matrosovich, Marburg, Germany and P. Puthavathana, Thailand, for providing MDCK-SIAT1 cells and the influenza virus A/Thailand/KAN-1/2004 (KAN-1, H5N1), respectively. We are very grateful to Stephan Ludwig, Muenster, Germany, for providing the complete reverse genetic system for HPAIV strain A/Thailand/KAN-1/2004 (KAN-1, H5N1). This work was supported in part by the Ministry of Education and Science of the Republica Serbia (Grant 173001), founded FluResearchNet (01 KI 07136 to S.P.), the DFG-funded Collaborative Research Center (SFB) 1021 (Projects A1, C1 to J.Z. and S.P., respectively), the German Centre for Infection Research (DZIF), partner site Giessen, Germany (TTU-Emerging Infections to S.P. and J.Z.) and a fellowship EuroFlu funded by the 6th Framework Program (FP6) of the EU (SP5B-CT-2007-044098, to S.P. and U.D.), the BMBF founded FluResearchNet (01 KI 07136 to S.P.) and a fellowship of the German-Egyptian Research Long-Term Scholarship “GERLS” program co-funded by the Egyptian government and the German Academic Exchange Service (DAAD, to A.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare no competing financial interests.
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Schmier, S., Mostafa, A., Haarmann, T. et al. In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses. Sci Rep 5, 11434 (2015). https://doi.org/10.1038/srep11434
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“Yeah, that’s why I just want to call it a project. An experience. I would say, it’s definitely bigger than anything I’ve ever done”.
As much as Jhené fans enjoyed the collaborative Twenty88 album with Big Sean, her solo project is well overdue, following the 2014 release of Souled Out. Though the wait continues, there is talk of working with John Mayer and short films even thought to be included in the project release.
“I feel like writing and acting and just expressing myself have always been things that I wanted to develop as an artist, so what I’ve been working on pretty much involves everything that I feel is my passion, rolled into one”.

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