Cold‐Adapted Reassortants of Influenza A Virus in MDCK Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/101846/1/mim03574.pd

Cold-adapted reassortants of influenza A virus: Pathogenicity of A/Ann Arbor/6/60×A/Alaska/6/77 reassortant viruses In vivo and In vitro

Cold-adapted reassortants of A/Ann Arbor/6/60×A/Alaska/6/77 viruses made in MDCK cells have recently been assessed genotypically and for temperature-sensitive and cold-adapted phenotypes. These reassortants were used to infect ferrets and hamsters and to inoculate organ cultures of hamster tracheal rings, in order to assess their degree of virulence. Virulence in the three model systems corresponded quite well, and a correlation between loss of virulence and particular A/AA/6/60 genes present in the reassortants was noted. Two different reassortants containing either RNA 2 or RNA 5 (NA gene) alone from A/AA/6/60 showed little attenuation from the wild-type parent. A reassortant containing both RNA2 and the NA gene from A/AA/6/60 and all remaining wild-type genes showed some small decrease in virulence compared to the wild-type virus. However a reassortant containing these two A/AA/6/60 genes and RNA 3 as an additional gene from this parent, had a level of attenuation comparable to that of the cold-adapted virus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41693/1/705_2005_Article_BF01316727.pd

Susceptibility of Influenza Viruses to the Novel Cap-Dependent Endonuclease Inhibitor Baloxavir Marboxil

The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza A and B virus infections in February 2018 in Japan. Because of the need to monitor influenza viruses for reduced susceptibility to this drug, we used two cell-based screening systems – a conventional plaque reduction assay and a focus reduction assay – to evaluate the susceptibility of influenza viruses to baloxavir. First, we generated a reference virus possessing an I38T substitution in the polymerase acidic subunit (PA), which is known to be associated with reduced susceptibility to baloxavir, and demonstrated the validity of our systems using this reference virus. We then determined the susceptibility of a panel of neuraminidase (NA) inhibitor-resistant viruses and their sensitive counterparts to baloxavir. No significant differences in baloxavir susceptibilities were found between the NA inhibitor-resistant and -sensitive viruses. We also examined seasonal influenza viruses isolated during the 2017–2018 influenza season in Japan and found that no currently circulating A(H1N1)pdm09, A(H3N2), or B viruses had significantly reduced susceptibility to baloxavir and none of the viruses possessed an amino acid substitution at PA residue 38. Use of a combination of methods to analyze antiviral susceptibility and detect amino acid substitutions is valuable for monitoring the emergence of baloxavir-resistant viruses

A humanized mouse model identifies key amino acids for low immunogenicity of H7N9 vaccines

Influenza vaccines of H7N9 subtype are consistently less immunogenic in humans than vaccines developed for other subtypes. Although prior immunoinformatic analysis identified T-cell epitopes in H7 hemagglutinin (HA) which potentially enhance regulatory T cell response due to conservation with the human genome, the links between the T-cell epitopes and low immunogenicity of H7 HA remains unknown due to the lack of animal models reproducing the response observed in humans. Here, we utilized a humanized mouse model to recapitulate the low immunogenicity of H7 HA. Our analysis demonstrated that modification of a single H7 epitope by changing 3 amino acids so that it is homologous with a known H3 immunogenic epitope sequence significantly improved the immunogenicity of the H7 HA in the humanized mouse model, leading to a greater than 4-fold increase in HA-binding IgG responses. Thus, we provide experimental evidence for the important contribution of this H7-specific T cell epitope in determining the immunogenicity of an influenza vaccine. Furthermore, this study delineates strategies that can be used for screening and selecting vaccine strains using immunoinformatics tools and a humanized mouse model

Influenza B associated paediatric acute respiratory infection hospitalization in central vietnam

Background: Influenza B is one of the major etiologies for acute respiratory infections (ARI) among children worldwide; however, its clinical-epidemiological information is limited. We aimed to investigate the hospitalization incidence and clinical-epidemiological characteristics of influenza B-associated paediatric ARIs in central Vietnam. Methods: We collected clinical-epidemiological information and nasopharyngeal swabs from ARI children hospitalized at Khanh Hoa General Hospital, Nha Trang, Vietnam from February 2007 through June 2013. Nasopharyngeal samples were screened for 13 respiratory viruses using Multiplex-PCRs. Influenza B-confirmed cases were genotyped by Haemagglutinin gene sequencing. We analyzed the clinical-epidemiological characteristics of influenza B Lineages (Victoria/Yamagata) and WHO Groups. Results: In the pre-A/H1N1pdm09 period, influenza B-associated ARI hospitalization incidence among children under five was low, ranging between 14.7 and 80.7 per 100 000 population. The incidence increased to between 51.4 and 330 in the post-A/H1N1pdm09. Influenza B ARI cases were slightly older with milder symptoms. Both Victoria and Yamagata lineages were detected before the A/H1N1pdm09 outbreak; however, Victoria lineage became predominant in 2010-2013 (84% Victoria vs 16% Yamagata).Victoria and Yamagata lineages did not differ in demographic and clinical characteristics. In Victoria lineage, Group1 ARI cases were clinically more severe compared to Group5, presenting a greater proportion of wheeze, tachypnea, and lower respiratory tract infection. Conclusions: The current results highlight the increased incidence of influenza B-related ARI hospitalization among children in central Vietnam in the post-A/H1N1pdm09 era. Furthermore,the difference in clinical severity between Victoria lineage Group1 and 5 implies the importance of influenza B genetic variation on clinical presentation

Oseltamivir-Resistant Influenza Viruses A (H1N1) during 2007–2009 Influenza Seasons, Japan

Prevalence of these viruses increased during the 2008–09 season

Selection of antigenically advanced variants of seasonal influenza viruses.

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.This work was supported by the Bill & Melinda Gates Foundation Global Health Grant OPPGH5383; National Institute of Allergy and Infectious Diseases (NIAID) Public Health Service research grants (USA); ERATO (Japan Science and Technology Agency); the Center for Research on Influenza Pathogenesis (CRIP) funded by the NIAID Contracts HHSN266200700010C and HHSN27 2201400008C; the Japan Initiative for Global Research Network on Infectious Diseases; Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; Grants-in-Aid from the Ministry of Health, Labour and Welfare, Japan; grants from the Strategic Basic Research Program of the Japan Science and Technology Agency; and by the Advanced Research & Development Programs for Medical Innovation from the Japan Agency for Medical Research and Development (AMED). C.A.R. was supported by a University Research Fellowship from the Royal Society. The authors acknowledge a Netherlands Organisation for Scientific Research (NWO) VICI grant, European Union (EU) FP7 programs EMPERIE (223498) and ANTIGONE (278976); Human Frontier Science Program (HFSP) program grant P0050/2008; Wellcome 087982AIA; and NIH Director's Pioneer Award DP1-OD000490-01. D.F.B and D.J.S. acknowledge CamGrid, the University of Cambridge distributed computer system. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nmicrobiol.2016.5

Molecular Evolutionary Analysis of the Influenza A(H1N1)pdm, May–September, 2009: Temporal and Spatial Spreading Profile of the Viruses in Japan

BACKGROUND: In March 2009, pandemic influenza A(H1N1) (A(H1N1)pdm) emerged in Mexico and the United States. In Japan, since the first outbreak of A(H1N1)pdm in Osaka and Hyogo Prefectures occurred in the middle of May 2009, the virus had spread over 16 of 47 prefectures as of June 4, 2009. METHODS/PRINCIPAL FINDINGS: We analyzed all-segment concatenated genome sequences of 75 isolates of A(H1N1)pdm viruses in Japan, and compared them with 163 full-genome sequences in the world. Two analyzing methods, distance-based and Bayesian coalescent MCMC inferences were adopted to elucidate an evolutionary relationship of the viruses in the world and Japan. Regardless of the method, the viruses in the world were classified into four distinct clusters with a few exceptions. Cluster 1 was originated earlier than cluster 2, while cluster 2 was more widely spread around the world. The other two clusters (clusters 1.2 and 1.3) were suggested to be distinct reassortants with different types of segment assortments. The viruses in Japan seemed to be a multiple origin, which were derived from approximately 28 transported cases. Twelve cases were associated with monophyletic groups consisting of Japanese viruses, which were referred to as micro-clade. While most of the micro-clades belonged to the cluster 2, the clade of the first cases of infection in Japan originated from cluster 1.2. Micro-clades of Osaka/Kobe and the Fukuoka cases, both of which were school-wide outbreaks, were eradicated. Time of most recent common ancestor (tMRCA) for each micro-clade demonstrated that some distinct viruses were transmitted in Japan between late May and early June, 2009, and appeared to spread nation-wide throughout summer. CONCLUSIONS: Our results suggest that many viruses were transmitted from abroad in late May 2009 irrespective of preventive actions against the pandemic influenza, and that the influenza A(H1N1)pdm had become a pandemic stage in June 2009 in Japan