TWiV 298: MV-NIS de myelo

17 August 2014

On episode #298 of the science show This Week in Virology, the TWiV gang answers follow-up questions about the Ebola virus outbreak in West Africa, then discuss treatment of  disseminated multiple myeloma with oncolytic measles virus.

You can find TWiV #298 at www.twiv.tv.

TWiV 300This Week in Virology, the podcast about viruses – the kind that may or may not make you sick, celebrates its 300th episode on Tuesday, August 26, 2014 with a live recording at the Washington, DC headquarters of the American Society for Microbiology. This special episode will be part of the ‘Microbes after Hours’ series, and will feature the TWiV hosts Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler recording together in person for the first time.

TWiV 300 will be live-streamed, but if you live in the Washington, DC area, you are welcome to join us and watch the episode in person. We have a limited number of seats available on a first come, first serve basis. Click the RSVP link below to register.

Date: Tuesday, August 26, 2014

Reception from 6-7 PM at ASM Headquarters, 1752 N Street, N.W. Washington, D.C. 20036-2904

TWiV 300th Episode live from 7-8 PM RSVP required to attend.

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On episode #297 of the science show This Week in Virology, the TWiVites present an all-ebolavirus episode, tackling virology, epidemiology, and approaches to prevention and cure that are in the pipeline.

You can find TWiV #297 at www.twiv.tv.

guinea-liberia-sierra-leone-2014As the epidemic of Zaire ebolavirus in Western Africa continues (1,779 cases and 961 deaths in four countries), many are questioning why there are no means of preventing or stopping infection. In the past two decades there has been substantial research into developing and testing active and passive vaccines and antiviral drugs, although none have yet been licensed for use in humans.

Using antibodies to treat infection with ebolaviruses with antibodies is probably the best known therapy, because it was used to treat a two Americans who were infected while working in Liberia. They received a mixture of three monoclonal antibodies (called ZMapp) which had been previously shown to block infection of cells with ebolaviruses, and prevent lethal infection of non-human primates when given within 24-48 hours after infection. These are mouse monoclonal antibodies that have been ‘humanized’ so that when given to people they do not induce an antibody response against the antibodies. Humanization involves changing the amino acids of the antibody molecule from mouse to human, except in the part of the antibody that binds antigen. The antibodies are then synthesized in tobacco plants and purified. Administering anti-viral antibodies to patients, also called passive immunization, was done long before vaccines were available. Serum from patients who had recovered from a particular disease would be given to others who had recently been infected, in order to prevent disease. Such therapy was used to save the life of virologist Jordi Casals, who had become infected with Lassa virus while isolating the virus from the blood of a patient, Penny Pinneo. The serum administered to Casals was obtained from Pinneo, who had recovered from the infection. The American doctor infected with Zaire ebolavirus while working in Liberia was also given serum from a boy who had recovered from infection.

As ZMapp has not yet been subjected to human clinical trials to determine its safety and efficacy, its use in an infected human is considered unusual. A phase I clinical trial needs to be done to ensure that the preparation of monoclonal antibodies is safe in humans. Determining whether monoclonal antibody therapy for ebolavirus infection is effective is more difficult. Such testing could only be done during an outbreak, during which it would not be ethical to withhold treatment from the control group. Nevertheless it is clear that such mixtures of monoclonal anti-viral antibodies could potentially save many lives during outbreaks.

While passive immunization has value in saving lives, its protection is temporary: the antibodies given to patients do not endure. A better approach is immunization, which not only induces anti-viral antibodies, but creates immune memory, so that subsequent infections are accompanied by another round of antibody production. The catch is that it takes about two weeks after immunization for antibodies to reach sufficient protective levels. Nevertheless, a vaccine would likely have had substantial impact on the current outbreak, which began in March 2014 and has continued for 5 months.

A number of experimental vaccines against ebolaviruses are in development. In one approach, the glycoprotein of vesicular stomatitis virus is replaced with the corresponding protein of different ebolaviruses. These vaccines protect non-human primates from lethal infection. A similar approach using an attenuated rabies virus to deliver the ebolavirus glycoprotein also protected non-human primates from infection, as did immunization with an adenovirus encoding the ebolavirus glycoprotein.  This vaccine candidate has been shown to be safe and immunogenic in phase I clinical trials. Another vaccine approach entails production of the ebolavirus glycoprotein in E. coli. Immunization of mice with the purified protein leads to the production of neutralizing antibodies. Because protein-based vaccines do not replicate, the immune response may need to be boosted by using an adjuvant that stimulates the innate immune system and leads to better antibody production. A double-stranded RNA adjuvant has been shown to augment the immune response against a non-infections, virus-like particle vaccine containing the Ebola virus glycoprotein but not the viral genome.

Antivirals certainly have a place in control of viral disease, and a number of promising candidates to control infection with ebolaviruses have been developed. One is a nucleoside analog which is incorporated into RNA by the viral RNA polymerase and leads to chain termination. It blocks replication of ebolaviruses in culture cells, and protects mice and nonhuman primates from lethal infection. This compound, called BCX4430, is a broad spectrum antiviral that inhibits the replication of not only members of the Filoviridae, but also Arenaviridae, Bunyaviridae, Orthomyxoviridae, Picornaviridae, Paramyxoviridae, Flaviviridae, Coronaviridae. Another inhibitor of viral RNA synthesis is favipiravir, which has the advantage of being in late stage clinical development for the treatment of influenza. This compound inhibits replication of ebolaviruses in cultured cells and reduces disease severity and mortality in a mouse model of disease.

It is likely that the extent of the current outbreak of Ebola virus disease, the largest to date, will provide impetus to move some of these treatments into human trials. But consider that all the research on active and passive vaccines and antivirals for ebolaviruses required work in BSL-4 laboratories. Those who call for the shuttering of BSl-4 laboratories need to take note and move away from their unrealistic and unreasonable position.

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Dr. Tom Solomon is Director of the Institute for Infection and Global Health at the University of Liverpool. Here he speaks with Vincent Racaniello about the 2014 outbreak of Zaire ebolavirus in West Africa. Dr. Solomon discusses why the epidemic has spread, how it might be curtailed, the return of two infected health workers back to the United States for treatment, and the possibility that he might be traveling to the affected region to assist with medical care.

 
 

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On episode #296 of the science show This Week in Virology, Vincent visits the Australian Animal Health Laboratory in Geelong, Australia and speaks with Linfa about his work on bats and bat viruses.

You can find TWiV #296 at www.twiv.tv.

After recording this episode, Vincent and Linfa drove to a nearby golf course where they watched a colony of bats awaken and fly into the night. Below is a video of that experience.

Scientists for Science

28 July 2014

Scientists for Science are confident that biomedical research on potentially dangerous pathogens can be performed safely and is essential for a comprehensive understanding of microbial disease pathogenesis, prevention and treatment. The results of such research are often unanticipated and accrue over time; therefore, risk-benefit analyses are difficult to assess accurately.

If we expect to continue to improve our understanding of how microorganisms cause disease we cannot avoid working with potentially dangerous pathogens. In recognition of this need, significant resources have been invested globally to build and operate BSL-3 and BSL-4 facilities, and to mitigate risk in a variety of ways, involving regulatory requirements, facility engineering and training. Ensuring that these facilities operate safely and are staffed effectively so that risk is minimized is our most important line of defense, as opposed to limiting the types of experiments that are done.

In contrast to recombinant DNA research at the time of Asilomar in 1975, studies on dangerous pathogens are already subject to extensive regulations. In addition to regulations associated with Select Agent research, experimental plans on other pathogens are peer reviewed by scientists and funding agencies, and the associated risk assessments are considered by biosafety experts and safety committees. Risk mitigation plans are proposed and then considered and either approved or improved by safety committees.

If there is going to be further discussion about these issues, we must have input from outside experts with the background and skills to conduct actual risk assessments based on specific experiments and existing laboratories. Such conversations are best facilitated under the auspices of a neutral party, such as the International Union of Microbiological Societies or the American Society for Microbiology, or national academies, such as the National Academy of Sciences, USA. We suggest they should organize a meeting to discuss these issues.

Scientists for Science have a range of opinions on how risk is best assessed. However, maintaining dogmatic positions serves no good purpose; only by engaging in open constructive debate can we learn from one another’s experience. Most importantly, we are united as experts committed to ensuring public health is not compromised and the reputation of science in general, and microbiology in particular, is defended.

Please visit the Scientists for Science website to view the supporters of this initiative.

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On episode #293 of the science show This Week in VirologyVincent 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 LaboratoriesRocky 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 resultsBig Creek Coffee 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 InstituteDoherty 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).

Vincent and LinfaAustralian 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, New York

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.

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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.