Monday, January 22, 2018

H9N2 Adaptation In Minks


Mink are a member of the Mustelidae family of carnivorous mammals, which also includes  badgers, otters, weasels, martens, ferrets, and wolverines. Many of these species are susceptible to flu viruses – most notably ferrets – which are often used in influenza research.
While small peridomestic animals like mink, skunks, and rabbits are known to be susceptible to certain types of influenza infection, we are still learning about their ability spread these viruses to other species, or how host adaptation might influence the evolution of flu viruses.
A little over 8 years ago, in 2009's That Touch Of Mink Flu, we looked at a story out of Denmark, where at least 11 mink farms in the Holstebro were reported to be infected with a variant of the human H3N2 virus.
Mink farming has become big business in China in recent years, with more than 60 million raised in 2012. Increasingly fox and raccoon dogs are raised on the same farms, increasing the odds of interspecies transmission of novel viruses.
In China, farmed animals are often fed a diet that includes raw poultry or poultry products (cite), which increases their risk of exposure to avian viruses. This practice inadvertently led to the deaths of hundreds of exotic tigers in Thailand in 2004 (see HPAI H5: Catch As Cats Can) from HPAI H5N1. 
In 2015, we revisited mink flu in That Touch Of Mink Flu (H9N2 Edition), after a study was published in the Virology Journal on a serological survey of antibodies to H9N2 (along with H5 & H7 viruses) in Chinese farmed minks, along with the results of experimental infection of minks with the H9N2 virus.

That 2015 study found that mink inoculated with the H9N2 subtype replicated the virus in their lungs (and to a lesser extent) heart, brain, and kidney. While H9N2 infection was non-fatal for mink, they developed lung lesions, edema, and shed the virus through their respiratory tract.

Last August, in That Touch Of Mink Flu (H9N2) - Revisited, we looked at yet another study - published in Nature's Scientific Reports - on the seroprevalence and transmissibility of H9N2 from minks to other peridomestic animals.

The authors wrote:
Transmission experiments showed that close contact between H9N2 infected mink and naïve contact mink, foxes and raccoon dogs resulted in spread of the virus to the sentinel animals as determined by virus isolation and/or seroconversion. H9N2-challenged foxes and raccoon dogs also showed H9N2 IAV could infect these animals without clinical signs and virus shedding, but with seroconversion.
A serosurvey in foxes and raccoon dogs demonstrated that H9N2 IAV circulated in these hosts. In some areas in China, mink, foxes and raccoon dogs are raised on the same farms, which could increase the chance for H9N2 IAV to cross the species barrier.
Our findings suggest that the potential exists for H9N2 IAV transmission to humans exposed to fur animals. Virological and epidemiological surveillance of IAVs in mink, foxes and raccoon dogs should be strengthened for public health.
Although farmed poultry and live bird markets are generally viewed as posing the greatest threat for producing a new pandemic strain, other animals - such as swine, which sparked the 2009 H1N1 pandemic - are also candidates (see Arch. Virology: Isolation & Characterization Of H5N1 In Swine - China 2015).
Somewhat further down the list are a number of lesser threats, including farmed mink, dogs, and foxes.
While H9N2 is regarded as having only moderate pandemic potential on its own (see CDC IRAT score), it more than makes up for that by its promiscuity - lending its internal genes to a long list of successful and dangerous HPAI & LPAI viruses, including H5N8, H5N6, and H7N9 (see graphic below).

H7N9 Genetic Structure - Credit Eurosurveillance

All of which serves as prelude to a short communications published last week in Transboundary and Emerging Diseases that characterizes two H9N2 viruses isolated from mink in 2014 in China.

One isolate caused no signs of illness in mice, while the other carried a mammalian adaptation (701N in PB2 protein) that  substantially increased its virulence in mice.  First the abstract (the full report is behind a paywall), then I'll return with more.

H9N2 influenza virus isolated from minks has enhanced virulence in mice
Xue R1, Tian Y1,2,3, Hou T2, Bao D2, Chen H2, Teng Q2, Yang J2, Li X2, Wang G4, Li Z2, Liu Q2.

First published: 14 January 2018Full publication history
DOI: 10.1111/tbed.12805 View/save citation


H9N2 is one of the major subtypes of influenza virus circulating in poultry in China, which has a wide host range from bird to mammals. Two H9N2 viruses were isolated from one mink farm in 2014. Phylogenetic analysis showed that internal genes of the H9N2 viruses have close relationship with those of H7N9 viruses.
Interestingly, two H9N2 were separated in phylogenetic trees, indicating that they are introduced to this mink farm in two independent events.
And further mice studies showed that one H9N2 caused obvious weight loss and 20% mortality in infected mice, while another virus did not cause any clinical sign in mice infected at the same dose.
Genetic analysis indicated that the virulent H9N2 contain a natural mutation at 701N in PB2 protein, which was reported to contribute to mammalian adaptation. However, such substitution is absent in the H9N2 avirulent to mice.
Circulation of H9N2 in mink may drive the virus to adapt mammals; continual surveillance of influenza virus in mink was warranted.

This 701N amino acid host adaptation is one of several we've been watching closely for several years in the evolution of H7N9 in China. Last October, in Cell Research: Another Cautionary H7N9 Study Out Of China, we looked at a study which warned of `alarming mutations' they saw occurring across an array of H7N9 viruses in China.

Mutations that can not only change LPAI H7N9 viruses into highly pathogenic ones, but that can also potentially make them a greater human pandemic threat.

An excerpt from the study by Hualan Chen et al.H7N9 virulent mutants detected in chickens in China pose an increased threat to humans - follows:
Importantly, some H7N9 viruses obtained an insertion of four amino acids in their hemagglutinin (HA) cleavage site and were lethal in chickens. The index strain was not lethal in mice or ferrets, but readily obtained the 627K or 701N mutation in its PB2 segment upon replication in ferrets, causing it to become highly lethal in mice and ferrets and to be transmitted efficiently in ferrets by respiratory droplet.
H7N9 viruses bearing the HA insertion and PB2 627K mutation have been detected in humans in China. Our study indicates that the new H7N9 mutants are lethal to chickens and pose an increased threat to human health, and thus highlights the need to control and eradicate the H7N9 viruses to prevent a possible pandemic.
Going back to 2014, in PLoS Path: Genetics, Receptor Binding, and Transmissibility Of Avian H9N2, we saw similar changes in the PB2 segment of the H9N2 virus when passaged through ferrets.
Perhaps most concerning, of 35 H9N2 viruses tested, all bound preferentially to alpha 2,6 receptor cells -  the type commonly found in the human upper respiratory tract - rather than to alpha 2,3 receptor cells which are found in the gastrointestinal tract of birds.
While this 701N mutation isn't new, finding it naturally occurring in a mink farm in China is another indication that H9N2 viruses are slowly accruing mammalian adaptations over time. 
The H9N2 virus is currently viewed as posing far less of a public health threat than H7N9 or H5N6, but its ability to readily lend its internal genes to other subtypes makes any move towards enhanced virulence or transmissibility something we need to watch. 
For more on the impact of this 701D mutation, you may wish to revisit:
Science: H7N9 Transmissibility Study In Ferrets