TWiV 62: Persistence of West Nile virus

December 13, 2009

The_Persistence_of_MemoryHosts: Vincent RacanielloDickson Despommier, and Alan Dove

Vincent, Dickson, and Alan discuss STEP HIV-1 vaccine failure caused by the adenovirus vector, presence of West Nile virus in kidneys for years after initial infection, adaptation of the influenza viral RNA polymerase for replication in human cells, and the significance of the D225G change in the influenza HA protein.

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Links for this episode:

Weekly Science Picks
Dick Smallpox – The Death of a Disease by DA Henderson
Alan Olympus Bioscapes Digital Imaging Competition
Vincent Microbe Magazine

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  • niman

    I heard the comments on D225G, and they don't even reach the level of extreme handwaving. No one has said that D225G is a requirement for the virus to reach the lungs. However, there are cell types in the lung that express gal 2,3 and a switch to 2,3 specificites could have fatal effects, as have been seen in MANY countries, including Ukraine, United States, Brazil, Norway, France, especially in cases since this summer. D225G is pretty rare (in about 1% of sequences), yet is 4/4 fatal casses in Ukraine. Another change at 225 (D225N) is even rarer, but is also linked to lethal cases (2/2 in Ukraine and 2/2 in Brazil).
    There are a couple of papers describing position 225 as an antigenic site in H1N1, and the position has already changed to three different amino acids (D225G, D225N, D225E) using 4 different codons. These changes are not accidents or coincidences.
    The low reactor status was by Mill Hill and declaring the isolate a low reactor was not taken lightly. Low reactor means that the titer is lowered by at least 4 fold, so your game of telephone where you ask questions that misrepresent the facts once again points for a need to actually looking at the data rather than going on air and spouting nonsense about fantasies

  • niman

    Speaking of low reactors, the Mill Hill designation has led to concerns of others (who are really dealing with H1N1 as opposed to selective readings and playing “telephone”). Canada in their latest FluWatch has noted that their low reactors do not have D222G (aka D225G). You guys really need to get up to speed before you spout nonsense on what is and isn't known about H1N1 and D225G:

    http://www.phac-aspc.gc.ca/fluwatch/09-10/w48_0

    Since September 1, 2009, the National Microbiology Laboratory (NML) has antigenically characterized 433 Pandemic (H1N1) 2009 viruses and eight seasonal influenza viruses (two influenza A/H1N1, five H3N2, and one B virus) that were received from Canadian laboratories. Of the 433 pandemic influenza A (H1N1) viruses characterized, 430 (99.3%) were antigenically related to A/California/7/2009, which is the pandemic reference virus selected by WHO as the Pandemic (H1N1) 2009 vaccine. Three viruses (0.7%) tested showed reduced titer with antisera produced against A/California/7/09. Sequence analysis of the HA showed that the two viruses with reduced titer did not have the mutation at amino acid position 222 as reported by Norway.

  • niman

    I have already run into internet sites quoting your nonsense on low reactors. You are setting an extremely BAD example of virologists, so lets get to the real data so maybe you can post something here, or issue a retraction.
    First, as seen in the Nature Biotechnology that you linked (do you really read these papers or just look for catchy titles) there were three non-synonymous changes in HA between California/4 and an isolate from Germany. The German isolate is really the consensus sequence so the vaccine starts out with three HA difference relative to the vast majority of sequences out there, so although the Ukraine sequence has only one ADDITIONAL change, it already has three changes with California/7, the vaccine target.
    The Mill Hill data shows a “low reactor” status for the Ukraine sequence tested with D225G. They are well aware of the importance of such a designation and have stood by their data. Low reactor means that the titer against the “low reactor” is at least 4 fold lower than the target, and is used to signal a move toward vaccine failure. Low reactor doesn't guarantee failure, but certainly is a concern for those who have borderline immunity, and a titer of 40 is well into the bordeline category, but is the standard for protective immunity. Thus, those with titers of 80 would fall below the “protective” mark if the titer fell four fold, and titers of 160 would fall to the borderline area. There are also many host factors that lead to infections in those who do have “protective” levels. so a four fold drop is not a good indicator.
    As noted, Canada is now mentioning D225G in their discussion of low reactors, so they must not have heard your telephone nonsense with Palese.
    I think most virologists would appreciate some homework on your part before you broadcast nonsense about H1N1, receptor binding domain changes, and low titers.
    Your comments are quite embarassing to the field.

  • JJackson

    Thank you for addressing the low reactor status (TWiV62) of A/Lviv/N6/ H1N1(2009) (PS Lviv/N2 is identical at the AA level on both HA & NA – two third position changes on HA – no low reactor status) but I note you said that 225 did not fall within one of the antigenic sites. As I thought it had I looked at http://peds.oxfordjournals.org/cgi/reprint/22/9… and while the numbering systems are confusing if I read the paper right they seem to be implying it falls within epitope D (Table 1).

    I am not a virologist – I mend computers – so I have probably misunderstood something.

    Thanks for the blog and TWiVs

    Jonathan Jackson.

  • Hogweed

    I listened to your broadcast with interest but I have difficulty fitting in Palese's comment with published papers. For instance.

    http://www.plosone.org/article/info:doi%2F10.13

    EXTRACT

    Evolutionary Trends of A(H1N1) Influenza Virus Hemagglutinin Since 1918

    The Pandemic (H1N1) 2009 is spreading to numerous countries and causing many human deaths. Although the symptoms in humans are mild at present, fears are that further mutations in the virus could lead to a potentially more dangerous outbreak in subsequent months. As the primary immunity-eliciting antigen, hemagglutinin (HA) is the major agent for host-driven antigenic drift in A(H3N2) virus. However, whether and how the evolution of HA is influenced by existing immunity is poorly understood for A(H1N1). Here, by analyzing hundreds of A(H1N1) HA sequences since 1918, we show the first evidence that host selections are indeed present in A(H1N1) HAs. Among a subgroup of human A(H1N1) HAs between 1918∼2008, we found strong diversifying (positive) selection at HA1 156 and 190. We also analyzed the evolutionary trends at HA1 190 and 225 that are critical determinants for receptor-binding specificity of A(H1N1) HA.Different A(H1N1) viruses appeared to favor one of these two sites in host-driven antigenic drift: epidemic A(H1N1) HAs favor HA1 190 while the 1918 pandemic and swine HAs favor HA1 225. Thus, our results highlight the urgency to understand the interplay between antigenic drift and receptor binding in HA evolution, and provide molecular signatures for monitoring future antigenically drifted 2009 pandemic and seasonal A(H1N1) influenza viruses.

    Jun Shen1, Jianpeng Ma1,2*, Qinghua Wang2*

    1 Department of Bioengineering, Rice University, Houston, Texas, United States of America, 2 Department of Biochemistry and Molecular Bi

    It was surprising that HA1 190, which is critical for receptor-binding specificity of A(H1N1) HAs, was also under positive selection. Through further analysis of HA1 190, together with HA1 225, the other critical determinant for receptor-binding specificity of A(H1N1), we found that the epidemic HAs and the 1918 pandemic and swine HAs favored one of these two sites for antigenic drift. Whether the 2009 pandemic A(H1N1) HA will adopt any of these two trends, or use a novel mechanism that does not involve HA1 190 and 225, will unfold in the coming months.

    HA1 138, 186, 190, 194, 225, 226 and 228 had been previously shown to affect receptor binding to H1 HA [75], [76]. Among them, two residues, HA1 190 and 225, play predominant roles in determining the receptor-binding specificity of H1 HA: D190/D225 for α(2,6) receptors in humans, D190/G225 for α(2,6) and α(2,3) receptors in swine, and E190/G225 for α(2,3) receptors in avian [11], [39], [40], [41], [42], [76]. Although changes at these two sites had been previously reported to cause antigenic drift in A(H1N1) epidemic strains [77], it was a somewhat common belief that key determinants of receptor-binding specificity are in general not subject to selection. Thus, the strong positive selection at HA1 190 within subgroup I-v is quite unexpected

    …The deviations from the ideal D190/D225 combination in A(H1N1) HAs might result in reduced binding to human receptors [11], [41], [42], [64], [79]. However, two possibilities, which are not mutually exclusive, may explain the fact that mutations are frequently observed at these two sites: one is that other concurring mutations at or around the receptor-binding site may sufficiently maintain the receptor binding affinity so that the overall binding affinity is largely unaffected; the second is that the gain in evading antibody neutralization far overweighs the reduction in receptor binding

    ====

    And

    The LAIV Flumist paper at http://jvi.asm.org/cgi/reprint/JVI.02106-09v1.pdf (See Table 3 Antigenicity of various A/California/7/2009 variants).

    I would appreciate comments

  • Rabadan

    Dear colleagues,

    I am reading the TWiV #62 comments about the D225G mutation in HA. I cannot disagree more with the form of the comments from Dr Niman. Personally, I share with Dr Niman an interest for possible changes within the virus, e.g. the D225G mutation. But I agree with the skepticism of Prof Racaniello and Prof Palese, as it has been remarkably difficult to link mutations to severity or vaccine resistance out of the laboratory, and all possible associations should be taken with a grain of salt.

    Disagreement is an important part of a healthy scientific dialogue, and I do not believe that the derogatory terms used by Dr Niman encourage this process.

    Best regards,

    Raul Rabadan, PhD

    Assistant Professor
    Department of Biomedical Informatics
    Center for Computational Biology and Bioinformatics

    Columbia University College of Physicians and Surgeons
    1130 St. Nicholas Ave
    ICRC Bldg 8th Floor, Office 803B
    New York, NY 10032

  • Hogweed

    Personally I disagree with the style of derogatory comments also but I've seen them aimed at Niman just as often (in fact more so) than he throws them. We're in the middle of a pandemic for goodness sake – not really a time for this. And despite Dr Niman's combative style I've found his commentary in general highly valuable. And this current broadcast was aimed at Niman's commentaries even if he wasn't mentioned by name – that was trailed in advance on the net.

  • Hogweed

    I should also add if the authors think Niman to be a fear mongerer they should read the multiple Niman vs. the “Whackadoodler” (his term) threads on http://fluboard.rhizalabs.com/forum/index.php where Dr Niman debunks virtually every conspiracy theory going. It's no surprise he gets wound up some times. Personally I don't know how he manages it. But he's there seemingly 24 hours a day to respond to questions and criticisms. You should feel free to drop over. Not many you can say that about.

  • niman

    I believe there is a bit of confusion on results posted by Mill Hill at GISAID. Although they desposited sequences from 10 isolates from Ukraine, only two were antigenic characterized. The sample from Kiev was labeled “California/7-like” while the sample from Lviv (with D225G) was labeled “California/7-like. Low reactor). The other 8, including three other isolates with D225G, were not tested (or test results were not posted), so those untested samples do not create discrepancies. Moreover, since the only non-synonymous change in the Lviv sample is D225G, it is clear that D225G is the main driver that pushes the sample into the “low reactor” status, which means the ferret reference titer generated by Mill Hill was at least 4 four four lower than the titer against California/7 (it is also worth noting that California/7 has several additional changes not present in the consensus sequence).

  • niman

    I agree that my comments should have left out derogatory terms, which were fueled by the rather misleading comments on the show (or in your remarks regarding “grain of salt”). Influenza is clearing a virus that generates a wide range of symtoms, even within a family, which I can speak from personal experience – lab confirmed H1N1 in myself and otehr fmily members with four developing symptoms within a 1 week period and all having the same characteristic cough, but fever ranging from 103.1 F to no fever at all).
    However, even though symptoms are diverse, and H1N1 flu sequences have been generated by many labs on isolates collected under a wide range of circumstances, the association of position 225 changes (especially D225G and D225N) remain compelling.
    D225G is only present in about 1% of the more than 2000 public HA sequences, yet was in four of four fatal cases in western Ukraine. In addition to these four sequneces, Mill Hill generated 5 other sequences fro survivors and althoug the sub-clade was the same, the milder cases had wild type 225. This result led other European countries to look at cases with D225G and a similar association with fatal or severe disease was noted. Norway had 3 cases – two had died and one was severe. France had two cases and both were fatal. This association was also supported by CDC data on patients in western Ukraine. They deposited data from 5 patients. 3 matched Mill Hill in sample number, demographics, and sequence from mild cases, while two were unique and almost certainly fatal cases and both had D225N. In the US, one patient had both D225G and D225N and that case (28F) was also fatal.
    Earlier sequences from the summer gave similar results (in Brazil 2/2 fatal cases with D225G and 2/2 fatal cases with D225N, while in China 1/1 severe cases with D225G).
    Last spring there were mild cases in the US, including California/7, which had D225G and wild type mixed signals, but when a virus jumps species and there is little or no protective immunity, many infections can be cause by low levels of virus, which are mild just based on viral load, and of course the VAST majority of H1N1 remain mild with infected individuals recovering with no treatment.
    Thus, although correlations of H1N1 genetic chnages with clinical presentation, the receptor binding domain chages at position 225 provide some rather compelling data linking D225G and D225N to fatal or severe disease, which was rather blatantly mispresented in teh broadcast as well as “grain of salt” comments.
    Canada now presents “low reactor” isolates with a mention of D225G, and the CDC is actively analyzing samples with D225G, so some clearly take exception with the “grain of salt” representation.

  • Rabadan

    Dear Dr Niman and other TWiV friends,

    Influenza, like many other pathogens, is a complex disease where a multiplicity of factors are thought to play an important role in its severity, including the genetics of the pathogen, the genetics of the host, previous exposures to similar pathogens in the past (the history of the host), co-infections with other pathogens, environmental factors, the presence of other preexisting health conditions, the age of the patient, the quality of the health care system, the climate (temperature and humidity), etc. The association of any of these factors to the phenotype (severity or lethality) rely on the selection of an appropriate cohort and a control set with similar characteristics, and a statistical evaluation of the significance of the results. Since 1918, many clinicians have been trying to disentangle these factors in influenza, but it has been proven to be a very complicated task. In particular, in the case at hand, the numbers are low, and there is not a well defined control group that could eliminate other factors. Most of the severe cases did not have the D225G mutation, and other factors like age, pregnancy and coinfections have been reported to play a major role in the severity of the disease.

    Unfortunately, it has not been uncommon for a study to report an association that could not be replicated by other groups. Some of these false associations were mentioned in TWiV, one of which is the link between vaccines and autism that have had a very negative effect in public health issues. Even with the best designed cohorts and the most competent scientists, there are unknown a priori factors that, a posteriori, could explain false associations.

    As Dr Niman is doing, one should always keep an alert eye on the evolution of this virus. However, I also believe, based on the existence of many previous unconfirmed associations (probably Prof Racaniello would agree with me), that caution is advisable.

    Raul Rabadan, PhD

    Assistant Professor
    Department of Biomedical Informatics
    Center for Computational Biology and Bioinformatics

    Columbia University College of Physicians and Surgeons
    1130 St. Nicholas Ave
    ICRC Bldg 8th Floor, Office 803B
    New York, NY 10032

  • erumkhan

    Hi, I listen to your discussions very regularly and its a real treat for scientists like me who are sitting at the end of world where experts in virology are virtually none. I truly appreciate the fantastic service you are providing to your fellow colleuges who would otherwise be left in isolation. Thanks a million. Erum from Pakistan

  • niman

    Although H1N1 is a very hetogeneous disease, the linkage of D225G and D225N to fatal cases continues to build. HA sequences from 10 fatal cases in Mexico were just released at Genbank, and 5 patients had D225G, D255N or both. Thus, position 225 changes to G or N have been reported in five cases in Mexico since this summer, and all 5 cases were fatal. Moreover, the two patients (25M and 40M) with both D225G and D225N were from San Luis Potosi and samples were collected within one day of each, once again signaling transmission. Thus, to date there have been 3 HA sequences from humans with D225G and D225N, and all three were fatal (the other case, 28F, died July 28 in Utah).

    Also of note were sequences from a swine outbreak in Queretaro, which is adjacent to San Luis Potosi. The HA sequence also had D225G and D225N as well as Q226R. All three positions were mixed signals, as were the fatal cases above which had mixed siganls at two poitions in the 225 codon (and the other three fatalities in Mexico also had mixed signals at codon 225.

    The swine outbreak was reported in December, but originated in April, once again highlighting limitations in the swine sequence database and abysmal surveillance. To my knowledge the swine sequence was the only public H1N1 sequence from swine in Mexico and it remains a candidate for the sequence circulating in swine prior to the jump to humans.

    The mixed signals for all position 225 chnages in the six isolates from Mexico also highlight potential differences in ratios of codon 225 polymorphisms, which can vary by collection site, time, and isolation procedure.

  • niman

    Although H1N1 is a very hetogeneous disease, the linkage of D225G and D225N to fatal cases continues to build. HA sequences from 10 fatal cases in Mexico were just released at Genbank, and 5 patients had D225G, D255N or both. Thus, position 225 changes to G or N have been reported in five cases in Mexico since this summer, and all 5 cases were fatal. Moreover, the two patients (25M and 40M) with both D225G and D225N were from San Luis Potosi and samples were collected within one day of each, once again signaling transmission. Thus, to date there have been 3 HA sequences from humans with D225G and D225N, and all three were fatal (the other case, 28F, died July 28 in Utah).

    Also of note were sequences from a swine outbreak in Queretaro, which is adjacent to San Luis Potosi. The HA sequence also had D225G and D225N as well as Q226R. All three positions were mixed signals, as were the fatal cases above which had mixed siganls at two poitions in the 225 codon (and the other three fatalities in Mexico also had mixed signals at codon 225.

    The swine outbreak was reported in December, but originated in April, once again highlighting limitations in the swine sequence database and abysmal surveillance. To my knowledge the swine sequence was the only public H1N1 sequence from swine in Mexico and it remains a candidate for the sequence circulating in swine prior to the jump to humans.

    The mixed signals for all position 225 chnages in the six isolates from Mexico also highlight potential differences in ratios of codon 225 polymorphisms, which can vary by collection site, time, and isolation procedure.