A Live-Attenuated Herpes Simplex Virus Vaccine Candidate

herpesvirusBy Gertrud U. Rey

There is currently no vaccine to prevent infection with herpes simplex virus type 1 or type 2 (HSV-1 or HSV-2). Infection with either of these viruses results in life-long viral latency. Sporadic reactivation and viral shedding may lead to painful oral and genital disease and an increased risk of HIV transmission.

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TWiV 233: We’re surrounded

On episode #233 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review aerosol transmission studies of influenza H1N1 x H5N1 reassortants, H7N9 infections in China, and the MERS coronavirus.

You can find TWiV #233 at www.microbe.tv/twiv.

Further defense of the Chinese H1N1 – H5N1 study

Robert Herriman of The Global Dispatch interviewed me this week on the H1N1 – H5N1 reassortant study that has been in the headlines:

There was much written concerning the research published earlier this month in Science, where researchers from China’s Harbin Veterinary Research Institute reported creating an  avian H5N1 (highly pathogenic) and pandemic 2009 H1N1 (easily transmissible) hybrid, that according to them, achieved airborne spread between guinea pigs.

Read the rest of the article at The Global Dispatch.

Influenza H5N1 x H1N1 reassortants: ignore the headlines, it’s good science

Those of you with an interest in virology, or perhaps simply sensationalism, have probably seen the recent headlines proclaiming another laboratory-made killer influenza virus. From The Independent: ‘Appalling irresponsibility: Senior scientists attack Chinese researchers for creating new strains of influenza virus’; and from InSing.com: ‘Made-in-China killer flu virus’. It’s unfortunate that the comments of several scientists have tainted what is a very well done set of experiments. Let’s deconstruct the situation with an analysis of the science that was done.

It is known that avian influenza H5N1 viruses can occasionally infect but not transmit among humans, while the 2009 pandemic H1N1 virus (which continues to circulate) readily transmits from person to person. The investigators asked whether reassortants of the two viruses – which could arise in nature – might confer transmissibility to H5N1 virus. To answer this question they produced 127 different reassortants of the two viruses, and tested their ability to transmit by aerosol among guinea pigs. The latter have been used for transmission studies on influenza, notably to understand the seasonality of infection. Ferrets have been more famously used for influenza virus transmission studies.

Rather than describe the results, I’ve made an illustration that shows what I believe to be the most important conclusions of the study (click for a larger version):

h1n1 h5n1 reassortants

The H5N1 virus (red RNAs) is not transmissible among guinea pigs, while the H1N1 virus (green RNAs) has highly efficient transmission. Exchange of the H5N1 RNA coding for PA or NS from H1N1 produces a highly transmissible virus. Exchange of the H5N1 RNA coding for NA or M produces a less efficiently transmitted virus. These are interesting and novel findings. It will be of great interest to determine how the PA, NS, NA, or M genes mechanistically enhance aerosol transmission. This is important information because our understanding of the determinants of transmission is very poor.

All the reassortant viruses shown in the figure have the H5 HA; when only the H1 of the H1N1 virus was substituted with the H5 HA, the reassortant virus transmitted efficiently among guinea pigs. In ferrets the H5 HA is not compatible with aerosol transmission. Therefore guinea pigs are clearly different from ferrets with respect to the determinants of transmissibility.

I cannot understand why some scientists have called these experiments ‘appallingly irresponsible’ and of no scientific use. I can only assume that they are not familiar with the literature on viral transmission and do not appreciate how the results advance our understanding of the field. It also seems irresponsible to predict that these viruses, should they escape from the laboratory, could kill millions of people. If you accept guinea pigs as a predictor of human pathogenicity – which I do not – then there is no reason for fear because none of the reassortants were lethal. I do not believe that any animal model predicts what will occur in humans, and so I am even less concerned about the safety of these experiments. I firmly believe that laboratory-constructed viruses do not have what it takes to be a human pathogen: only viral evolution in nature can produce the right combination of RNA segments and mutations. I also believe that scientists are quite responsible when it comes to safe handling of pathogens. If we worry about every type of transmission experiment involving influenza H5N1 virus, we will never make progress in understanding why this virus does not transmit among humans. The moratorium on H5N1 transmission research is over; let’s move beyond the sensational headlines and get back to the science.

In summary, I believe that these are well designed experiments which show that single RNA exchanges with H1N1 virus can produce an H5N1 virus that transmits via aerosol among guinea pigs. The relevance of these findings to humans is not known; nevertheless understanding how the individual viral proteins identified in this study enhance transmission may be mechanistically informative. I believe that the news headlines depicting these experiments as irresponsible and dangerous are based on uninformed statements made by scientists who are not familiar with the literature on influenza virus transmission. I wonder if they even read the paper in its entirety before making their comments.

Oseltamivir resistance decreases influenza aerosol transmission

tamifluThe isolates of influenza virus obtained in the current global outbreak have proven to be resistant to the adamantane antivirals, but susceptible to oseltamivir (Tamiflu) and zanamivir (Relenza). Consequently the two neuraminidase inhibitors will likely be used extensively to control the outbreak until a vaccine is available. Extended use of the antiviral drugs will undoubtedly lead to the selection of drug resistant influenza virus mutants. However, there might be a silver lining to this otherwise dismal prediction.

Transmission of oseltamivir-resistant influenza viruses was studied in a guinea pig model for infection. Viruses with either one or two amino acid changes that lead to drug resistance multiplied normally in guinea pigs, but the viruses were transmitted very poorly to other animals by aerosols. Oseltamivir-sensitive viruses were transmitted efficiently by aerosol among animals. Interestingly, both oseltamivir-sensitive and oseltamivir-resistant viruses transmitted efficiently by the contact route among guinea pigs placed in the same cage.

These observations indicate that mutations that cause resistance to oseltamivir reduce aerosol, but not contact transmission of influenza virus. The authors speculate that the amino acid changes associated with drug resistance also decrease the enzymatic activity of the viral NA (neuraminidase) protein. One of the functions of this viral protein is to facilitate release of newly synthesized virus particles from the surface of the infected cell. Mutations that cause drug resistance may lead to impaired virus release, and therefore poor transmission by the aerosol route.

Although these results are encouraging, it is not clear if drug resistance mutations would have a similar effect on aerosol transmission of the current A/Mexico/2009 (H1N1) influenza viruses. A high proportion of the influenza H1N1 viruses from the previous season were resistant to the drug, yet the viruses spread extensively. One possible explanation for the discrepancy is that influenza in guinea pigs does not lead to coughing or sneezing. Hence, the virus-laden aerosols produced by guinea pigs are a consequence of normal breathing. In humans, air turbulence caused by coughing and sneezing might be sufficient to dislodge virus particles that in the guinea pig would remain firmly attached to the epithelial surface. Nevertheless, the results suggest that simple hygienic measures could slow the spread of a drug resistant virus that transmits by efficiently by contact and poorly by aerosol.

Bouvier, N., Lowen, A., & Palese, P. (2008). Oseltamivir-Resistant Influenza A Viruses Are Transmitted Efficiently among Guinea Pigs by Direct Contact but Not by Aerosol Journal of Virology, 82 (20), 10052-10058 DOI: 10.1128/JVI.01226-08

Influenza virus transmission

sneezeInfluenza virus may be transmitted among humans in three ways: (1) by direct contact with infected individuals; (2) by contact with contaminated objects (called fomites, such as toys, doorknobs); and (3) by inhalation of virus-laden aerosols. The contribution of each mode to overall transmission of influenza is not known. However, CDC recommendations to control influenza virus transmission in health care settings include measures that minimize spread by aerosol and fomite mechanisms.

Respiratory transmission depends upon the production of aerosols that contain virus particles. Speaking, singing, and normal breathing all produce aerosols, while coughing and sneezing lead to more forceful expulsion. While coughing may produce several hundred droplets, a good sneeze can generate up to 20,ooo. Aerosolized particles produced by these activities are of different sizes. The largest droplets fall to the ground within a few meters and will transmit an infection only to those in the immediate vicinity. Other droplets travel a distance determined by their size. Those droplets 1-4 microns in diameter are called ‘droplet nuclei’; these remain suspended in the air for very long periods and may not only travel long distances, but can reach the lower respiratory tract. Inhalation of droplets and droplet nuclei places virus in the upper respiratory tract, where it may initiate infection.

The importance of aerosol transmission is illustrated by an outbreak of influenza aboard a commercial airplane in the late 1970s. The plane, carrying 54 persons, was delayed on the ground for three hours, during which time the ventilation system was not functional. Most of the travelers remained on board. Within 72 hours, nearly 75% of the passengers developed influenza. The source of the infection was a single person on the airplane with influenza.

Nasal secretions, which contain virus particles, are responsible for transmission by direct contact or by contaminated objects. An infected person will frequently touch their nose or conjunctiva, placing virus on the hand. Intimate or non-intimate contact (e.g. shaking hands) will transfer the virus to another person, who will then infect themselves by touching their nose or eyes. When contaminated hands touch other objects, the virus is transferred to them. In one study, 23-59% of objects from homes and day care facilities were shown to harbor influenza viral RNA. Others have shown that infectious influenza virus may be persist on paper currency for several weeks.

Influenza transmission can be reduced by covering your nose and mouth when coughing or sneezing, and by washing hands often with soap and water or alcohol-based hand cleaners. Note that CDC does not recommend the use of face masks for reducing viral spread. It is important to recognize that, in human infections, maximum levels of virus shedding may occur about a day before the peak of symptoms.

Ethical considerations preclude controlled influenza virus transmission studies in humans, and therefore animal models must be used. Ferrets are susceptible to many strains of influenza virus, and develop symptoms similar to those in humans. However these animals are costly and difficult to house, precluding their use in most large scale transmission studies. The guinea pig has recently been described as an alternative animal model for studying influenza virus transmission. These animals are susceptible to different viral strains, and the virus replicates to high titers in the respiratory tract. The guinea pig was used to show that transmission of influenza virus occurs by aerosols and through contaminated environmental surfaces. The efficiency of aerosol transmission in the guinea pig model is regulated by temperature and humidity.

Fabian, P., McDevitt, J., DeHaan, W., Fung, R., Cowling, B., Chan, K., Leung, G., & Milton, D. (2008). Influenza Virus in Human Exhaled Breath: An Observational Study PLoS ONE, 3 (7) DOI: 10.1371/journal.pone.0002691

Carrat, F., Vergu, E., Ferguson, N., Lemaitre, M., Cauchemez, S., Leach, S., & Valleron, A. (2008). Time Lines of Infection and Disease in Human Influenza: A Review of Volunteer Challenge Studies American Journal of Epidemiology, 167 (7), 775-785 DOI: 10.1093/aje/kwm375

Mubareka, S., Lowen, A., Steel, J., Coates, A., García‐Sastre, A., & Palese, P. (2009). Transmission of Influenza Virus via Aerosols and Fomites in the Guinea Pig Model The Journal of Infectious Diseases, 199 (6), 858-865 DOI: 10.1086/597073

Seasonality of influenza revisited

239997306_60c718f6c7_mThe cute guinea pig returns for another installment on why influenza is prevalent during winter months in temperate climates.

We previously discussed work by Palese and colleagues in which a guinea pig model for influenza virus transmission was used to conclude that spread of influenza virus in aerosols is dependent upon temperature and relative humidity. They found that transmission of infection was most efficient when the humidity was 20-35%; it was blocked at 80% humidity. The authors concluded that conditions found during winter, low temperature and humidity, favor spread of the infection. Lower humidity favors virion stability and smaller virus-laden droplets, which have a better chance to travel longer distances.

Another group re-analyzed Palese’s data and found that relative humidity explains only a small amount of the variability in influenza virus transmission and survival – 12% and 36%, respectively. When they converted the measurements of moisture to absolute humidity, the results were striking: 50% of the variability in transmission and 90% of the variability in survival could be explained by absolute humidity.

Changes in relative humidity, the authors argue, do not match the seasonal patterns of influenza transmission. Although relative humidity indoors is low in the winter, outdoor levels peak during this season. Absolute humidity, on the other hand, has a seasonal cycle with low values both indoors and outdoors during winter months, consistent with the increased transmission of influenza. 

The conclusion of both papers is the same: humidification of indoor air during the winter might be an effective means of decreasing influenza virus spread. 

On the weather report, the amount of moisture in the air is given as a percentage. This is relative humidity – the ratio of the partial pressure of water vapor in a gaseous mixture of air and water vapor to the saturated vapor pressure of water at a given temperature. Absolute humidity is the actual amount of water vapor in a liter of gas. Any easy way to remember what measurement of humidity is important for influenza transmission is that it’s not what you hear on the weather report.

J. Shaman, M. Kohn (2009). Absolute humidity modulates influenza survival, transmission, and seasonality Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0806852106