On episode #293 of the science show This Week in Virology, Vincent visits Melbourne, Australia and speaks with Melissa, Alex, Gilda, and Paul about their work on HIV infection of the central nervous system, West Nile virus, microbicides for HIV, and the Koala retrovirus.
You can find TWiV #293 at www.twiv.tv.
Experiments with the most dangerous human viruses, such as Ebola virus and Lassa virus, are carried out in biosafety level 4 (BSL-4) laboratories. Since visiting the National Infectious Diseases Laboratory BSL-4 and releasing the documentary video Threading the NEIDL, I was given the opportunity to tour three BSL-4 laboratories in the United States and Australia. My impressions of each facility might be of interest to readers of this blog.
Rocky Mountain Laboratories, Hamilton, Montana
Rocky Mountain Laboratory (RML) is located in the small and sleepy town of Hamilton, Montana (population 4,508), in the Bitterroot Vally two hours drive from Missoula and its airport. RML is the answer to those who feel that BSL4 laboratories should not be located in large cities. The downside is that it is more difficult to accommodate two careers in such a small town. The laboratory began as a shack in 1928 to study Rocky Mountain fever, and subsequently expanded to today’s size.
RML is made up of numerous independent buildings in which research on viruses, fungi, and bacteria is conducted under BSL-2 conditions. One of the larger buildings houses a BSL-3 and a BSL-4 laboratory. I was invited to visit RML to present a seminar and was offered the opportunity to tour the BSL-4. In contrast to the NEIDL, the RML BSL-4 is a functioning laboratory and therefore it was not possible for me to enter the facility.
RML is surrounded by a high black metal fence reminiscent of the fence at NEIDL. My host drove to the front entrance and parked on the street. The laboratory is in a residential neighborhood, nestled among one and two story homes. We walked through the front gate and proceeded through a security cottage which is manned by an armed guard. I walked through an airport-style metal detector and my suitcase containing a portable TWiV studio was passed on a conveyor belt through an imager. I had already received security clearance to enter the facility and upon showing the guard my drivers license, I was signed in and provided with an identification card. I was also asked if I had any guns, weapons, or explosives.
After leaving the guard house it is possible to walk freely to and from the various laboratory buildings, although entry to any of them requires proper electronic identification. The BSL-4 laboratory is in a separate building and entry requires passing through another security check point with armed guard, metal detector, and more questions about guns, weapons, or explosives. After this procedure I received a second identification card.
The BSL-4 laboratory is contained within a larger building that also houses BSL-3 and BSL-2 laboratories. A large multistory atrium in the building interior separates the office and BSL-2 space from the BSL-4 and BSL-3 laboratories. The RML BSL-4 is very much like the NEIDL in that it is a concrete box that sits within the outer building. I entered BSL-4 perimeter space with a guide, who used his ID to enter a corridor which surrounds the concrete box, much like the NEIDL (a separate iris scan is needed to pass through the guard house at the entrance to the NEIDL grounds). Entrance to this corridor did not require an iris scan as in the NEIDL; however entry into the BSL-4 laboratory does require an iris scan. On the wall near the entrance was a LCD panel showing the air pressure in each of the BSL-4 rooms. Negative pressure is maintained to ensure that air enters but does not leave each room. As in the NEIDL, individual labs within the BSL-4 may be activated or deactivated as needed.
Both the BSL-4 cube and the corridor surrounding it have windows, so it is possible for workers in the BSL-4 to look across the atrium at the BSL-2 laboratories, and vice versa. I did not visit the BSL-3 lab, which was a smaller facility at one end of the BSL-4.
The first BSL-4 room that we passed was occupied by two workers. They wore sealed suits supplied with air through coiled hoses dropped at intervals from the ceiling, the same arrangement I found at the NEIDL. The suits at RML are different: the head portion is completely clear, providing what should be excellent peripheral vision. The NEIDL suits in contrast have a clear portion only on the front. While at the NEIDL high slip-on boots are worn over the suit, in the RML BSL-4 the workers wore Crocs.
Both workers in the BSL-4 recognized me and waved. One is a postdoctoral scientist in the laboratory of Elke Muhlberger; she is working at RML because the NEIDL is not yet operational. The other person picked up a cellphone and dialed my guide to tell me that I do a ‘great podcast’. I suppose this cell phone always stays in the facility and is used for outside communication. The worker was able to use the phone by holding it next to his suit headpiece. My guide told me that this was possible because the suits are quiet. Later I was told that a variety of devices are employed for communication within the BSL-4 lab itself, including headsets, landlines and cell phones. Learning how to use these devices is obviously crucial for proper emergency response and is a critical part of the training that everyone going into the lab must undergo.
The workers also wore wireless headsets with microphones for communicating with each other. This type of communication was being planned for the NEID but was not yet implemented during my visit. The NEIDL now has a fully functional in-suit communication system. It permits persons in the BSL-4 suites to communicate with each other as well as communicate with persons outside of the BSL-4 labs. Persons working in the laboratory always have someone outside the labs (but within the building) whom they communicate with while working in the BSL-4 spaces.
Also on the perimeter of the BSL-4 is the clothing room, where those heading for the BSL-4 select surgical scrubs, socks, and underwear. I was provided with a souvenir: a pair of disposable underwear. No personal clothing can be worn into the BSL-4. In the NEIDL I was allowed to wear my underwear only because I was on a tour; normally no personal clothing may be worn into the facility.
Entry into the RML BSL-4 is similar to that of the NEIDL: workers enter a locker room, change from street clothes to scrubs, and then pass into a second room where they don the sealed suits. They then pass through an airlock into the BSL-4. The outer door is opened by pushing a button on the door which releases the gaskets only if the second door is sealed. After entering the lock the door seals, and then a second sealed door is opened to enter the BSL-4. The same procedure is used to exit the lab except that a chemical shower is taken on the way out.
Like the NEIDL, the RML BSL-4 can support experiments with animals. Through one window I could see a technician weighing mice. The results
are sent to the outside world via fax. Material may be passed to the outside through dunk tanks containing a disinfectant. For example, if sequence analysis of a sample is to be done, the material is placed in a tube containing a chemical that inactivates infectivity and extracts nucleic acids. The tube is then tightly sealed and dropped into a tank which passes through the wall. I was told that the tanks were designed to prevent a contortionist from passing through.
Overall the RML BLS-4 is very similar to the NEIDL. It exists to allow the safe conduct of experiments on the most dangerous human pathogens in cells and in animals. I did not request permission to photograph the RML. We were allowed to film ‘Threading the NEIDL’ to help demystify this type of laboratory, but the film was reviewed by their security team to ensure that nothing was filmed that could compromise security.
If you are in Hamilton, I strongly suggest you have coffee at Big Creek Coffee Roasters. The young lady at right made me one of the best cups of coffee I have ever had.
Doherty Institute, Melbourne, Australia
The Doherty Institute is a new multistory research facility built on the campus of the University of Melbourne, not far from the city center. It combines university research laboratories with diagnostic and WHO reference laboratories. The building contains a BSL-3 and BSL-4 laboratory. I entered the front door and passed by a security guard who was not armed and did not question my entrance. There were security gates by the elevators but they were not activated. I was told that the building security level is currently being negotiated between the university, who wants public access to the building, and the other laboratories who would like restricted accesss.
Any visitor to the Doherty can take the elevator to any floor, but access to the laboratories is restricted to individuals with ID cards that open locked doors. The exception is the fourth floor which has the cafeteria which is not locked. My host brought me to an upper floor where a guide showed us the BSL-4 laboratory. Although the BSL-4 is not operational I could not enter or take photographs because I had not requested proper permission.
The Doherty BSL-4 is quite different from those at RML and the NEIDL. It consists of one room where clinical specimens from suspected cases of hemorrhagic fever within Australia will be brought in and studied to determine if they contain any of the known hemorrhagic fever viruses. No research will be done in this BSL-4.
The Doherty BSL-4 is not housed in a concrete box. The facility is a separate entity within the outer building but it is built of panels of what appeared to be white metal with an insulated core. These are fitted together to make an airtight cube. The entire BSL-4 ‘box’ fits on one floor of the institute, with a narrow hallway between it and the outer walls of the building. There is a mechanical space of about four feet tall between the top of the BSL-4 box and the ceiling.
Entry into this BSL-4 takes place through airlocks similar to those in the RML and NEIDL facilities, and exit is through a chemical shower. The suits used in the facility resemble those in RML with completely clear head covers. We observed the BSL-4 from a command room which abuts the laboratory and has a clear view through large panes of glass. I was told that standard procedure will be that two workers must always be in the facility, and there must also be someone in the command center observing the workers. Communication between workers in the BSL-4, and also with the command center, is via headsets within the helmet. Video observation is part of the operating procedure in RML and NEIDL. These BSL-4 laboratories have numerous cameras which permit live viewing of the activities in the laboratories, and permit review at a later time.
The Doherty BSL-4 is the smallest and least flexible of those I visited. Its small size is consistent with its primary function, to diagnose viral hemorrhagic fevers. However it is not clear to me why clinical specimens could not be sent to the far more capable AAHL (below).
Australian Animal Health Laboratory, Geelong, Australia
The AAHL is the oldest and largest biosafety laboratory that I have visited. It is operated by the Commonwealth Scientific and Industrial Research Organisation (CSIRO). It was opened in 1985 after a long construction period, and its original purpose was to house research on animal pathogens. Therefore the emphasis was on preventing animal pathogens from leaving the facility. Later it was expanded to include work on human pathogens and the BSL-4 capability was added. The impetus for the expansion was the discovery of Hendra virus in Australia in 1994.
The AAHL is in the city of Geelong, about an hour drive south of Melbourne. My host picked me up at my Melbourne hotel and drove south though a heavily industrialized area. However Geelong is a very pleasant coastal town and the AAHL is located in a bucolic setting near the sea. We drove up to the front gate which my host opened with an ID card. There is a guard shack at the entry gate but my host told me that the guard was removed some time ago as it was deemed unnecessary. The laboratory is a huge concrete building several stories high, dominated by a tall water tower that from the distance resembles a cross. My host opened the front door of the building with his ID card. I signed in at the front desk but there were no metal detectors or armed guards.
When I asked my host if I could take photographs he said of course, anything that I liked. He said only in the US do BSL-4 facilities worry about bioterrorism.
Before entering the laboratory I was required to take a brief training session. I watched a 10 minute video which detailed entry and exit procedures, and then took a 15 question quiz. I am sure I got all the answers right. Then my host and I entered the facility.
A large command center stands at the entrance of the facility. There a single individual monitors, on large displays, the status of each room and who has passed from section to section based on swiping of IDs on wall mounted sensors. I placed my valuables in a safe, and then we swiped our cards to unlock a revolving door and moved into the secure area.
The first step was to change our clothing. We reached a room lined with doors behind which were small locker rooms. I had been assigned to room 16, locker 16B. We had been instructed to knock first to be sure that no one else was in the room. Inside I found a narrow room with 10 small lockers. I removed all of my clothes, including my ID, and placed them in the locker. The only item I was allowed to bring in was my glasses.
At one end of the room was a metal door sealed with a gasket. To release the gasket I pushed a large, black rubber switch on the wall. In a few seconds the heavy metal door could be pushed open. As the door swung shut after me the gasket automatically sealed. Then I pressed another switch to open the second door and passed into a second locker room. Here I found locker #16B filled with clothing that had been selected according to my measurements, which I had sent several weeks earlier. I donned underpants, a blue short sleeved shirt, white overalls, socks, sneakers, eye guard, and another ID card. Outside of this room I met my host who then lead me around the laboratory.
My first impression was the imposing size of the facility: the ceilings are very high and the hallways are over 10 feet wide. My host told me that the hallways were made large by the original planners because they realized that workers would be spending a lot of time in the facility and did not want them to feel claustrophobic.
The walls of this facility are very thick concrete. To pass wiring through the walls, it was necessary to embed pipes in the concrete. These are visible at multiple locations and the wires going in and out of them are secured to the concrete surface. My host was excited to point out that the original planners had great foresight and installed numerous such conduits, not all of which are yet being used.
The AAHL is a multi-story facility divided roughly into three parts. On one side of building is a laboratory for large animals such as horses or cows, which can be configured as BSL3 or BSL4 space as needed. I did not see this part of the laboratory as the lights were off and nothing was visible through the windows.
The middle building section contains BSL3 and BSL4 laboratories which also can be configured as needed. The BSL4 space was like that in RML and the NEIDL – a concrete block within the building, with windows cut in the walls. I saw two scientists working in one of the laboratories. Here during working hours it is not necessary to have two workers in a BSL4, as long as there is an observer on the outside. After working hours and on weekends the two worker rule is in effect.
Procedures for entering and exiting the BSL4 are similar to those in RML and NEIDL, with airlocks and chemical showers. The AAHL BSL4 is used for research on all of the most deadly human viruses, both in cell culture and in animals. The BSL4 can also conduct work on smaller animals, such as mice and ferrets. One of the floors of the AAHL houses 25,000 mice.
We also visited the mechanical space in the basement. One floor consisted largely of HEPA filters – over 1,000 of them are needed to service the laboratory. Another floor contained the ‘cook tanks’, where liquid waste from sinks and showers is autoclaved. I saw over a dozen tanks, far more than the three at the NEIDL (the 3 NEIDL cook tanks are each over 5600 liters in size, which permits the facility to have one in use (filling with liquid waste) while another is in sterilization mode, and keeping one in reserve). The engineering support for the AAHL facility is impressive – 70 full time engineers are needed to run the laboratory at a cost of $36 million per year.
The last third of the AAHL contains rooms to make people happy, such as an exercise room and a cafeteria. The latter has a very large glass window looking out onto a golf course. In the center of the room was a shelf full of plastic boxes labeled with last names, and containing mustard, ketchup, salt, pepper, and other things to put on your food. These can be brought in to the laboratory but cannot be brought out.
I had lunch with many of the scientists who work in the facility. One of them had been the first person to isolate Hendra virus in 1994. I told him about the rising concern of some in the US over the perceived lack of safety in biosecurity labs. He laughed and said that the labs are completely safe. Who would know better than someone who has spent their life working in one?
To leave the laboratory, I went back to the locker room area, removed all of the AAHL clothing, and put it back in the locker. Then I dipped my glasses in a glutaraldehyde solution for two minutes, followed by a rinse in tap water. I was assured that this treatment would not damage the plastic lenses. Next I passed through the air lock door into the shower. I turned on the shower which runs for two minutes, during which time the doors cannot be opened. This time period is indicated by a flashing yellow light, and I was told to use shampoo and soap. After the shower I punched the rubber switch and the sealed door opened into the next locker area, where towels and my clothing awaiting me. Then we left the secure area back into the office and administration area.
My visits to BSL4 laboratories enforce my belief that these are very safe places to work with very dangerous pathogens. Well defined procedures are in place for every operation, and there are many levels of redundancy built into the laboratories to deal with mechanical failures of any type. Most importantly, the workers are well trained and dedicated to carrying out their mission. I strongly believe that we must investigate organisms of both high and low pathogenicity, because no one knows where the next revelation will come from.
Plum Island is a small island off the tip of Long Island, New York, and the home of a biosecurity facility for work on animal pathogens. Its mission is similar to that of AAHL before BSL4 capability was added to that Australian laboratory. I worked at Plum Island briefly as a PhD student in the 1970s and have only a vague recollection of the laboratory. I’ll be visiting Plum Island in the near future and I’ll be sure to update my memory. However one aspect of Plum Island stands out in my mind.
On Plum Island, as at AAHL, it is necessary to change from street clothes to laboratory wear provided by the facility. However unlike AAHL, where there is only one person in a locker room at any given time, the changing rooms at Plum Island were communal. I recall entering the men’s locker room where dozens of scientists were disrobing and placing their clothes in lockers. Here I was introduced to people I had not met before – a disconcerting experience because everyone was naked! You were sure to keep your eyes raised while shaking hands. I remember then passing into a second locker room where we donned white labwear – but I don’t remember any airlocks. We worked without masks or suits – the threat was to animals, not to humans. Viruses such as foot and mouth disease virus African swine fever virus are typical research subjects at Plum Island.
Updated 21 July 2014 to correct facts about RML ires scans, communication, and access to buildings, and NEIDL cook tanks, clothing allowed in the BSL-4, photography and communication.
On episode #292 of the science show This Week in Virology, Vincent visits Medimmune and speaks with Wade, Matt, Nicole, and Ken about why they work in industry and their daily roles in a biotechnology company.
You can find TWiV #292 at www.twiv.tv.
Vincent, Rich, and Kathy and their guests Clodagh and Ron recorded episode #291 of the science show This Week in Virology at the 33rd annual meeting of the American Society for Virology at Colorado State University in Ft. Collins, Colorado.
You can find TWiV #291 at www.twiv.tv.
Wild poliovirus has been detected in the sewers of Brazil and Israel. Fortunately, no cases of poliomyelitis have been reported in either country. Why is poliovirus present in these countries and what are the implications for the eradication effort?
Wild type poliovirus (e.g. not vaccine-derived virus) was detected in sewage samples that had been collected in March 2014 at Viracopos International Airport in the State of Sao Paulo. Wild type poliovirus had not been detected in Brazil since 1989 when the last case of poliomyelitis was reported in that country, and has not been found since March 2014.
Sequence analysis of the RNA genome of the wild type poliovirus found in the Brazilian sewer indicates that it is closely related to an isolate from a case of poliomyelitis in Equatorial Guinea. It seems likely that this virus was carried to Brazil in the intestine of an infected person who did not have symptoms of paralytic disease (only 1 in 100 poliovirus infections lead to paralysis). This individual might have traveled from Equatorial Guinea to the Brazilian airport where use of the bathroom lead to introduction of poliovirus into the sewer.
There have been 8 reported cases of poliomyelitis in Equatorial Guinea in 2014, from which we can extrapolate that there have been approximately 800 infected individuals. Given the number of cases of poliomyelitis that have been reported globally over the past 20 years, it is surprising that virus has not been detected previously in Brazilian sewage, especially at the airport. I suspect that wild type poliovirus would be detected in sewage in the US, given the number of individuals who enter that country each day. However the US does not conduct routine surveillance for poliovirus in sewage.
Brazil utilizes the Sabin vaccine to control poliomyelitis, and in the past 8 years over 95% immunization coverage has been achieved. The Sabin vaccine is taken orally and replicates in the intestine where it induces mucosal immunity. The intestine of Brazilians do not support the replication of wild type poliovirus, which is why the presence of wild type virus in sewage is not a threat – it is unlikely to spread in the population.
The isolation* of wild type poliovirus from sewage and from stool samples in Israel is a far more serious matter. As with Brazil, there have been no reported cases of poliomyelitis in Israel since 1989. Yet ten different sites in central and south Israel have been persistently positive for wild type poliovirus since February 2013. Wild type poliovirus has been found intermittently at 8 of 47 different sampled sites in southern and central Israel, and in stool from healthy persons collected in July 2013.
Two major lineages of wild type polioviruses currently circulate in endemic countries: the South Asian (SOAS) lineage in Pakistan and Afghanistan, andthe West African lineage in Nigeria. Nucleotide sequence analysis of the wild type poliovirus isolates from Israel indicate that they are closely related to the South Asian lineage, and in particular to polioviruses that circulated in Pakistan in 2012 and in Egypt in 2012. Molecular clock analysis of the sequences indicate that poliovirus was probably transmitted in 2012 from Pakistan into Egypt and Israel, and then spread in the latter country.
The central point of poliovirus circulation is within Bedouin communities in the south of Israel. The main virus reservoir within this community is children less than 9 years of age who had been immunized with inactivated poliovirus vaccine (IPV). This vaccine has been exclusively used in Israel since 2005, with overall vaccination coverage between 92-95%, and 81-100% within individual districts. The last nine birth cohorts in this country have been immunized solely with IPV.
The response to isolation of wild type poliovirus in Israeli sewers was to complete IPV immunization of all children in the south, raising coverage to above 99%. Then from August 2013 onwards, all children up to the age of nine years old were given a dose of bivalent oral poliovirus vaccine (OPV) containing types 1 and 3 poliovirus. All children who received OPV had previously been immunized with IPV, a strategy that prevents vaccine-associated poliomyelitis.
The finding of sustained circulation of wild type poliovirus in Israel shows that the virus can circulate silently in a population that has been well immunized with IPV. Such circulation occurs because IPV does not sufficiently protect the intestinal tract against poliovirus infection. However poliomyelitis does not occur in such populations because IPV-induced antibodies in the blood prevent virus invasion into the central nervous system. The US now exclusively uses IPV and it is likely that wild polioviruses are present in US sewage, although as mentioned above the US does not search for poliovirus in sewage. Silent circulation of wild type poliovirus in countries that use IPV poses a threat to other countries where immunization coverage is low.
These findings indicate that immunization with IPV will not lead to eradication of wild type poliovirus. This observation is problematic because the World Health Organization has recommended a gradual shift from OPV to IPV. In the past I have also supported such a transition, but I have also remained cautious about the ability of IPV to immunize the human gut. The experience in Israel confirms my suspicions.
The US shifted from using OPV to IPV because the associated vaccine-associated poliomyelitis was not acceptable in a country with no paralytic disease caused by wild type poliovirus. Now it seems that eradication cannot be achieved with IPV. What can be done about this conundrum? OPV should be used to eradicate remaining pools of wild type poliovirus in endemic countries (Nigeria, Afghanistan, Pakistan). At the same time environmental surveillance must be done in all countries that exclusively use IPV. If wild type poliovirus is found in the sewage of such countries, then introduction of OPV, in children previously immunized with IPV, should be considered to eliminate the reservoir of will type virus. It will be important to observe the effect of the distribution of OPV in Israel on the circulation of wild type poliovirus.
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*Infectious poliovirus was isolated by adding sewer and stool filtrates to monolayers of L20B cells, which are mouse fibroblasts that produce the cellular receptor for poliovirus. These cells were produced in my laboratory, and are useful for isolating polioviruses because they are not susceptible to infection with non-polio enteroviruses. I am pleased to be able to contribute to efforts to control poliomyelitis. [reference]
On episode #290 of the science show This Week in Virology, Vincent meets up with Janet Butel and Rick Lloyd at Baylor College of Medicine to talk about their work on polyomaviruses and virus induced stress.
You can find TWiV #290 at www.twiv.tv.
The gain of function experiments in which avian influenza H5N1 virus was provided the ability to transmit by aerosol among ferrets were met with substantial outrage from both the press and even some scientists; scenarios of lethal viruses escaping from the laboratory and killing millions proliferated (see examples here and here). The recent publication of new influenza virus gain of function studies from the laboratories of Kawaoka and Perez have unleashed another barrage of criticism. What exactly was done and what does it mean?
According to critics, virologists should not be entrusted to carry out gain of function studies with influenza virus; they are dangerous and of no scientific value. The headline of a Guardian article is “Scientists condemn ‘crazy, dangerous’ creation of deadly airborne flu virus” (the headline is at best misleading because the influenza virus that was reconstructed by Kawaoka and colleagues is not deadly when transmitted by aerosol). The main opponents of the work appear to be Lord May*, former President of the Royal Society; Harvard epidemiologist Mark Lipsitch; and virologist Simon Wain Hobson. They all have nasty things to say about the work and the people doing it. To his credit, author of the Guardian article Ian Sample (who likely did not write the headline) does present both sides of the study, and attempts to explain what was done. He even quotes Kawaoka on the value of the work. But much is left unsaid, and without a detailed analysis of the study, its importance is not readily apparent.
The work by Kawaoka and colleagues attempts to answer the question of whether an influenza virus similar to that which killed 50 million people in 1918 could emerge today. First they identified in the avian influenza virus sequence database individual RNA segments that encode proteins that are very similar to the 1918 viral proteins.
Next, an infectious influenza virus was produced with 8 RNA segments that encode proteins highly related to those of the 1918 virus. Each RNA segment originates from a different avian influenza virus, and differs by 8 (PB2), 6 (PB1), 20 (PB1-F2), 9 (PA), 7 (NP), 33 (HA), 31 (NA), 1 (M1), 5 (M2), 4 (NS1), and 0 (NS2) amino acids from the 1918 virus.
The 1918-like avian influenza virus was less pathogenic in mice and ferrets compared with the 1918 virus, and more pathogenic than a duck influenza virus isolated in 1976. Virulence in ferrets increased when the HA or PB2 genes of the 1918-like avian influenza virus were substituted with those from the 1918 virus.
Aerosol transmission among ferrets was determined for the 1918-like avian influenza virus, and reassortants containing 1918 viral genes (these experiments are done by housing infected and uninfected ferrets in neighboring cages). The 1918 influenza virus was transmitted to 2 of 3 ferrets. Neither the 1918-like avian influenza virus, nor the 1976 duck influenza virus transmitted among ferrets. Aerosol transmission among ferrets was observed after infection with two different reassortant viruses of the 1918-avian like influenza virus: one which possesses the 1918 virus PB2, HA, and NA RNAs (1918 PB2:HA:NA/Avian), and one which possesses the 1918 virus PA, PB1, PB2, NP, and HA genes (1918(3P+NP):HA/Avian).
It is known from previous work that amino acid changes in the viral HA and PB2 proteins are important in allowing avian influenza viruses to infect humans. Changes in the viral HA glycoprotein (HA190D/225D) shift receptor specificity from avian to human sialic acids, while a change at amino acid 627 of the PB2 protein to a lysine (627K) allows avian influenza viruses to efficiently replicate in mammalian cells, and at the lower temperatures of the human upper respiratory tract.
These changes were introduced into the genome of the 1918-like avian influenza virus. One of three contact ferrets was infected with 1918-like avian PB2-627K:HA-89ED/190D/225D virus (a mixture of glutamic acid and aspartic acid at amino acid 89 was introduced during propagation of the virus in cell culture). Virus recovered from this animal had three additional mutations: its genotype is 1918-like avian PB2-627K/684D:HA-89ED/113SN/190D/225D/265DV:PA-253M (there are mixtures of amino acids at HA89, 113, and 265). This virus was more virulent in ferrets and transmitted by aerosol more efficiently than the 1918-like avian influenza virus. The virus recovered from contact ferrets contained yet another amino acid change, a T-to-I mutation at position 232 of NP. Therefore ten amino acid changes are associated with allowing the 1918-like avian influenza virus to transmit by aerosol among ferrets. Aerosol transmission of these viruses is not associated with lethal disease in ferrets.
Previous studies have shown that changes in the HA needed for binding to human sialic acid receptors reduced the stability of the HA protein. Adaptation of these viruses to aerosol transmission among ferrets required amino acid changes in the HA that restore its stability. Similar results were obtained in this study of the 1918-like avian influenza virus, namely, that changes that allow binding to human receptors (HA-190D/225D) destabilize the HA protein, and changes associated with aerosol transmission (HA-89D and HA- 89D/113N) restore stability.
The ability of current influenza virus vaccines and antivirals to block replication with ferret transmissible versions of the 1918-like avian influenza virus was determined. Sera from humans immunized with the 2009 pandemic H1N1 strain poorly neutralized the virus, indicating that this vaccine would likely not be protective if a similar virus were to emerge. However replication of the ferret transmissible 1918-like avian influenza virus is inhibited by the antiviral drug oseltamivir.
Examination of influenza virus sequence databases reveals that avian viruses encoding PB2, PB1, NP, M, and NS genes of closest similarity to those of the 1918-like avian virus have circulated largely in North America and Europe. The PB2-627K change is present in 168 of 4,293 avian PB2 genes (4%), and the HA-190D change is in 9 of 266 avian H1 HA sequences (3%), and one also had HA-225D.
Most of the viral sequences used in this work were obtained quite recently, indicating that influenza viruses encoding 1918-like proteins continue to circulate 95 years after the pandemic. Now that we have discussed the work, we can summarize why it is important:
Do these experiments constitute an unacceptable risk to humans? Whether or not the 1918-like avian influenza virus, or its transmissible derivatives, would replicate, transmit, and cause disease in humans is unknown. While ferrets are a good model for influenza virus pathogenesis, they cannot be used to predict what will occur in humans. Nevertheless it is prudent to work with these avian influenza viruses under appropriate containment, and that is how this work was done. The risk is worth taking, not only because understanding transmission is fundamentally important, but also because of unanticipated results which often substantially advance the field.
The Guardian quotes Lipsitch as saying that “Scientists should not take such risks without strong evidence that the work could save lives, which this paper does not provide”. The value of science cannot only be judged in terms of helping human health, no matter what the risk. If we only did work to improve human health, we would not have most of the advances in science that we have today. One example is the biotechnology industry, and the recombinant DNA revolution, which emerged from the crucial discovery of restriction enzymes in bacteria – work that was not propelled by an interest in saving lives.
The results obtained from the study of the reconstruction of a 1918-like avian influenza virus are important experiments whose value is clear. They are not without risk, but the risk can be mitigated. It serves no useful purpose to rail against influenza virus gain of function experiments, especially without discussing the work and its significance. I urge detractors of this type of work to carefully review the experiments and what they mean in the larger context of influenza virus pathogenesis. I understand that the papers are complex and might not be easily understood by those without scientific training, and that is why I have tried to explain these experiments as they are published (examples here and here).
In the next post, I’ll explain the gain of function experiments recently published by the Perez laboratory.
*May’s objection is that the scientists carrying out the work are ‘grossly ambitious people’. All scientists are ambitious, but that is not what drives Kawaoka and Perez to do this work. I suggest that Lord May read the papers and base his criticism on the science.
On episode #289 of the science show This Week in Virology, Vinny and the capsids answer listener questions about the definition of life, state vaccination laws, the basic science funding problem, viral ecology, inactivation of viruses by pressure, and much more.
You can find TWiV #289 at www.twiv.tv.
Segmented genomes abound in the RNA virus world. They are found in virus particles from different families, and can be double stranded (Reoviridae) or single stranded of (+) (Closteroviridae) or (-) (Orthomyxoviridae) polarity. Our recent discussion of the advantages of a segmented viral genome, compared with monopartitie genomes, generated a good discussion. Another interesting question concerns the evolutionary relationship between the two genome types. Did monopartite viral genomes emerge first, then later fragmented to form segmented genomes? Some recent experiments provide insight into this question.
Insight into how a monopartite RNA genome might have fragmented to form a segmented genome comes from studies with the picornavirus foot-and-mouth disease virus (FMDV). The genome of this virus is a single molecule of (+) RNA. Serial passage of the virus in baby hamster kidney cells led to the emergence of genomes with two different large deletions (417 and 999 bases) in the coding region. Each mutant genome is not infectious, but when introduced together into cells, infectious virus is produced. This virus stock consists of a mixture of the two mutant genomes packaged separately into virus particles. Infection takes place because of complementation: each genome provides the proteins missing in the other.
Further study of the deleted FMDV genomes revealed the presence of point mutations in other regions of the genome. These mutations had accumulated before the deletions appeared, and increased the fitness of the deleted genome compared with the wild type genome.
These results illuminate the first steps in fragmentation of monopartite viral RNA, possibly a pathway to a segmented genome. It is very interesting that the point mutations that gave the fragmented RNAs a fitness advantage over the standard RNA arose before fragmentation occurred – further evidence that mutations occur in a specific sequence. As the authors write:
Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.
While the fragmentation of the FMDV genome may represent a step on the path to segmentation, its relevance to what occurs in nature is unclear, because the results were obtained in cell culture.
A compelling picture of the genesis of a segmented RNA genome comes from the discovery of a new tick borne virus in China, Jingmen tick virus (JMTV). The genome of this virus comprises four segments of (+) stranded RNA. Two of the RNA segments have no known sequence homologs, while the other two are related to sequences of flaviviruses. The RNA genome of flaviviruses is not segmented: it is a single strand of (+) sense RNA. The proteins encoded by RNA segments 1 and 3 of JMTV are non-structural proteins which are clearly related to the flavivirus NS5 and NS3 proteins.
The genome structure of JMTV suggests that at some point in the past a flavivirus genome fragmented to produce the RNA segments encoding the NS3 and NS5-like proteins. This fragmentation might have initially taken place as shown for FMDV in cell culture, by fixing of deletion mutations that complemented one another. Next, co-infection of this segmented flavivirus with another unidentified virus took place to produce the precursor of JMTV.
Both sets of findings were accidents, made while investigating unrelated problems. The results provide new clues about the origins of segmented RNA viruses, and are examples of the value and unpredictable nature of basic science research.
The value of influenza aerosol transmission experiments
4 July 2014
In early 2013 Lipsitch was the subject of a piece in Harvard Magazine about avian influenza H5N1 virus entitled The Deadliest Virus. I have previously criticized this article in which Lipsitch calls for more stringent H5N1 policies. More recently Lipsitch published an opinion in PLoS Medicine in which he called for alternatives to experiments with potential pandemic pathogens. We discussed this piece thoroughly on This Week in Virology #287. The arguments he uses in both cases are similar to those in the OpEd.
The Times OpEd is entitled Anthrax? That’s not the real worry. The title is a reference to the possible exposure to anthrax bacteria of workers at the Centers for Disease Control. Even worse than anthrax, argues Lipsitch, would be accidental exposure to a pathogen that could transmit readily among humans. He then argues that such a pathogen is being created in laboratories that study avian influenza H5N1 transmission.
Lipsitch tells us ‘These experiments use flu strains like H5N1, which kills up to 60 percent of humans who catch it from birds.’ As an epidemiologist Lipsitch knows that this statement is wrong. The case fatality ratio for avian H5N1 influenza virus in humans is 60% – the number of deaths divided by the cases of human infections that are diagnosed according to WHO criteria. The mortality rate is quite different: it is the number of fatalities divided by the total number of H5N1 infections of humans. For a number of reasons the H5N1 mortality ratio in humans has been a difficult number to determine.
Next Lipsitch incorrectly states that the goal of experiments in which avian influenza H5N1 viruses are given the ability to transmit by aerosol among ferrets is ‘to see what gives a flu virus the potential to create a pandemic.’ The goal of these experiments is to identify mechanistically what is needed to make an avian influenza virus transmit among mammals. Transmission of a virus is required for a pandemic, but by no means does it assure one. I do hope that Lipsitch knows better, and is simply trying to scare the readers.
He then turns to the experiments of Kawaoka and colleagues who recently reconstructed a 1918-like avian influenza virus and provided it with the ability to transmit by aerosol among ferrets. These experiments are inaccurately described. Lipsitch writes that the reconstructed virus was ‘both contagious and comparably deadly to the 1918 flu that killed tens of millions of people worldwide’. In fact the reconstructed virus is less virulent in ferrets than the 1918 H1N1 virus that infected humans. In the same sentence Lipsitch mixes virulence in ferrets with virulence in humans – something even my virology students know is wrong. Then he writes that ‘Unlike experiments with anthrax, creating such flu strains in the lab presents a danger that affects us all, because once it is out, such a strain would be extremely hard to control.’ This is not true for the 1918-like avian influenza virus assembled by the Kawaoka lab: it was shown that antibodies to the 2009 pandemic H1N1 influenza virus can block its replication. The current influenza virus vaccine contains a 2009 H1N1 component that would protect against the 1918-like avian influenza virus.
The crux of the problem seems to be that Lipsitch does not understand the purpose of influenza virus transmission experiments. He writes that ‘The virologists conducting these experiments say that by learning about how flu transmits in ferrets, we will be able to develop better vaccines and spot dangerous strains in birds before they become pandemic threats.’ This justification for the work is wrong.
Both Kawaoka and Fouchier have suggested that identifying mutations that improve aerosol transmission of avian influenza viruses in ferrets might help to detect strains with transmission potential, and help vaccine manufacture. I think it was an error to focus on these potential benefits because it detracted from the real value of the work, to provide mechanistic information on what allows aerosol transmission of influenza viruses among mammals.
In the Kawaoka and Fouchier studies, it was found that adaptation of H5N1 influenza virus from avian to mammalian receptors lead to a decrease in the stability of the viral HA glycoprotein. This property had to be reversed in order for these viruses to transmit by aerosol among ferrets. Similar stabilization of the HA protein was observed when the reconstructed 1918-like avian influenza virus was adapted to aerosol transmission among ferrets. It is not simply coincidence when three independent studies come up with the same outcome: clearly HA stability is important for aerosol transmission among mammals. This is one property to look for in circulating H5N1 strains, not simply amino acid changes.
Lipsitch mentions nothing about the mechanism of transmission; he focuses on identifying mutations for surveillance and vaccine development. He ignores the fundamental importance of this work. In this context, the work has tremendous value.
The remainder of the Times OpEd reminds us how often accidents occur in high security biological labortories. There are problems with these arguments. Lipsitch cites the emergence of an H1N1 influenza virus in 1977 as ‘escaped from a lab in China or the Soviet Union’. While is seems clear that the 1977 H1N1 virus probably came from a laboratory, there is zero evidence that it was a laboratory accident. It is equally likely that the virus was part of a clinical trial in which it was deliberately administered to humans.
Lipsitch also cites the numerous incidents that occur in American laboratories involving select agents. I suggest the reader listen to Ron Fouchier explain on TWiV #291 how a computer crash must be recorded as an incident in high biosecurity laboratories, but does not lead to the release of infectious agents.
Lipsitch clearly feels that the benefits of aerosol transmission research do not justify the risks involved. I agree that the experiments do have some risk, but it is not as clear cut as Lipsitch would suggest. Although ferrets are a good model for influenza virus pathogenesis, like any animal model, they are not predictive of what occurs in humans. An influenza virus that transmits by aerosol among ferrets cannot be assumed to transmit in the same way among humans. This is the assumption made by Lipsitch, and it is wrong.
I agree that transmission work on avian H5N1 influenza virus must be done under the proper containment. Before these experiments can be done they are subject to extensive review of the proposed containment and mitigation procedures. There is no justification for the additional regulation proposed by Lipsitch.
In my opinion aerosol transmission experiments on avian influenza viruses are well worth the risk. We know nothing about what controls aerosol transmission of viruses. The way to obtain this information is to take a virus that does not transmit by aerosol, derive a transmissible version, and determine why the virus has this new property. To conclude that such experiments are not worth the risk not only ignores the importance of understanding transmission, but also fails to acknowledge the unpredictable nature of science. Often the best experimental results are those which were never anticipated.
Lipsitch ends by saying that ‘There are dozens of safe research strategies to understand, prevent and treat pandemic flu. Only one strategy — creating virulent, contagious strains — risks inciting such a pandemic.’ Creating a virulent strain is not part of the strategy. Lipsitch conveniently ignores the fact that Fouchier’s H5N1 strain that transmits by aerosol among ferrets is not virulent when transmitted by that route. And of course we do not know if these strains would be transmissible in humans.
I am very disappointed that the Times chose to publish this OpEd without checking Lipsitch’s statements. He is certainly entitled to his own opinion, but he is not entitled to his own facts.
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