A Computational-Experimental Approach Identifies Mutations That Enhance Surface Expression of an Oseltamivir-Resistant Influenza Neuraminidase

The His274 → Tyr (H274Y) oseltamivir (Tamiflu) resistance mutation causes a substantial decrease in the total levels of surface-expressed neuraminidase protein and activity in early isolates of human seasonal H1N1 influenza, and in the swine-origin pandemic H1N1. In seasonal H1N1, H274Y only became widespread after the occurrence of secondary mutations that counteracted this decrease. H274Y is currently rare in pandemic H1N1, and it remains unclear whether secondary mutations exist that might similarly counteract the decreased neuraminidase surface expression associated with this resistance mutation in pandemic H1N1. Here we investigate the possibility of predicting such secondary mutations. We first test the ability of several computational approaches to retrospectively identify the secondary mutations that enhanced levels of surface-expressed neuraminidase protein and activity in seasonal H1N1 shortly before the emergence of oseltamivir resistance. We then use the most successful computational approach to predict a set of candidate secondary mutations to the pandemic H1N1 neuraminidase. We experimentally screen these mutations, and find that several of them do indeed partially counteract the decrease in neuraminidase surface expression caused by H274Y. Two of the secondary mutations together restore surface-expressed neuraminidase activity to wildtype levels, and also eliminate the very slight decrease in viral growth in tissue-culture caused by H274Y. Our work therefore demonstrates a combined computational-experimental approach for identifying mutations that enhance neuraminidase surface expression, and describes several specific mutations with the potential to be of relevance to the spread of oseltamivir resistance in pandemic H1N1

Multiple reassortment events in the evolutionary history of H1N1 influenza A virus since 1918

The H1N1 subtype of influenza A virus has caused substantial morbidity and mortality in humans, first documented in the global pandemic of 1918 and continuing to the present day. Despite this disease burden, the evolutionary history of the A/H1N1 virus is not well understood, particularly whether there is a virological basis for several notable epidemics of unusual severity in the 1940s and 1950s. Using a data set of 71 representative complete genome sequences sampled between 1918 and 2006, we show that segmental reassortment has played an important role in the genomic evolution of A/H1N1 since 1918. Specifically, we demonstrate that an A/H1N1 isolate from the 1947 epidemic acquired novel PB2 and HA genes through intra-subtype reassortment, which may explain the abrupt antigenic evolution of this virus. Similarly, the 1951 influenza epidemic may also have been associated with reassortant A/H1N1 viruses. Intra-subtype reassortment therefore appears to be a more important process in the evolution and epidemiology of H1N1 influenza A virus than previously realized

Respiratory and systematic humoral and cellular immune response of pigs to a heterosubtypic influenza A virus infection

The level of heterosubtypic immunity (Het-I) and the immune mechanisms stimulated by a heterosubtypic influenza virus infection were investigated in pigs. Pigs are natural hosts for influenza virus and, like humans, they host both subtypes H1N1 and H3N2. Marked Het-I was observed when pigs were infected with H1N1 and subsequently challenged with H3N2. After challenge with H3N2, pigs infected earlier with H1N1 did not develop fever and showed reduced virus excretion compared with non-immune control pigs. In addition, virus transmission to unchallenged group-mates could be shown by virus isolation in the non-immune control group but not in the group infected previously with H1N1. Pigs infected previously with homologous H3N2 virus were protected completely. After challenge with H3N2, pigs infected previously with H1N1 showed a considerable increase in serum IgG titre to the conserved extracellular domain of M2 but not to the conserved nucleoprotein. These results suggest that antibodies against external conserved epitopes can have an important role in broad-spectrum immunity. After primary infection with both H1N1 and H3N2, a long-lived increase was observed in the percentage of CD8 T cells in the lungs and in the lymphoproliferation response in the blood. Upon challenge with H3N2, pigs infected previously with H1N1 again showed an increase in the percentage of CD8 T cells in the lungs, whereas pigs infected previously with H3N2 did not, suggesting that CD8 T cells also have a role in Het-I. To confer broad-spectrum immunity, future vaccines should induce antibodies and CD8 T cells against conserved antigens

Cross-protection between antigenically distinct H1N1 swine influenza viruses from Europe and North America

Background An avian-like H1N1 swine influenza virus (SIV) is enzootic in swine populations of Western Europe. The virus is antigenically distinct from H1N1 SIVs in North America that have a classical swine virus-lineage H1 hemagglutinin, as does the pandemic (H1N1) 2009 virus. However, the significance of this antigenic difference for cross-protection among pigs remains unknown. Objectives We examined protection against infection with a North American triple reassortant H1N1 SIV [A/swine/Iowa/H04YS2/04 (sw/IA/04)] in pigs infected with a European avian-like SIV [A/swine/Belgium/1/98 (sw/B/98)] 4 weeks earlier. We also examined the genetic relationships and serologic cross-reactivity between both SIVs and with a pandemic (H1N1) 2009 virus [A/California/04/09 (Calif/09)]. Results After intranasal inoculation with sw/IA/04, all previously uninfected control pigs showed nasal virus excretion, high virus titers in the entire respiratory tract at 4 days post-challenge (DPCh) and macroscopic lung lesions. Most pigs previously infected with sw/B/98 tested negative for sw/IA/04 in nasal swabs and respiratory tissues, and none had lung lesions. At challenge, these pigs had low levels of cross-reactive virus neutralizing and neuraminidase inhibiting (NI) antibodies to sw/IA/04, but no hemagglutination-inhibiting antibodies. They showed similar antibody profiles when tested against Calif/09, but NI antibody titers were higher against Calif/09 than sw/IA/04, reflecting the higher genetic homology of the sw/B/98 neuraminidase with Calif/09. Conclusions Our data indicate that immunity induced by infection with European avian-like H1N1 SIV affords protection for pigs against North American H1N1 SIVs with a classical H1, and they suggest cross-protection against the pandemic (H1N1) 2009 virus

Health-Care Providers' Preparedness for H1N1/09 Influenza Prevention and Treatment in Dar es Salaam, Tanzania.

\ud In Tanzania, the first case of H1N1/09 influenza was reported in September 2009. By March 2010, the reported number of cases was 770 with one death. Due to shortage of qualified human resources, essential medicines and laboratory supplies in health facilities, it was not known how well health-care providers in the country were prepared to deal with the pandemic. The study was conducted from December 2009 to May 2010 in public hospitals and private community pharmacies in Dar es Salaam, Tanzania. Fifty-three prescribers and 200 dispensers were interviewed to assess their knowledge on the prevention and treatment of H1N1/09 influenza, and dissemination of correct information to the public regarding the pandemic. Most participants had inadequate knowledge on the transmission, prevention and management of H1N1/09 influenza. The majority of prescribers knew neither the antiviral drugs recommended for the treatment of H1N1 influenza (64%) nor their adverse effects (92.5%). The drug dispensers' knowledge was also inadequate regarding the transmission, prevention and treatment of H1N1/09 influenza. Forty-one (20.5%) salespersons without formal training in pharmaceutical or medical sciences were found dispensing and supervising the pharmacies. Most participants had misconceptions on how H1N1/09 virus was transmitted. The results of the study revealed gaps in the knowledge of the health-care providers on the prevention and treatment of H1N1/09 influenza infection. This indicates the need for strengthening of health-care systems and provision of continuing education and professional development for health-care providers on new and re-emerging diseases in the community.\u

Prediction of specific virus outbreaks made from the increased concentration of a new class of virus genomic peptides, replikins.

Advance warning of pathogen outbreaks has not been possible heretofore. A new class of genomic peptides associated with rapid replication was discovered and named replikins. Software was designed to analyze replikins quantitatively. Replikin concentration changes were measured annually prior to, and “real time” every few days during, the 2009 H1N1 influenza pandemic. Replikins were seen by both linear sequence representation and three-dimensional X-ray diffraction, and found to expand on the virus hemagglutinin surface prior to and during the H1N1 pandemic.

A highly significant increased concentration of virus replikins was found a) retrospectively in three pandemics from 1918 to 1999 (14,227 sequences)(p<0.001), and b) prospectively before the H1N1 2009 pandemic (12,806 sequences) (in the hemagglutinin gene (N=8,046), p values by t-test = 1/10130, by linear regression = 1/1024 and 1/1029, by Spearman correlation < 2/1016, by Wilcoxon rank sum<1/1016, by multiple regression adjusting for correlation between consecutive years = 2/1022. Rising replikin concentration in H1N1 from 2006 to 2008, predicted one year in advance the H1N1 outbreak of 2009; and in H5N1, predicted the lethal outbreaks of H5N1 1997-2010. 

The possible combination of influenza strains H1N1 (high infectivity) and H5N1 (high lethality) is a matter of global concern (1,2). The risk of a combined H1N1 (high infectivity) - H5N1 (high lethality) outbreak may have increased because first, the Replikin Counts of the two virus strains have risen simultaneously, not seen previously; second, the rise is to the highest levels recorded since 1918 for H1N1, in Mexico (16.7), and since 1957 for H5N1, in Egypt (23.3); and third, clinical outbreaks of each strain are occurring in 2011. These simultaneous conditions may increase the risk that the two virus strains might come into contact with each other more frequently, facilitating transfer of genomic material to form a hybrid

A Novel Sequence-Based Antigenic Distance Measure for H1N1, with Application to Vaccine Effectiveness and the Selection of Vaccine Strains

H1N1 influenza causes substantial seasonal illness and was the subtype of the 2009 influenza pandemic. Precise measures of antigenic distance between the vaccine and circulating virus strains help researchers design influenza vaccines with high vaccine effectiveness. We here introduce a sequence-based method to predict vaccine effectiveness in humans. Historical epidemiological data show that this sequence-based method is as predictive of vaccine effectiveness as hemagglutination inhibition (HI) assay data from ferret animal model studies. Interestingly, the expected vaccine effectiveness is greater against H1N1 than H3N2, suggesting a stronger immune response against H1N1 than H3N2. The evolution rate of hemagglutinin in H1N1 is also shown to be greater than that in H3N2, presumably due to greater immune selection pressure.Comment: 26 pages, 7 figures, 2 tables, supplemen

Pandemic (H1N1) 2009 Cluster Analysis: A Preliminary Assessment

Pandemic (H1N1) 2009 virus has been causing major concerns around the world because of its epidemic potential, rapid dissemination, rate of mutations, and the number of fatalities. One way to gain an advantage over this virus is to use existing rapid bioinformatics tools to examine easily and inexpensively generated genetic sequencing data. We have used the protein sequences deposited with the National Center for Biotechnology Information (NCBI) for data mining to study the relationship among the Pandemic (H1N1) 2009 proteins. There are 11 proteins in the Pandemic (H1N1) 2009 virus, and analysis of sequences from 65 different locations around the globe has resulted in two major clusters. These clusters illustrate the Pandemic H1N1 2009 virus is already experiencing significant genetic drift and that rapid worldwide travel is affecting the distribution of genetically distinct isolates

Unusual association of ST-T abnormalities, myocarditis and cardiomyopathy with H1N1 influenza in pregnancy: two case reports and review of the literature.

Introduction Myocarditis is rarely reported as an extra-pulmonary manifestation of influenza while pregnancy is a rare cause of cardiomyopathy. Pregnancy was identified as a major risk factor for increased mortality and morbidity due to H1N1 influenza in the pandemic of 2009 to 2010. However, to the best of our knowledge there are no previous reports in the literature linking H1N1 with myocarditis in pregnancy. Case presentation We report the cases of two pregnant Caucasian women (aged 29 and 30), with no pre-existing illness, presenting with respiratory manifestations of H1N1 influenza virus infection in their third trimester. Both women developed evidence of myocarditis. One woman developed acute respiratory distress syndrome, almost reaching the point of requiring extra-corporeal membrane oxygenation, and subsequently developed persistent cardiomyopathy; the other recovered without any long-term consequence. Conclusions While it is not possible to ascertain retrospectively if myocarditis was caused by either infection with H1N1 virus or as a result of pregnancy (in the absence of endomyocardial biopsies), the significant association with myocardial involvement in both women demonstrates the increased risk of exposure to H1N1 influenza virus in pregnant women. This highlights the need for health care providers to increase awareness amongst caregivers to target this 'at risk' group aggressively with vaccination and prompt treatment