Nature Communications: Respiratory Transmission of Avian H3N8 In Ferrets

Photo Credit Wikipedia

 

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For those who aren’t completely overwhelmed by the plethora of zoonotic pathogens that have been coming out of the woodwork over the past few years, we’ve a new study today on an avian influenza virus – first isolated in New England seals back in 2011 (see New England Seal Deaths Tied to H3N8 Flu Virus)  –  that finds it is not only well adapted to mammalian hosts, that it can also be easily transmitted between ferrets via the respiratory route.

 
This H3N8 virus is of a different lineage than the Equine/Canine H3N8 virus, although they are cousins. 

 

Until it landed in seals, this H3N8 strain was an avian adapted virus.  That is, that it bound preferentially to the kind of receptor cells commonly found in the digestive and respiratory tracts of birds; alpha 2,3 receptor cells.

 

In 2010, in mBio: A Mammalian Adapted H3N8 In Seals, we saw the first hints that this virus had recently adapted to bind to alpha 2,6 receptor cells, the type found in the human upper respiratory tract.

From the accompanying mBio press release New influenza virus from seals highlights the risks of pandemic flu from animals.

 

The mBio® study analyzed the DNA of a virus associated with a die-off of 162 New England harbor seals in 2011. Autopsies of five of the seals revealed they apparently died from infection with a type of influenza called H3N8, which is closely related to a flu strain that has been circulating in North American birds since 2002. Unlike the strain in birds, this virus has adaptations to living in mammals and has mutations that are known to make flu viruses more transmissible and cause more severe disease. The virus also has the ability to target a receptor called SAα-2,6, a protein found in the human respiratory tract.

Moscana says the study raises two concerns about flu. First, this strain is a novel virus that infects mammals and may well pass from animal to animal, a combination of traits that make it a potential threat to humans. Also, the possibility that a bird flu virus would infect seals hadn't been widely considered before, highlighting the fact that pandemic influenza can crop up in unexpected ways. She emphasizes the need for readiness.

 

Fast forward a couple of years, and today we have a study conducted by the USGS and St. Jude Children’s Research Hospital  appearing  in Nature Communications, that looks at the transmissibility of this emerging virus. 

 

In their words, they found  `the virus has an increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells.’

 

A triple threat, in any league. And if that weren’t enough, testing of human sera for neutralizing antibodies finds little evidence of population wide immunity to this strain. 

 

All of this becomes even more concerning when you consider the history of H3N8 in humans.

 

The H3N8 virus is thought to have sparked the 1900 influenza pandemic. Since then it has continued to circulate in birds and pigs, and is considered a prime candidate to mount a return engagement someday again (see Are Influenza Pandemic Viruses Members Of An Exclusive Club?).

 

First the Abstract (the full article is behind a pay wall), then a press release with additional details from the USGS.

 

Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal

Erik A. Karlsson, Hon S. Ip, Jeffrey S. Hall, Sun Woo Yoon, Jordan Johnson, Melinda A. Beck, Richard J.  Webby & Stacey Schultz-Cherry Article number:

doi:10.1038/ncomms5791

Published 03 September 2014

The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells.

Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

 

From the USGS we have the following press release.

 

 

Avian Flu in Seals Could Infect People

Released: 9/3/2014 5:00:00 AM

Contact Information:
U.S. Department of the Interior, U.S. Geological Survey
Office of Communications and Publishing
12201 Sunrise Valley Dr, MS 119
Reston, VA 20192
 

The avian flu virus that caused widespread harbor seal deaths in 2011 can easily spread to and infect other mammals and potentially humans.

A new study by the U.S. Geological Survey and St. Jude Children’s Research Hospital shows that the avian influenza H3N8 strain that infected New England harbor seals could be transmitted to other mammals through the air without physical contact. Transmission by respiratory droplets through coughing, for example, is the main way influenza viruses spread among people. The study also showed that current seasonal flu vaccines do not protect against this seal virus, meaning a new vaccine would be necessary if there ever was an outbreak in humans.

"The ability to transmit through the air is an important step in the path toward any influenza virus becoming pandemic," said USGS scientist Hon Ip. "The lack of protection against the seal virus from the annual seasonal vaccine highlights the risks posed by this H3N8 group of viruses."

The article, led by St. Jude in collaboration with the USGS and the University of North Carolina at Chapel Hill, was published today in the journal Nature Communications and is available online.

The scientists tested a sample of the influenza virus taken from an infected harbor seal in New Hampshire in 2011, and found that the virus was closely related to influenza viruses from wild birds. However, the H3N8 virus isolated from the seal contained mutations that allowed it to reproduce efficiently in human lung cells, cause disease in mice and infect ferrets through the air.

"Findings from this study highlight the need for continued surveillance and study of avian influenza genetics, particularly in areas like coastal regions where wild birds, wild mammals and human populations come into contact with each other,” said USGS scientist Jeff Hall.

H3N8 viruses, common in wild birds, have been associated with ongoing outbreaks in dogs and horses and have also been detected in pigs, donkeys and now seals. Beginning in September 2011, more than 160 young harbor seals were found dead or dying along the New England coast as a result of this infection. In previous H3N8 mortality events, up to 20 percent of the local seal population died.

For more information on zoonotic diseases, or diseases that spread between animals and humans, please visit the USGS National Wildlife Health Center website.

 

Up until a few years ago most researchers would have said that birds and pigs are the most likely source of the next global health threat. Today, we look at bats, horses, dogs, camels and yes – even seals - as possibly sparking the next pandemic.

 

For more on the different strains of H3N8 (including canine and equine versions), and influenza in seals,  you may wish to revisit:

 

EID Journal: Equine H3N8 In Mongolian Bactrian Camel

Study: Dogs As Potential `Mixing Vessels’ For Influenza

The 2009 H1N1 Virus Expands Its Host Range (Again)

EID Journal: Equine H3N8 In Mongolian Bactrian Camel

 

Bactrian Camel – Credit Wikipedia

 

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Mongolia is home to recurring epizootic outbreaks of Equine H3N8 – an avian influenza that jumped to horses about a half century ago, and to dogs a decade ago -  among their horse populations (see Isolation and characterization of H3N8 equine influenza A virus associated with the 2011 epizootic in Mongolia). 

 

These outbreaks, which occur roughly once a decade, not only impact the lives and economy of nomadic Mongolians, it has the potential of exposing other mammals to the virus.

 

While not as plentiful as horses, the Bactrian (two humped) camel is an important domesticated beast of burden for Mongolians, and as such is frequently exposed to both humans and horses.  It is also on the critically endangered list, and may even be extinct in the wild.

 

In the fall of 1979 a severe epizootic influenza broke out among Mongolian camels, which turned out to be a reassortant of the recently re-emerged (1977) H1N1 (aka `Russian flu’) virus, showing that this species of camels was susceptible to at least some strains of influenza.

 

Virology. 1993 Dec;197(2):558-63.

A reassortant H1N1 influenza A virus caused fatal epizootics among camels in Mongolia.

Yamnikova SS¹, Mandler J, Bekh-Ochir ZH, Dachtzeren P, Ludwig S, Lvov DK, Scholtissek C. 

Abstract

In the autumn of 1979 a severe influenza epizootic started among camels in Mongolia (Lvov et al., 1982; Viprosi Virusol. 27, 401-405.) Between 1980 and 1983 13 independent isolates of H1N1 viruses were obtained from diseased camels, which were virtually indistinguishable from the human A/USSR/90/77 strain by serological means. Two hundred and seventy-one samples of camel sera collected between 1978 and 1983 contained antibodies against the human A/USSR/90/77 isolate. After experimental infection of camels with some of these isolates, the animals developed similar symptoms as those found during natural infection: coughing, bronchitis, fever, discharge from nose and eyes. A genetic sequence analysis revealed that among the eight segments (genes) the PB1, HA, and NA genes were almost identical with allelic genes of the USSR/77 strain, and the PB2, PA, NP, M, and NS genes were almost identical with those of the A/PR/8/34 strain.

 

Today the EID Journal carries a report on the detection of the Equine H3N8 influenza virus in a Mongolian Bactrian camel, likely acquired through exposure to infected horses.  First the link and some excerpts from the study, after which I’ll have a bit more:

 

Volume 20, Number 12—December 2014

Dispatch

Equine Influenza A(H3N8) Virus Isolated from Bactrian Camel, Mongolia

Myagmarsukh Yondon, Batsukh Zayat, Martha I. Nelson, Gary L. Heil, Benjamin D. Anderson, Xudong Lin, Rebecca A. Halpin, Pamela P. McKenzie, Sarah K. White, David E. Wentworth, and Gregory C. Gray

ABSTRACT

Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission.

CONCLUSIONS

The phylogeny indicates that A/camel/Mongolia/335/2012 probably represents a relatively recent horse-to-camel transmission event. Without additional isolates from camels or corresponding epidemiologic data, and given the close genetic relationship between A/camel/Mongolia/335/2012 and related equine viruses, it is impossible to determine at this time whether the virus has been successfully transmitted from camel to camel.

In recent years, enhanced surveillance has detected influenza A viruses across a wider range of mammalian hosts, including horses, swine, dogs (14), seals (15), cats, and now camels, providing a more complete picture of the ecology of influenza A viruses beyond their presence in birds. How influenza A viruses successfully jump from 1 host species to another, and what the constraints on interspecies transmission are, remain key questions about influenza virus ecology and assessments of pandemic threats. Our findings highlight the need to further elucidate the ecology of influenza viruses and other pathogens in free-ranging camel populations.

 

As noted before, anytime an influenza virus jumps to a new host, we tend to take notice.  Influenza viruses as a rule are promiscuous, but until 2003 when it jumped to dogs, the H3N8 virus had only impacted birds and horses.

 

Last week, in Study: Dogs As Potential `Mixing Vessels’ For Influenza, we looked at a study that examined the susceptibility of canine tracheal cells to infection by canine, equine, and human influenza strains – including equine H3N8.

 

Any species that is susceptible to multiple, and diverse, flu strains has at least the potential to serve as a `mixing’ vessel for viral reassortment. Dogs, given their massive numbers and close contact with humans, are a far greater concern in this regard than Mongolian camels.

 

While the discovery of a single equine H3N8 virus among Bactrian camels may seem like an obscure, perhaps even insignificant bit of research, it reveals another piece of the influenza puzzle.

 

Learning how influenza viruses jump and adapt to new species – even among relatively small population of animals in a remote areas of the world – may very well lead to a better understanding of how the next pandemic (or epizootic) virus will emerge.

Study: Dogs As Potential `Mixing Vessels’ For Influenza

 

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Although influenza A viruses are believed to have originated in avian species (primarily waterfowl), over time they’ve managed to infect, and adapt to, a wide variety of mammals.  Humans, obviously, and swine . . .  but we’ve also seen `human’  flu viruses documented in  turkeys, skunks, ferrets, cats, elephant seals and dogs.

 

In That Touch Of Mink Flu I wrote about 11 farms in Holstebro, Denmark that were reported to be infected with a variant of the human H3N2 virus.

 

Less commonly reported - camels, whales and seals have all been shown to be susceptible to influenza viruses (cite Evolution and ecology of influenza A viruses R.G. Webster et al.)  In  2012, an avian flu strain was identified in New England seals (see mBio: A Mammalian Adapted H3N8 In Seals).

 

While influenza viruses are generally categorized as being avian, swine, human, or equine – their host range is far greater than that. 

 

And about a decade ago, we saw that host range formally expand to canines as well.

 

In 2004, the H3N8 equine influenza – a strain that has been around nearly a half century – suddenly jumped, and adapted to dogs, creating a new dog-specific (canine) lineage of H3N8.  Since then, H3N8 has continued to spread  among dogs, although it has never been known to infect humans.

But, as noted above, on rare occasions humanized influenza viruses have been shown to infect dogs as well, along with avian flu strains like H5N1 and H5N8 (see Korea Finds More Dogs With H5N8 Antibodies). 

 

This ability to be infected by more than one type of influenza virus opens the door to the possibility that dogs could serve as a `mixing vessel’ for influenza viruses, through a process called Reassortment. 

 

Reassortment, or `Shift’ occurs when a single host is simultaneously infected with two different influenza viruses.   When that happens, it is possible for viruses to swap parts of their genetic sequence, and create a new, hybrid virus.

 

While most of the time, this leads to an evolutionary failure, every once in awhile this process produces a viable, even highly competitive, new flu strain. 

 

It was a series of reassortments in swine, over a period of years, that led to the creation of the 2009 H1N1 pandemic virus, and the recent outbreak of H7N9 in China, and the H5N8 outbreaks in Korea, are apparently due to viral reassortment in avian species.

 

We normally think of birds, or swine, as prime `mixing vessels’ for influenza, but the truth is, any host that can support dual infection has the potential to produce a `hybrid’ flu virus. 

 

In the past we’ve looked at a number of species with at least theoretical potential to act as mixing vessels.

 

Mixing Vessels For Influenza

A Host Of Reservoirs

Virology Journal: Receptor Cells In Minor Poultry Species

The (Swine) Influenza Reassortment Puzzle

 

All of which serves as prelude to a new study, which appears ahead of print in the Journal of Virology, that looks at the ability of different influenza strains to infect, and replicate in, canine tracheal tissues.  

 

First a link to the study (PDF), a link to the abstract, followed by excerpts from a press release from the American Society for Microbiology, and then some closing comments.

 

Infection and pathogenesis of canine, equine and human influenza viruses in canine tracheas.

Gaelle Gonzaleza, John F. Marshallb, Joanna Morrella, David Robbc, John W. McCauleyd, Daniel R. Pereze, Colin R. Parrishf and Pablo R. Murciaa#

ABSTRACT

Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism and virulence. Canine influenza virus (CIV, H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza emergence.

 

Here we characterize the replication of canine, equine and human IAVs in the trachea of the dog, a species to which humans are heavily exposed.

 

We defined a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionary distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that those infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity.

(Continue . . . )

This from the American Society for Microbiology.

Evolution of Equine Influenza Led to Canine Offshoot Which Could Mix With Human Influenza

CONTACT:  Jim Sliwa
jsliwa@asmusa.org

WASHINGTON, DC – June 19, 2014 – Equine influenza viruses from the early 2000s can easily infect the respiratory tracts of dogs, while those from the 1960s are only barely able to, according to research published ahead of print in the Journal of Virology. The research also suggests that canine and human influenza viruses can mix, and generate new influenza viruses.

Canine influenza is a relatively new disease.  The first appearance is believed to be in 2003, as a result of direct transfer of a single equine influenza virus to dogs in a large greyhound training facility and was subsequently carried to many states by the infected greyhound, say the researchers.  Similar transfers have occurred among foxhounds in the UK, and in dogs kept near infected horses during a 2007 outbreak in Australia, they report.  

In the study, investigators from the United States and the United Kingdom infected dog tracheal explant cultures—essentially pieces of trachea cultured in the laboratory to mimic the cellular complexity and the host physiology of the host—with canine influenza virus, equine influenza virus, and human influenza viruses. (The use of explants has increased in recent years, and they have been shown to be useful for studies of viral pathogenesis.) They then compared the growth of the viruses, and the damage they wrought.

(Continue . . .)

 

Worthy of note, in Korea a different canine influenza virus emerged in 2007.  Unlike the canine H3N8 virus seen in the United States, this canine H3N2 appears to have jumped directly from an avian source.  In 2008 the CDC’s EID Journal carried the following report on this newly emerging canine flu: 

 

 

Transmission of Avian Influenza Virus (H3N2) to Dogs

 

In 2011, we saw the plot thicken yet again, when it was announced that this canine H3N2 had jumped to cats (see Korea: Interspecies Transmission of Canine H3N2).  In their abstract, the authors wrote:

 

Our study for the first time shows that cats are susceptible to canine influenza H3N2 infection, suggesting that cats may play an intermediate host role in transmitting the H3N2 virus among feline and canine species, which could lead to the endemic establishment of the virus in companion animals.

Such a scenario raises a public health concern, as the possibility of the emergence of new recombinant feline or canine influenza viruses in companion animals with the potential to act as zoonotic infection cannot be excluded.

 

While pigs and birds may be far better `mixing vessels’ for influenza than dogs or cats will ever be, companion animals are of particular concern because of how closely humans interact with them.  

 

All of this just shows how remarkable influenza viruses are at adapting to new hosts, and re-inventing themselves. 

 

And why we ignore their progress in the wild at our own peril.

mBio: A Mammalian Adapted H3N8 In Seals

Photo Credit Wikipedia

 

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Even though the actual study has not appeared online (now online) in the journal mBio, overnight we’ve seen more than a half dozen stories on soon-to-be published research into a novel influenza virus that infected and killed more than 160 seals along the New England coast last year.

 

You’ll find an excellent report from Carl Zimmer in the New York Times (see Flu That Leapt From Birds to Seals Is Studied for Human Threat), as well as this reportage from the BBC (see New flu virus found in seals concerns scientists)

 

Regular readers may recall this story from last fall (see New England Seal Deaths Tied to H3N8 Flu Virus & NOAA: New England Dead Seals Test Positive For Flu), but in brief:

 

In early October of 2011 media reports indicated that scores of dead seals had been discovered along the shoreline of New England, predominantly from the North Shore of Massachusetts to the southern coast of Maine.

 

In November NOAA declared these deaths an “Unusual Mortality Event”, cautioned the public to avoid contact with seals, and directed more resources to study the event.

 

Although the cause of these deaths was still undetermined, some researchers saw similarities to a seal die off that had occurred 30 years earlier.

 

In that instance, the culprit turned out to be an H7N7 influenza. (see Isolation of an influenza A virus from seals G. Lang, A. Gagnon and J. R. Geraci)

 

In December it was announced that the pathogen behind this latest seal die off was a variant of the H3N8 avian flu strain, versions of which are known to also infect horses and dogs.

 

Science team identifies influenza virus subtype that infected five dead seals

Risk to humans and pets low; tests continue

 

Harbor Seals (Credit NOAA)

A virus similar to one found in birds but never before in harbor seals was the cause of five of 162 recent deaths of the animals  in New England, according to a group of federal agencies and private partners.

 

This Influenza A virus subtype, H3N8, appears to have a low risk of transmission to humans. Experts continue to analyze this virus, and any findings of public health significance will be immediately released. The virus is not the infamous H5N1  virus that caused a global pandemic in 2007, or the H1N1 virus from 2009.

(Continue . . . )

 

While we tend to think of humans, pigs, and birds as the main hosts of influenza (and to a lesser extent dogs and horses), a number of other species are susceptible to some kinds of influenza strains as well.

 

• In That Touch Of Mink Flu I wrote about 11 farms in Holstebro, Denmark that were reported to be infected with a variant of the human H3N2 virus back in 2009.
• The AVMA Pandemic Flu page documents species – including dogs, cats, turkeys, skunks, ferrets – that tested positive for the 2009 H1N1 pandemic virus.
• While less commonly reported - camels, whales and seals have all been shown to be susceptible to influenza (cite Evolution and ecology of influenza A viruses R.G. Webster et al.)

 

 

Fast forward eight months and today research is being published to show that the H3N8 virus collected from dead seals last fall has mutated from its avian origins to become better adapted to mammalian hosts. 

 

First stop, excerpts from a press release from the American Society for Microbiology, publishers of mBio.

 

 

New influenza virus from seals highlights the risks of pandemic flu from animals

A new strain of influenza virus found in harbor seals could represent a threat to wildlife and human health, according to the authors of a study appearing July 31 in mBio®, the online open-access journal of the American Society for Microbiology. It is crucial to monitor viruses like this one, which originated in birds and adapted to infect mammals, the authors say, so that scientists can better predict the emergence of new strains of influenza and prevent pandemics in the future.

 

"There is a concern that we have a new mammalian-transmissible virus to which humans haven't been exposed yet. It's a combination we haven't seen in disease before," says Anne Moscona of Weill Cornell Medical College in New York City, the editor of the report.

 

<SNIP>

 

The mBio® study analyzed the DNA of a virus associated with a die-off of 162 New England harbor seals in 2011. Autopsies of five of the seals revealed they apparently died from infection with a type of influenza called H3N8, which is closely related to a flu strain that has been circulating in North American birds since 2002. Unlike the strain in birds, this virus has adaptations to living in mammals and has mutations that are known to make flu viruses more transmissible and cause more severe disease. The virus also has the ability to target a receptor called SAα-2,6, a protein found in the human respiratory tract.

(Continue . . . )

Another press release, from Columbia University's Mailman School of Public Health, titled An avian flu that jumps from birds to mammals is killing New England's baby seals cautions:

 

Based on full genome sequencing and phylogenetic analysis, seal H3N8 descended from an avian strain that has been circulating in North American waterfowl since 2002, which implies recent transmission from wild birds to seals.

 

Accordingly, seal H3N8 has acquired the ability to bind sialic acid receptors that are commonly found in the mammalian respiratory tract. Mutations in the HA and PB2 genes – required for cell entry and replication, respectively – suggest enhanced virulence and transmission in mammals, but these putative attributes require further investigation. Given these findings along with the long history of the spread of avian influenza to humans—most notably H1N1 and H5N1—seal H3N8 could pose a threat to public health.

 

 

Regular readers of this blog are aware that avian influenza strains bind preferentially to the kind of receptor cells commonly found in the digestive and respiratory tracts of birds; alpha 2,3 receptor cells.

 

Human (and mammalian adapted) influenzas – on the other hand - bind to the kind of receptor cells that line the surfaces of the human upper respiratory system; alpha 2,6 receptor cells.

 

 

In both the BBC and NYTs report, one of the authors of this study – celebrated virus hunter Professor Ian Lipkin of Columbia University – expressed concerns over the discovery that seals – like pigs – have both types of receptor cells.

 

If infected by two different strains simultaneously, they could act as a `mixing vessel’ for influenza strains, and produce a hybrid (reassorted) virus. 

 

 

Every day, it seems, we learn more about how remarkably well these influenza viruses evolve and adapt. Despite decades of work by thousands of researchers, there is still so very much we are just beginning to understand.

 

Although the public health threat from H3N8 is hard to quantify, this is yet another example of how Nature’s laboratory is open and operating 24/7, and why we need to remain alert and prepared for the next pandemic threat that may emerge.

 

When the mBio study is published, I’ll post the link here.

 

NOW ONLINE:

 

Emergence of Fatal Avian Influenza in New England Harbor Seals

S. J. Anthony, J. A. St. Leger, K. Pugliares, H. S. Ip, J. M. Chan, Z. W. Carpenter, I. Navarrete-Macias, M. Sanchez-Leon, J. T. Saliki, J. Pedersen, W. Karesh, P. Daszak, R. Rabadan, T. Rowles and W. I. Lipkin

doi:10.1128/mBio.00166-12