H7N9 Influenza Virus: A Perfect Pathogen?

Artist’s rendition of a virus particle.

It’s late October and here in Wisconsin, like many of you, we are experiencing a change of seasons, with the associated drop in temperatures, changes in leaf color and later this week, Halloween.

Another thing that comes with fall is the start of cold and flu season. By “flu”, I mean influenza, caused by avian influenza viruses of the H-N type. Recent research results by teams at UWI-Madison and in Japan, makes the coming influenza season potentially more scary than usual.

In a recent Cell Host & Microbe paper, M. Imai et al. study a seemingly more virulent version of H7N9 avian influenza virus that is startling in its ability to spread from infected to healthy animal models. Based on a current epidemic of H7N9, human-to-human transmission with this strain is increasing.

When I was in graduate school studying microbial pathogenesis, a favorite class exercise was to design the ‘perfect pathogen’. This was a perfect pathogen on paper only—we were not creating such in the laboratory—just thinking of the properties that would make the best microbial pathogen.

In this exercise, some of us thought that the ‘perfect pathogen’ would be a microbe that caused an infection characterized by excessive purulent discharge, phlem production and/or bleeding. Perfect meant most gory, right?

Others designed a pathogen that caused death immediately upon infection, a real killer of an organism.

But in our exuberance to conjur a superlative pathogen, we ignored the most important trait—transmission.
At the end of the exercise, the instructor reminded us that a pathogen that caused too much bleeding and/or quick death was not a perfect pathogen. The most important trait of pathogenicity is a subtle yet effective ability to spread.

Contagion wins the day.

In the Cell Host & Microbe paper, “A highly pathogenic avian H7N9 influenza virus isolated from a human is lethal in some ferrets infected via respiratory droplets” and from the Centers for Disease Control (CDC) we learn that there have been some startling changes in H7N9 infection rates and fatalities in China since 2013.

If contagion wins the day, H7N9 might be on it’s way to perfect pathogen status.

Let’s take a look at some general H7N9 information from the CDC and some details from the paper that includes work from Yoshihiro Kawaoka, a researcher at the University of Wisconsin, here in Madison.

About H7N9 Influenza Virus
The CDC notes that human infections with avian influenza A H7N9 virus were first reported in China in early 2013 and are believed to have resulted from human contact with infected birds. Annual epidemics of sporadic human infections with H7N9 viruses in China have been reported since 2013, with the 5th and largest epidemic of H7N9 human infection currently underway. During this most recent epidemic, nearly 40 percent of people confirmed to have H7N9 infection have died.

The Cell Host & Microbe authors state:

“Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmission.”

To characterize this particular H7N9 virus, the researchers examined molecular markers associated with pathogenicity, replication and antiviral resistance. Results showed that the H7N9 virus first seen in 2013 was a low pathogenic avian influenza virus (LPAI) and that this was the virus circulating from 2013–2017, during the first four epidemics seen in China. These epidemics were characterized by mild or asymptomatic disease in poultry.

However, the number of reported human cases increased significantly during the fifth epidemic (starting approximately February of 2017), along with a sudden increase in fatalities, upwards of 39-40%, as noted in the CDC report.

Paper Results
Imai et al. isolated and studied H7N9 virus from a highly pathogenic influenza (HPAI) virus that caused a human fatality in China. Incidentally, this human had been treated with the antiviral oseltamivir (a neuraminidase or NA inhibitor). Their characterization of this virus found that it: 1) did not encode the mammalian-adaptive PB2-62K marker; 2) but was positive for presence of A588V substitution in PB2, a substitution known to enhance viral polymerase activity, replication in mammalian cells and virulence in mice.

Deep sequencing analysis of virus stock showed a mixed population of viruses encoding arginine at amino acid 294 of NA (in 94% of the sequence reads) or at lysine (in 6% of sequence reads). Although the original H7N9 virus from this region did not show the NA-294K (lysine) substitution, the fact that this isolate showed the lysine substitution and came from a NA-inhibitor treated patient was of concern to these researchers.

They next created two recombinant viruses, rGD/3-NA294R and rGD/3-NA294K, and performed studies comparing these two recombinant strains to GD/3, the original HPAI H7N9 strain as well as a LPAI H7N9 strain.

It’s important to note that the recombinant influenza strains were developed and studied in Japan, as such recombinant influenza virus research was banned in the US beginning in 2016, due to fears of spread of these recombinant viruses beyond the laboratory. Study author and internationally known influenza expert Y. Kawaoka has commented publicly against the ban on recombinant virus research due to its use in understanding more about influenza viruses.

The authors studied ability of the recombinant and HPAI and LPAI viruses to replicate in cultured human bronchial epithelial cells, at 33°C and 37°C (mimicking upper and lower respiratory track temperatures, respectively). They found that LPAI strains and the rGD/3-NA294R strains grew similarly, while the HPAI GD/3 and rGD/3-NA294K strain showed delayed growth at 33°C.

The authors evaluated viral replication and pathogenicity in established animal models for influenza virus. The GD/3 and NA294R strains were highly pathogenic in BALB/c mice, while the NA294K mutation showed lowered pathogenicity in mice; the lysine mutation appeared to reduce pathogenicity somewhat.

Viral replication was further studied in ferrets, where all four virus strains showed relatively high replication levels in nasal turbinates.

In cynomolgus macaques infected with HPAI GD/3 H7N9 strain, there were no signs of serious infection, similar to previous infection of macaques with LPAI strain; however, lungs of the GD/3-infected macaques did show viral antigen positive cells and pulmonary edema, demonstrating ability of this H7N9 strain to establish a robust infection.

Since host-to-host spread of H7N9 virus is a critical aspect of transmission in pandemic influenza viruses, the authors examined infection of the four strains via respiratory droplet transmission to ferrets. Nasal wash titers were calculated for ferrets inoculated with each of the four strains. The washes showed no significant difference between the GD/3, NA294R and LPAI strains, while the NA294K mutation showed lower virus titers from nasal wash. The inoculated ferrets all showed lowered appetite and lethargy, and 1-2 animals from each inoculation group died, including those infected with the NA294K mutation viral strain.

Most intriguing perhaps, was a separate study of viral transmission via respiratory droplets. The authors established transmission pairs of ferrets, each composed of a naive ferret housed adjacent to an infected ferret 1-day post-infection. Transmission was observed to one GD/3-exposed ferret, three NA294R and two NA294K exposed ferrets. (Other similar studies have established that only 0/4 and 4/4 infections by this means can be considered significantly different results.) Ferrets exposed to GD/3, NA294K and the LPAI virus recovered, while 2/3 ferrets exposed to NA294R died of their infections.

For the sake of brevity, I’ll leave further results of these studies for your inspection—Imai, et al. is available online in its entirety as linked in this blog.

The authors summarize by noting that HPAI H7N9 virus requires a small amount of virus to initiate lethal infection of exposed ferrets via respiratory droplets. It can successfully spread from one host to another. The statistics on infection rate and deaths during the current epidemic in China seem to further show that spread and pathogenicity in humans has increased considerably.

This paper was published online October, 19, 2017, so these results are new. Author Y. Kawaoka has stated that studies are ongoing and that this is just the first report from their work.

Here is the paper:
Imai, M. et al. (2017) A highly pathogenic avian H7N9 influenza virus isolated from a human is lethal in some ferrets infected via respiratory droplets. Cell Host & Microbe pii: S1931-3128(17)30396-7.

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Kari Kenefick

Kari Kenefick

Kari has been a science writer/editor for Promega since 1996. Prior to that she enjoyed working in veterinary microbiology/immunology, and has an M.S. in Bacteriology, U of WI-Madison. Favorite topics include infectious disease, inflammation, aging, exercise, nutrition and personality traits. When not writing, she enjoys training her dogs in agility and obedience. About the practice of writing, as we say for cell-based assays, "add-mix-measure".

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