Virology: Pathogenesis Of Avian A/H5N8 In Ferrets

 

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Despite being closely related to avian virus subtypes (H5N1, H5N6) that have produced serious morbidity and mortality in humans, the recently emerged H5N8 virus which has spread to both Europe and North America (see EID Journal: Novel Eurasian HPAI A H5 Viruses in Wild Birds – Washington, USA)  has never been linked to human infection or illness. 

Although not currently considered a serious threat to human health threat, the CDC has issued specific guidance documents for dealing with those who may have been exposed (see CDC Interim Guidance For Testing For Novel Flu & CDC Interim Guidance On Antiviral Chemoprophylaxis For Persons With Exposure To Avian Flu).

 

Despite the reassuring anecdotal data to date, it is important to understand the pathogenesis of this virus in birds and mammals, with an eye towards understanding how the threat may change over time.  

 

While humans have been so far unaffected, we have seen reports of dogs being infected with H5N8 (see MAFRA: H5N8 Antibodies Detected In South Korean Dogs (Again)).   The ability to infect canines does not automatically make it a human threat, but it does raise some concerns (see Study: Dogs As Potential `Mixing Vessels’ For Influenza).

 

Although not perfect, ferrets are considered a reasonable mammalian substitute for humans when testing respiratory viruses because their lung physiology is similar, and because they cough and sneeze when infected, very much as humans do.

 

All of which brings us to a new study (most of which, alas, is behind a pay wall) that looks at the pathogenesis of two Korean strains of H5N8 challenged ferrets.  What we can tell from the abstract, however, is that ferrets intranasally inoculated suffered no mortality or serious respiratory symptoms, but that ferrets intratracheally infected with one of the H5N8 strains showed `dose-dependent mortality’.

 

Pathogenesis of novel reassortant avian influenza virus A (H5N8) Isolates in the ferret 

Heui Man Kim^{1, 2,} , Chi-Kyeong Kim^{1, 3,} , Nam-Joo Lee, Hyuk Chu, Chun Kang, Kisoon Kim, Joo-Yeon Lee^,

doi:10.1016/j.virol.2015.02.042

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Highlights

•  Outbreaks of HAPI H5N8 occurred in 2014, and spread to poultry farms in Korea.

• We evaluated the pathogenesis of H5N8 viruses in ferrets.

• Two Korean H5N8 strains did not induce mortality in intranasal challenged ferrets.

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Abstract

Outbreaks of avian influenza virus H5N8 first occurred in 2014, and spread to poultry farms in Korea. Although there was no report of human infection by this subtype, it has the potential to threaten human public health. Therefore, we evaluated the pathogenesis of H5N8 viruses in ferrets. Two representative Korean H5N8 strains did not induce mortality and significant respiratory signs after an intranasal challenge in ferrets. However, ferrets intratracheally infected with A/broiler duck/Korea/Buan2/2014 virus showed dose-dependent mortality. Although the Korean H5N8 strains were classified as the HPAI virus, possessing multiple basic amino acids in the cleavage site of the hemagglutinin sequence, they did not produce pathogenesis in ferrets challenged intranasally, similar to the natural infection route. These results could be useful for public health by providing the pathogenic characterization of H5N8 viruses.

 

While it isn’t easy to infect a ferret, apparently if you provide a high enough dose, and place it far enough down the respiratory tract, it is possible.  Whether these same (unlikely to occur) conditions would elicit the same response in humans is unknown, but it is possible.

 
Although it is tempting to relegate H5N8 to the `only a threat to poultry’ column, the simple fact is: viruses constantly change. 

 

We’ve already seen multiple strains, new clades, and additional reassortants (H5N2, H5N3, H5N1) emerge since the H5N8 virus first appeared in early 2014, and more are undoubtedly on the horizon.  How they will behave, and the threat they pose to humans, may change as well. 

For now,  the news remains good, at least in so far as human infection is concerned. 

 

For the poultry industry, however, there is considerably less to celebrate. 

 

H5N8 and it’s reassortant H5N2 progeny appear to spread faster, and more easily by wild and migratory birds than any HPAI virus previously observed.  It is highly pathogenic in chickens, turkeys, and ducks - and if Taiwan’s outbreak is any indication (875 farms & counting) - once it enters a region’s poultry population, it is capable of spreading with alarming speed. 

All of which means that H5N8 (and its descendents) have staying power, and are likely to be with us for some time.

mBio: The Pathogenicity Of Avian H7N9 In Cynomolgus Macaques

Credit CDC

 

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The open access journal mBio today has published a study today looking at the replication, and pathogenicity of, the avian H7N9 virus in both the upper and lower respiratory tracts of cynomolgus macaques. 

 

Although ferrets and other non-primate animals are often used for influenza transmission  research, when you want to study the pathogenicity of a virus, non-human primates offer the closest analog to humans.

The   Wisconsin National Primate Research Center explains the rationale behind their use:

Why use macaques?

Macaques are genetically very similar to humans. They especially share analogous neurological, reproductive and immunological systems with humans. Rhesus and cynomolgous macaques are not endangered in the wild and adapt well to captive housing. Research with rhesus and cynomolgous monkeys, as well as with other nonhuman primates, tells us a great deal about primate biology. Animal studies can be better controlled and can garner more consistent results than human studies, and are often precursors to human studies.

 

Shortly after the outbreak of H7N9 in Eastern mainland China in the spring of 2013 we learned of some of the research studies that were quickly set in motion to better understand this emerging avian flu virus (see ScienceInsider: Laboratory Plans For H7N9 Virus).

 

At that time we knew little about the pathogenicity and transmissibility of the virus in humans, and a top stated priority was to test H7N9 on a variety of lab animals, including ferrets and cynomolgus macaques.

Since then, we’ve seen a steady procession of studies, many of which have suggested that the H7N9 virus – perhaps more than any other avian influenza virus we’ve seen to date – has worrying pandemic potential.

 

• Last year we saw studies suggested H7N9 is unusually well-adapted to mammalian physiology (see mBio: H7N9 Naturally Adapted For Efficient Growth in Human Lung Tissue)  – but thus far this virus has failed to demonstrate the ability to transmit efficiently among humans.
• Last May, in  EID Journal: H7N9 As A Work In Progress, we looked at a study that found the H7N9 avian virus continues to reassort with local H9N2 viruses, making the H7N9 viruses that circulated in wave 2 genetically distinct from those that were seen during the 1st wave.
• While last June, in Eurosurveillance: Genetic Tuning Of Avian H7N9 During Interspecies Transmission, we saw even more evidence of the genetic diversity, and continual evolution, of the H7N9 virus in Mainland China.  Researchers found that at least 26 separate genotypes had emerged, mostly during the first wave, through a process they called `genetic tuning’.

 

While this virus is apparently on summer hiatus, few expect it to remain so once temperatures begin to decline this fall and winter.  Last winter’s outbreak came after an exceptionally quiet summer, and was twice the size of the first wave.

 

All of which makes learning as much as we can about the pathogenicity, and pandemic potential, of this virus a high priority. 

 

Influenza Virus A/Anhui/1/2013 (H7N9) Replicates Efficiently in the Upper and Lower Respiratory Tracts of Cynomolgus Macaques

Emmie de Wit^a, Angela L. Rasmussen^b, Friederike Feldmann^c, Trenton Bushmaker^a, Cynthia Martellaro^a, Elaine Haddock^a, Atsushi Okumura^b, Sean C. Proll^b, Jean Chang^b, Don Gardner^c, Michael G. Katze^b,d, Vincent J. Munster^a, Heinz Feldmann^a,e

ABSTRACT

In March 2013, three fatal human cases of infection with influenza A virus (H7N9) were reported in China. Since then, human cases have been accumulating. Given the public health importance of this virus, we performed a pathogenicity study of the H7N9 virus in the cynomolgus macaque model, focusing on clinical aspects of disease, radiographic, histological, and gene expression profile changes in the upper and lower respiratory tracts, and changes in systemic cytokine and chemokine profiles during infection.

Cynomolgus macaques developed transient, mild to severe disease with radiographic evidence of pulmonary infiltration. Virus replicated in the upper as well as lower respiratory tract, with sustained replication in the upper respiratory tract until the end of the experiment at 6 days after inoculation. Virus shedding occurred mainly via the throat.

Histopathological changes in the lungs were similar to those observed in humans, albeit less severe, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, pneumocyte hyperplasia, and fibroproliferative changes. Analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage during H7N9 infection as well as leads for development of therapeutics targeting host responses rather than virus replication.

Overall, H7N9 infection was not as severe in cynomolgus macaques as in humans, supporting the possible role of underlying medical complications in disease severity as discussed for human H7N9 infection (H. N. Gao et al., N. Engl. J. Med. 368:2277–2285, 2013, doi:10.1056/NEJMoa1305584).

 

Curiously, while more pathogenic to macaques than most strains of the 2009 H1N1 pandemic virus, and seasonal H3N2, the H7N9 virus produced less severe symptoms in these non-human primates than has been common observed in humans. 

 

The authors suggest that the high rate of co-morbidities in the – mostly elderly – cohort of cases in China could explain this difference.  

 

And indeed, the chart below from Dr. Ian Mackay’s VDU Blog shows a pronounced demographic shift towards older, predominantly male, victims in China. Children, and young adults – when they were found to be infected – were more likely to have mild, moderate, or even asymptomatic infections (see Mild Influenza A/H7N9 Infection among Children in Guangdong Province).

 

Compared to infection with the 1918 H1N1 and H5N1 avian flu viruses (see my three part review of the 2009 Baskin Influenza in Primates Study), H7N9 produced far less lung damage and illness in macaques. 

 

You’ll want to read the entire study for details on methods and materials, and a closer look at the results.   In their concluding remarks, the authors summed up their work by saying:

 

Thus, the emerging H7N9 influenza virus is more pathogenic than seasonal influenza A virus and most isolates of the pandemic H1N1 virus but not as pathogenic as the 1918 Spanish influenza virus and HPAI H5N1 virus in cynomolgus macaques.

However, the pathogenicity of the H7N9 virus may decrease if the virus adapts further to solely using α2,6-linked sialic acids as the receptor for entry, as pandemic influenza viruses to date have done (52–55).

Exclusive attachment to α2,6-linked sialic acids would most likely result in a shift to replication mainly in the upper respiratory tract of humans, likely resulting in less severe disease, as has been described for the 2009 pandemic H1N1 virus (56) and upon adaptation of HPAI H5N1 virus to efficient transmission via respiratory droplets or aerosols (57).

 

The switch to an `exclusive attachment to α2,6-linked sialic acids’  is one of the evolutionary changes that is believed would make this virus far more easily transmitted between humans, and is probably required if  H7N9 were to become a viable pandemic virus.

 

And while a commensurate decrease in severity would be a welcome result, it is worth noting that a reduction of even a full order of magnitude (30% CFR down to 3%) would still put this virus in the same league as the 1918 Spanish flu.

 

While the tragedy of Ebola has captured the world’s attention this summer, the most serious pandemic threats are those posed by respiratory viruses, such as influenza, SARS, and MERS.

Science: H7N9 Transmissibility Study In Ferrets

 

Photo Credit Wikipedia

 

UPDATED: CIDRAP NEWS now has an extensive review of this paper up on their website, with expert commentary provided by Dr. Ian MacKay. 

 

Study: H7N9 highly transmissible by airborne route

  Highly recommended.

 

 

 

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While the welcomed lull in avian influenza cases in Eastern China over the past six weeks has taken H7N9 largely out of the daily headlines, history has shown that avian flu viruses often go `quiet’ during the warm summer months, only to reappear again in the fall or winter.

 

Complicating matters, we still don’t know the source of this virus, or exactly how it managed to spread across tens of thousands of square kilometers of Eastern China - and infect at least 132 people - in a matter of a few weeks.

 

So over the summer researchers have continued to examine the H7N9 virus, trying to gauge its pandemic potential should it return with colder weather this fall.

 

Last week we saw two studies in the Journal Nature  (see Nature: H7N9 Pathogenesis and Transmissibility In Ferrets & Mice & CIDRAP NEWS  article New studies on H7N9 raise pandemic concerns) that suggested the H7N9 virus might be better equipped to spark a pandemic than previously thought.

 

Researchers determined that while the virus did not appear to transmit as easily as seasonal flu via respiratory droplets between ferrets, once acquired, the virus replicated at a much higher rate than one normally sees with seasonal flu.

 

Today, we’ve a complex and fascinating new study by scientists from Harbin Veterinary Research Institute and the Gansu Agricultural University appearing in the Journal Science that gives us a detailed look at two critical issues; the pathogenicity and transmissibility of the virus (in mice & ferrets). 

 

Ferrets – whose respiratory system (and susceptibility to flu) are reasonably close to that of humans – are often used in transmissibility and pathogenicity studies of influenza. 

 

Today’s study tested several different H7N9 isolates (acquired from birds, and from humans) for transmissibility, and even though their HA and NA proteins were genetically quite similar, at least one H7N9 isolate transmitted readily via respiratory droplets among ferrets.

 

The abstract (see below), only scratches the surface of research conducted in this study.

 

H7N9 Influenza Viruses Are Transmissible in Ferrets by Respiratory Droplet

Science DOI: 10.1126/science.1240532

Qianyi Zhang, Jianzhong Shi, Guohua Deng, Jing Guo, Xianying Zeng, Xijun He, Huihui Kong, Chunyang Gu,  Xuyong Li, Jinxiong Liu, Guojun Wang, Yan Chen,  Liling Liu,  Libin Liang, Yuanyuan Li, Jun Fan, Jinliang Wang,  Wenhui Li, Lizheng Guan,  Qimeng Li, Huanliang Yang,  Pucheng Chen,  Li Jiang,Yuntao Guan, Xiaoguang Xin, Yongping Jiang, Guobin Tian, Xiurong Wang, Chuanling Qiao, Chengjun Li,  Zhigao Bu, Hualan Chen

Abstract (EXCERPT)

We systematically analyzed H7N9 viruses isolated from birds and humans. The viruses were genetically closely related and bound to human airway receptors; some also maintained the ability to bind to avian airway receptors. The viruses isolated from birds were nonpathogenic in chickens, ducks, and mice; however, the viruses isolated from humans caused up to 30% body weight loss in mice.

 

Most importantly, one virus isolated from humans was highly transmissible in ferrets by respiratory droplets. Our findings indicate nothing to reduce the concern that these viruses can transmit between humans.

 

 

While I’m sure CIDRAP NEWS will have a more detailed look later today (I’ll post a link), a few of the high points in this study include:

 

• Researchers tested more than 10,700 samples taken from poultry farms, live market birds, wild bird habitats, and even poultry and swine slaughter houses and only managed to find 52 samples that were H7N9 positive (all but 2 were found in live-markets)
• Chickens intravenously inoculated with two early strains of the virus showed no signs of illness indicating the H7N9 is largely non-pathogenic in chickens
• Isolates taken from humans appeared to be more pathogenic in ferrets and mice than those taken from birds, suggesting adaptation of the virus may occur after the virus jumps to humans.
• All of the human isolates had either the 627K or 701N amino acid changes in their PB2 (both associated with increased virulence and transmission in mammals) - while the avian isolates did not - again suggesting that adaptive mutations may have occurred during replication of the virus in the human host.
• Mice inoculated with avian H7N9 isolates showed no signs of disease signs or deaths.
• Mice inoculated with human derived H7N9 isolates experienced significant weight loss & pathogenesis.
• In ferret transmissibility studies, four of the five viruses tested could be transmitted between ferrets in direct contact with each other, and one transmitted with high efficiency via respiratory droplets.

 

Being non-pathogenic in chickens, this virus has the potential to spread stealthily, and its rapid spread across multiple provinces in China suggests it transmits efficiently among poultry species.

 

This research suggests that the H7N9 virus already binds pretty well to human-like (a2,6) receptor cells, and it replicates efficiently in a mammalian (ferret) host.

 

Additionally, the H7N9 virus appears capable of accruing adaptive mutations that enhance both its virulence and transmissibility once it starts to replicate within a human host.

 

And perhaps most worrisome of all, at least one isolate tested already demonstrates the ability to transmit readily via respiratory droplets in ferrets.

 

Complicating matters, earlier this week we saw a study (see mBio: Antiviral Resistance In H7N9) suggesting that antiviral resistance may form quickly in patients infected with the H7N9 virus.

 

All qualities that could help make the H7N9 virus a formidable foe should we see it return in the fall.

H5N1: A Rite Of Passage

 

 

 

 

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Simplified Illustration of a Serial Passage Experiment. 

 

 

We’ve a study today that appeared recently in the Journal of Animal and Veterinary Advances that looks at increases in pathogenicity (in mice) of two H5N1 viruses after six serial passages in quail.

 

While the H5N1 virus remains primarily a disease of birds, concerns persist that the virus may someday acquire the ability to effectively infection – and transmit between – humans.

One of the primary barriers (but perhaps, not the only one) to this happening is the avian virus’s preference to bind to the type of receptor cells commonly found in the digestive and respiratory tracts of birds; alpha 2,3 receptor cells.

 

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

 

Humans do have avian-like a2,3 receptor cells, but mainly deep in the lungs where it is difficult for the virus to reach.

 

There are some species that have an abundance of both types of receptor cells (most notably pigs, along with some varieties of poultry,the North American striped skunk, some small carnivores), and researchers worry that these could serve as `mixing vessels’ and give the virus an opportunity to adapt to the human receptor cells.

 

And according to today’s study, we can add quail to the list.

 

The study is called:

 

The Pathogenicity Variation of Two Quail-Origin H5N1 HPAV to BALB/c Mice after Six Passages in Quail

Hailiang Sun, Peirong Jiao, Yuqiang Cheng, Runyu Yuan, Pengfei Cui, Liming Jin, Chaoan Xin and Ming Liao

Abstract: H5N1 Highly Pathogenic Avian Influenza (HPAI) viruses have posed a serious threat to poultry, wild birds and mammals including humans since 1997. Quails are a potential reservoir in which influenza viruses might mutate to a mammalian transmissible form.

 

To investigate the molecular changes that occur in H5N1 HPAI viruses following passage in quail, two isolates, A/Quail/Guangdong/342/2008 (QL342) and A/Qquail/Guangdong/176/2004 (QL176) were selected. QL342 (clade 2.3.2) and QL176 (clade 7) viruses were high pathogenic to quail with a mortality rate of 18.3-100% and could be transmitted between naive contact quails.

 

After six passages in Japanese quails, researchers obtained two viruses, F6QL342 and F6QL176. Compared with QL342, F6QL342 had six animo acid substitutions in polymerases PB1 and PA, Nucleoprotein (NP) and Hemagglutinin (HA) but there was little difference in their pathogenicity to mice.

 

Compared with QL176, F6QL176 virus had 10 amino acid substitutions in PB2, NA, HA proteins. F6QL176 showed an increased pathogenicity towards mice causing more severe weight loss and higher lethality compared with QL176.

 

The findings showed that quails might play an important role in the adaptation of H5N1 avian influenza viruses to mammals. Therefore, researchers should enhance surveillance of H5N1 HPAI viruses in the quail population especially in live-bird markets.

 

The entire study is available HERE.

 

Essentially researchers infected a series of quail – taking the virus from one quail to the next – and then tested the viruses after six serial passages to see what, if any, changes to the virus had occurred.

 

The also tested the pathogenicity in mice of these viruses before and after the serial passage.

 

 

Mice, while convenient to work with, are not the best substitutes for human physiology (they lack the a2,6 airway receptor cells). They are highly susceptible to H5N1, however, and are often used to determine the pathogenicity of avian influenza viruses in small mammals.

 

Genetic sequencing F6QL342 and F6QL176 end viruses showed that the receptor binding domain of the two viruses were unchanged after six passages in quails.

 

That doesn’t mean that it couldn’t happen.  Only that it didn’t happen in the course of this particular research project.

 

There were significant changes however, both in pathogenicity in mice, and amino acid substitutions.

 

The authors conclude:

 

CONCLUSION

This study suggested that avian influenza virus receptor-linked specificity could change by passages in the host in vivo. The researchers want to clarify if QL342 or QL176 had gained human receptor-linked specificity following passage in quails. However, the study showed that the amino acids present in the HA receptor-binding sites of the two viruses were not changed after six passages in quails.

 

Interestingly, the substitution N170D in HA protein in the F6Ql176 isolate resulted in the absence of a potential N-linked glycosylation site. Glycosylation can affect hemagglutinin receptor affinity and also the efficiency of release of new virus from host cells (Gallagher et al., 1992; Abe et al., 2004).

 

There were nine animo acids changes in NA and HA proteins from the F6Ql176 isolate that could potentially change the virus membrane characteristics.

 

 

Nature’s laboratory has unlimited time, no budgetary constraints, and a staggering array of hosts and pathogens to work with.

 

Which is why scientists like to remind us that `Nature always bats last’.