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Thoughts on India's [epic 2015] flu season

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  • #16


    Influenza A viruses have been responsible for four influenza pandemics in last century viz., Spanish influenza (H1N1) in 1918, Asian influenza (H2N2) in 1957, Hong Kong influenza (H3N2) in 1968 and pandemic influenza (H1N1) in 2009, which was caused by influenza A(H1N1)pdm09. During the 2009 pandemic period (2009?2010), India was affected with around 50,000 cases and a case fatality of 6% [12]. After the end of the 2009 pandemic, the virus continued to circulate at low level in the population, and during the period from 2011 to 2014 the circulation of the virus declined [13]. From January to May 2015 however, over 39,000 persons in India were affected by a new epidemic of influenza A(H1N1)pdm09, with more than 2,500 deaths [1]. The outbreak spread across 22 of the 29 states in the country, making it the largest since 2009. This sudden re-emergence and wide spread simultaneous reporting of influenza A(H1N1)pdm09 along with higher number of hospitalisations and deaths was a major public health concern.
    By further characterising the strains infecting patients positive for influenza A(H1N1)pdm09 through HA phylogeny, this study finds that sequences of genogroup 6B were circulating during the 2015 epidemic. The genogroup 6B was found to evolve from a Russian isolate (A/Moscow-Oblast/CRIE-08/2013) and is since then circulating in many parts of the world. However, this is the first report from India regarding circulation of genogroup 6B, coinciding with a large scale outbreak [1].
    Researchers from Massachusetts Institute of Technology (MIT) have recently reported mutations D225N, and T200A in a 2014 Indian strain (A/India/6427/2014, which also clusters with genogroup 6B sequences in the phylogenetic treeFigure 1) making the virus more infectious [14]. Although we did not find these two mutations in our study, all the sequences that we characterised harboured five mutations (P83S, I321V in HA1, as well as E47K, S124N, and E172K in HA2), which although previously described, have not been reported in combination. Moreover, two isolates from patients with severe disease harboured a N129D mutation in HA1 and two isolates had a mutation in HA2, E164G, that has not been observed to date. These unique features of the viruses found here may have played a role in shaping the large scale epidemic with cases of severe disease. On the other hand, the 2015 epidemic in India may be attributed to lack of immunity among an immune-na?ve population. It is also noteworthy that seasonal influenza vaccination is not very common in India.
    Some limitations of the study include that the samples were only tested for influenza A(H1N1)pdm09 virus, whereby only 38% of samples tested were positive. Therefore, co-circulation of other influenza subtypes or types could not be ruled out. Moreover the sequence analysis was conducted with only few positive samples that did not cover other gene segments than the HA and NA genes.
    The influenza A(H1N1)pdm09 virus represents a quadruple reassortment of two swine, one human, and one avian strain of influenza virus [15]. The largest proportion of genes comes from swine influenza viruses (30.6% from North American swine strains, 17.5% from Eurasian swine strains), followed by North American avian strains (34.4%) and human influenza strains (17.5%). It will be interesting to investigate the involvement of any gene reassortment in the 2015 outbreak in India through complete genome sequencing.
    Two of 12 strains from fatal cases were found to harbour a mutation conferring resistance to oseltamivir. Learning more about the 2015 strains circulating in India could help public health officials determine treatment options and inform on vaccines for the next influenza season, which is likely to include currently circulating strains [16].
    Our findings show the importance of systematic molecular surveillance to provide insight into strains circulating during influenza epidemics.
    Twitter: @RonanKelly13
    The views expressed are mine alone and do not represent the views of my employer or any other person or organization.


    • #17
      Ronan I have no relevant formal training in virology or sequence analysis, but for what it's worth, I think the C1, C2 part of the reply relates to the passages and passage medium and I have pasted below part of a reply I made to a question of Mixin's in this thread on vaccines in my work shop.
      The collected flu sample is grown in the lab in chicken egg cells (C) or some other cell line like the mammalian line (MDCK) where C1 would be the first passage in an egg medium and its product would be the input for C2 etc.
      It is important in understanding the trends in the sequences while they may drift during passage there will be limits to how far.

      Flu is famous for its ability to mutate so if you have a swab and grow it in X – where X is one of MDCK, SIAT, egg or some other cell line – you are going to get a whole range of variations around whatever was in the swab. All of the virions produced will then have to find a new cell to infect in the growth medium but which of this generation succeed will depend, at least in part, on how well adapted the RBS of their HA is to whatever X we happened to choose. The reason we have to passage the virus at all is the swab sample size is too small. Each generation is termed a passage and each passage produces more virus but it also increases the chance that the virus will drift away from the sequence we would have got if we had been able to exactly clone the original sample (I am ignoring the whole quasi species aspect of flu to keep it simple – think of it as adding an extra passage).

      From the above we can see that what comes out is not necessarily identical to what went in. The question is how different is it? That depends on two main variables how many passages were performed and how strong the selection pressure was. To minimise the drift we can reduce the number of passages – for which we either need a bigger initial sample or we can increase the diagnostic sensitivity (needs less product) – and/or we can reduce the selection pressure by making sure we match, as accurately as possible, our passage cell line to the samples host species. There is no perfect match for a human flu virus sample; egg would be worse than MDCK which would not be as good as SIAT. There is a limit to the amount of drift you are likely to get so you will end up near the ideal but not exactly on target.
      Now if you use your product in an HI test then original samples which were already further away from the test antisera are more likely to have drifted over the border into 'low reactor' territory. Conversely on remote branches of the phylogenic tree could – which would have been low reactor – samples may passage back into the more central zone.
      The extract is from post #7 and there is more relevant information in the thread but some of it is more technical and relating to Mixin's specific question.

      On the more general aspect of 'mutations' or 'no mutation' on the HA.
      There are always mutations in the sequence data the question is are they significant? Some areas on some of the RNA strands are known to correlate effects on biological function however, sadly, these are only guides and do not always have the same degree of influence on all genetic backgrounds. H1N1(2009), for example, has a very well know avian sequence on its PB2 strand which should make it reproduce poorly in mammals and I expected it to mutate rapidly to the mammalian form but it has remained stable proving that on this strain the change is not critical.
      Over the last decade I, and many others, have written extensively here on many aspects of problems relating to the sequence databases and the interpretation of sequence data. If you, or anyone else, have questions relating to this area please ask them below and I will try and write something or find a link to a remembered post covering that aspect.
      As always, in awe of your work on collating data on India's disease burden. JJackson.
      If it would be helpful to your work I would be willing to attempt an analysis of any Indian H1N1 data at GISAID & Genebank but I am a little doubtful there will be firm conclusions - I suspect the CDC analysis is correct there were mutations but none of known significance. Markers for Oseltamivir resistance are well known, easy to spot and would have been reported. PM me if you think I could help along with any time periods and specific areas of the genome. Even if anything genetic had occurred it would seem to have been out performed by another wild-type and removed from the gene pool as the spike has not persisted or spread geographically.
      Last edited by JJackson; March 7, 2016, 11:23 AM.


      • #18
        Thanks JJackson - your thoughts are very much appreciated. For a while there, India was blanketing all contacts of H1N1 cases with Tamiflu. In 2015, at least 68,500 patients were prescribed Tamiflu in Maharashtra alone. I wonder how many of those courses of treatment were not completed or shared. I'd be curious to see to what extent Oseltamivir resistance has changed over time. From the paper at the top of this page 2 of 12 fatal cases showed resistance. I agree that the effect of many mutations to the influenza genome are insignificant. I'd also welcome expansion of India's surveillance system to include public reporting of other influenza strains so that we could compare the effect of an H1N1 dominant season to an H3N2 dominant season for example. If wishes were fishes...
        Twitter: @RonanKelly13
        The views expressed are mine alone and do not represent the views of my employer or any other person or organization.