Tuesday, May 02, 2017

Sci Rpts:Reassorted H9N2:pH1N1 Virus Transmission After Serial Passage In Swine


We've a new study - published today in Nature's Scientific Reports - that ticks so many of the boxes we've covered before, its hard to know where to begin. You may want to grab a fresh cup of coffee, as this may take a while.
Today's study uses the time honored technique of serial passage of a virus through a series of hosts (in this instance, pigs), to determine what host-species adaptations occur over time.
We've looked at serial passage experiments many times, most recently in a Sci Rpts study from last January H5N8 - Rapid Acquisition of Virulence Markers After Serial Passage In Mice. The concept (see graphic below) is simple. 

You inoculate a naive host with a strain of a virus, let it replicate awhile, then take the virus from the first host and inoculate a second, and then repeat the process five, ten, fifteen times or more.
Over time, the virus may better adapt to the new host (assuming there are no species barriers to prevent it).
You are simulating, in a controlled way, pretty much what goes on in the `wild' all the time. The advantage is you can compare the original virus to the downstream `adapted' virus at specific intervals to find out what genetic changes have occurred and document any behavioral changes.

The virus in today's study is a reassortment of avian H9N2  genes (HA & NA), with a backbone of internal genes from the 2009 pH1N1 virus.  

While this reassorted H9N2 virus was engineered in the laboratory, with H9N2 viruses recently reported in Chinese swine herds, circulating alongside pH1N1 virus, a naturally occurring reassortment of this type is a real possibility.

Not only does pH1N1 have a record of sharing its (already mammalian adapted) internal genes with other viruses, the H9N2 virus is very promiscuous, something we looked at in some depth just a couple of days ago (see Vet. Microb.: Analysis Of Korea Lineage Of Avian H9N2 viruses).

As the authors explain in their Introduction, previous experiments had shown limited transmissibility of H9N2 and H9N2:pH1N1 reassortants in pigs, but at levels significantly below that of swine-adapted viruses like pH1N1. The authors write (bolding mine):

In the present study, we have examined the replication and transmissibility after ten serial pig passages of a reassortant virus containing A/quail/Hong Kong/G1/1997 (H9N2) surface-protein genes and NP gene within the A/California/04/2009 (pH1N1) background to evaluate if the pH1N1 backbone confers an advantage in H9N2 adaptation to pigs.
This serial passaging of the H9N2:pH1N1 reassortant resulted in a virus that replicated in and transmitted between pigs at high rates. The predominant mutation in the passaged reassortant virus was an aspartic acid to glycine at position 225 in the HA RBS. Therefore, our results showed that the combination of reassortment and mutations induced by the serial passages generated a virus with a predominant mutation at position 225 in HA RBS that replicated and transmitted at high rates in pigs.

If a switch to glycine at position 225 in the HA gene sounds familiar, it is because it was a  source of concern during the 2009 pandemic (see Norway Reports An H1N1 Mutation), and while only rarely reported, has been linked to more severe disease.
This mutation involves an amino acid substitution at position 225 (222 using H1 Numbering) from aspartic acid (D) to glycine (G) and allows the virus to bind to receptors found deeper in the lungs, and is linked to the development of more severe pneumonia.
The development of this mutation after only 7 passages is linked to improved replication and transmission of the H9N2:pH1N1 reassortant virus in pigs. For a more detailed look at this mutation, you may wish to revisit my 2015 blog EID Journal: Emergence of D225G Variant A/H1N1, 2013–14 Flu Season, Florida.

Then entire study, while lengthy and at times complex,  is well worth reading in its entirety. I've only covered the highlights, the Abstract, and an excerpt from the discussion section follow:

A reassortant H9N2 influenza virus containing 2009 pandemic H1N1 internal-protein genes acquired enhanced pig-to-pig transmission after serial passages in swine

José Carlos Mancera Gracia, Silvie Van den Hoecke, Juergen A. Richt, Wenjun Ma, Xavier Saelens & Kristien Van Reeth

Scientific Reports 7, Article number: 1323 (2017)
Received:03 February 2017
Accepted:30 March 2017
Published online:02 May 2017


Avian H9N2 and 2009 pandemic H1N1 (pH1N1) influenza viruses can infect pigs and humans, raising the concern that H9N2:pH1N1 reassortant viruses could emerge. Such reassortants demonstrated increased replication and transmissibility in pig, but were still inefficient when compared to pH1N1. 

Here, we evaluated if a reassortant virus containing the hemagglutinin and neuraminidase of A/quail/Hong Kong/G1/1997 (H9N2) in the A/California/04/2009 (pH1N1) backbone could become better adapted to pigs by serial passaging. The tropism of the original H9N2:pH1N1 (P0) virus was restricted to the nasal mucosa, with no virus detected in the trachea or lungs.

Nevertheless, after seven passages the H9N2:pH1N1 (P7) virus replicated in the entire respiratory tract. We also compared the transmissibility of H9N2:pH1N1 (P0), H9N2:pH1N1 (P7) and pH1N1. While only 2/6 direct-contact pigs showed nasal virus excretion of H9N2:pH1N1 (P0) ≥five days, 4/6 direct-contact animals shed the H9N2:pH1N1 (P7). Interestingly, those four animals shed virus with titers similar to those of the pH1N1, which readily transmitted to all six contact animals. 

The broader tissue tropism and the increased post-transmission replication after seven passages were associated with the HA-D225G substitution. Our data demonstrate that the pH1N1 internal-protein genes together with the serial passages favour H9N2 virus adaptation to pigs.


In line with previous studies17, 28, this report demonstrated that avian H9N2 influenza viruses can reassort with pH1N1 internal genes, resulting in enhanced virus transmission in pigs when compared to the parental, non-reassorted H9N2 virus. The present study also underscores that repeated introduction of reassortant H9N2 viruses into the swine or humans may result in the selection of virus variants with a transmission efficiency close to that of the endemic swine or human influenza viruses.
However, the lack of transmission detected in one of the contact groups used for transmission of passage seven virus, emphasizes the complexity of the adaptation process of an avian virus to a mammalian species and the need for additional research to better understand this crucial step in cross-species transmission of influenza viruses.
         (Continue . . . )


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