Monday, July 21, 2014

Serial Passage Of H5N2 In Mice



# 8854


Influenza viruses are prolific, but sloppy, replicators.  They make millions of copies of themselves inside every host, but in the process, often make small transcription errors – amino acid substitutions – that can change the way the replicated virus acts.  Often, these changes are of little or no effect, or are even detrimental to the survival of the virus.

Those that favor replication in the host, however, tend to carry on to produce more progeny, advancing their newfound lineage forward, often drowning out the earlier `wild type’ virus in the host.


This process is called host adaptation, and while it can (and does) happen in the wild, it can be easily simulated in the laboratory as well via a classic serial passage study (see chart above).  You simply inoculate a host with a `wild type’ 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 tends to adapt to the new host (assuming there are no species barriers to prevent it). 

This tendency to adapt over long chains of infection is one of the reasons we watch for clusters of novel flu infections in humans, as it could either indicate adaptation, or provide more opportunities for the virus to do so.


Although regrettably most of this article is behind a pay wall, the journal Veterinary Microbiology has published a study wherein researcher performed this serial passage experiment in mice using a naturally H5N2 reassortant virus.


One should note that the H5N2 virus (A/chicken/Hebei/1102/2010 (HB10)) used was a reassortant of the H5N1 virus and H9N2 avian flu.


Unlike it’s highly pathogenic, and infamous, sibling  H5N1 – there is only limited evidence suggesting that H5N2 viruses can infect mammals.



Still, we haven’t seen any solid evidence that H5N2 has produced significant or serious human illness, although the possibility was explored in the J. Chinese Medical Association in 2012 in  The threat of highly pathogenic avian influenza H5: will H5N2 infections occur in humans?.


First the abstract from this latest study, which produced a virus with increased virulence and replication efficiency after just 15 serial passages,  then I’ll return with a bit more.


Adaptation of a natural reassortant H5N2 avian influenza virus in mice.

Li Q1, Wang X1, Zhong L1, Wang X2, Sun Z1, Gao Z1, Cui Z1, Zhu J1, Gu M2, Liu X2, Liu X3.


It is reported that the H5N2 highly pathogenic avian influenza virus A/chicken/Hebei/1102/2010 (HB10) is a natural reassortant between circulating H5N1 and endemic H9N2 influenza viruses. To evaluate the potential of its interspecies transmission, the wild-type HB10 was adapted in mice through serial lung passages.

Increased virulence was detectable in 5 sequential lung passages in mice and a highly virulent mouse-adapted strain (HB10-MA) with a 50% mouse lethal dose of 102.5 50% egg infectious dose was obtained in 15 passages. The virulence and the replication efficiency of HB10-MA in mice were significantly higher than those of HB10 while HB10-MA grew faster and to significantly higher titers than HB10 in MDCK and A549 cells.

Only five amino acid mutations in four viral proteins (HA-S227N, PB2-Q591K, PB2-D701N, PA-I554V and NP-R351K) of HB10-MA virus were found when compared with those of HB10, indicating that they may be responsible for the adaptation of the novel reassortant H5N2 avian influenza virus in mice with increased virulence and replication efficiency.

The results in this study provide helpful insights into the pathogenic potential of novel reassortant H5N2 viruses to mammals that deserves further attentions.


Although I know better than to anthropomorphize viruses (they hate when you do that), as time goes on I’ve begun to think of H9N2 as the Professor Moriarty of avian flu viruses.  Whenever something worrisome happens with an avian flu strain – if you look deep enough – you often find clues that H9N2 was behind it all.


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.


As we’ve discussed before, the genetic contributions from the avian H9N2 virus appear to be significant.


Of the three avian flu viruses we are currently watching with the most concern – H5N1, H7N9, and H10N8 – all  share several important features (see Study: Sequence & Phylogenetic Analysis Of Emerging H9N2 influenza Viruses In China):

    • They all first appeared in  Mainland China
    • They all  have come about through viral reassortment in poultry
    • And most telling of all, while their HA and NA genes differ - they all carry the internal genes from the avian H9N2 virus

This ubiquitous, yet fairly benign H9N2 virus, is promiscuous, as we keep finding bits and pieces of it turning up in new reassortant viruses.  Most recently, in Eurosurveillance: Genetic Tuning Of Avian H7N9 During Interspecies Transmission, we saw evidence of even more influence of H9N2 on the ongoing evolution of H7N9.


For now, H5N2 is generally regarded as posing a low level of threat to human health. 


Of course, the same thing could have been said about all of the H7 family of avian flu viruses 18 months ago, before H9N2 lent its internal genes to H7N9 in China, sparking a serious threat to public health.

If there is one constant with influenza viruses, it is that they continually change.

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