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We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells., Competing Interests: Declaration of competing interest Takao Fujimoto, Fumiaki Horikoshi, and Shuhei Chida were employees of BIKEN Co., Ltd., Kenji Tanaka is an employee of Daiichi Sankyo Biotech Co., Ltd., Kenji Minari, and Yoshimi Tanimoto are employees of Takeda Pharmaceutical Company Limited., The other coauthors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

In Japan, the Ministry of Health, Labour and Welfare (MHLW) designates one specific virus strain for each component of the quadrivalent seasonal influenza vaccine, and four domestic manufacturers produce egg-based influenza vaccines with the same formulation (inactivated, split-virus) using uniform vaccine strains. Thus, discussions of the development of effective seasonal influenza vaccines so far has focused solely on the antigenic match between the vaccine strains and epidemic viruses. However, in 2017, the Japanese selection system of vaccine viruses demonstrated that even a candidate vaccine virus that is antigenically similar to the predicted circulating viruses is not necessarily suitable for vaccine production, given lower productivity of the vaccine. Taking this experience into account, the MHLW reformed the scheme of vaccine strain selection in 2018, and instructed the Vaccine Epidemiology Research Group created by the MHLW to probe how the virus strains for the seasonal influenza vaccine should be selected in Japan. In this context, a symposium, entitled "Issues of the Present Seasonal Influenza Vaccines and Future Prospects", was held as part of the 22nd Annual Meeting of the Japanese Society for Vaccinology in 2018, and subjects related to the influenza vaccine viruses were discussed among relevant administrators, manufacturers, and researchers. This report summarizes the presentations given at that symposium in order to convey the present scheme of vaccine virus selection, the evaluation of the resulting vaccines, and the efforts at new vaccine formulation in Japan. Notably, from March 2022, the MHLW has launched a discussion of the merits of the seasonal influenza vaccines produced by foreign manufacturers., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Takao Hozawa is an employee of Denka Co., Ltd. in Tokyo, Japan. All other authors declare that they are free of competing interests.’, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Manufactured influenza vaccines have to contain a defined amount of hemagglutinin (HA) antigen. Therefore, vaccine viruses with a high HA antigen yield (HAY) are preferable for manufacturing vaccines, particularly vaccines in response to a pandemic, when vaccines need to be rapidly produced. However, the viral properties associated with a high HAY have not yet been fully clarified. To identify the HAY-associated traits, we first propagated 26 H5 candidate vaccine viruses (CVVs) in eggs, which were previously developed based on genetic reassortment methods using master viruses, to determine their total protein yield (TPY), ratio of HA to total viral protein (%-HA content) and HAY. The results revealed that the HAY was correlated with the TPY but not with the %-HA content. We further found that altering the sequences of the 3' noncoding region of HA vRNA or replacing the master virus improved the HAYs and TPYs of the low-HAY CVVs to approximately double the values of the original CVVs but did not change the %-HA content, which a previous study suggested was associated with the HAY. Analyses based on real-time PCR assays and scanning electron microscopy revealed that the virus samples with an improved HAY contained more copies of the virus genome and viral particles than the original samples. The results suggest that an improvement in virus growth (i.e., an increase in the amount of viral particles) leads to an increase in the TPY and thus in the HAY, regardless of the %-HA content. The approximately twofold increase in the HAY shown in this study may not appear to represent a large improvement, but the impact will be significant given the millions of chicken eggs used to produce vaccines. These findings will be informative for developing high-HAY vaccine viruses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Arita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 10 8 copies, declined to 10 2 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses., (© 2022 The Societies and John Wiley & Sons Australia, Ltd.)

In Japan, the practical application of completely cell-based seasonal influenza vaccines is under consideration. Considering the good correlation between the immunogenicity of egg-based influenza vaccines and the hemagglutinin (HA) content determined by the single radial immunodiffusion (SRD) assay, we determined the potency of the first cell-based quadrivalent vaccine experimentally generated in Japan using the SRD assay in this study. A primary liquid standard (PLS) and reference antigen were generated from the purified vaccine virus, and a sheep antiserum was produced against the HA of the vaccine virus. Since the purity of the PLS affects the reliability of vaccine potency testing, the purification steps are significant. We successfully prepared a purified PLS nearly free of cell debris. The HA content in the PLS was first estimated from the total amount of viral protein and the percentage of HA content determined by SDS-PAGE analysis. The HA content in the reference antigen was calibrated to that in the PLS via the SRD assay. The vaccine potency, that is, the HA content in each vaccine, was finally measured using the corresponding reference antigen. Ultimately, the measured vaccine potency of the monovalent vaccine was similar to that of the quadrivalent vaccine., (Copyright © 2020. Published by Elsevier Ltd.)

The H1N1 influenza pandemic vaccine has been developed from the A/California/07/09 (Cal) virus and the well-known high-yield A/Puerto Rico/8/34 (PR8) virus by classical reassortment and reverse genetics (RG) in eggs. Previous studies have suggested that Cal-derived chimeric hemagglutinin (HA) and neuraminidase (NA) improve virus yields. However, the cell-based vaccine of the H1N1 pandemic virus has been less investigated. RG viruses that contained Cal-derived chimeric HA and NA could be rescued in Madin-Darby canine kidney cells that expressed α2,6-sialyltransferase (MDCK-SIAT1). The viral growth kinetics and chimeric HA and NA properties were analyzed. We attempted to generate various RG viruses that contained Cal-derived chimeric HA and NA, but half of them could not be rescued in MDCK-SIAT1 cells. When both the 3'- and 5'-terminal regions of Cal HA viral RNA were replaced with the corresponding regions of PR8 HA, the RG viruses were rescued. Our results were largely consistent with those of previous studies, in which the N- and C-terminal chimeric HA slightly improved virus yield. Importantly, the chimeric HA, compared to Cal HA, showed cell fusion ability at a broader pH range, likely due to amino acid substitutions in the transmembrane region of HA. The rescued RG virus with high virus yield harbored the chimeric HA capable of cell fusion at a broader range of pH.

Antibodies against influenza virus neuraminidase (NA) protein prevent releasing of the virus from host cells and spreading of infection foci and are considered the 'second line of defence' against influenza. Haemagglutinin inhibition antibody-low responders (HI-LRs) are present among influenza split vaccine recipients. The NA inhibition (NAI) antibody response in vaccinees is worth exploring, especially those in the HI-LRs population. We collected pre- and post-vaccination sera from 61 recipients of an inactivated, monovalent, split vaccine against A/H1N1pdm09 and acute and convalescent sera from 49 unvaccinated patients naturally infected with the A/H1N1pdm09 virus during the 2009 influenza pandemic. All samples were subjected to haemagglutinin inhibition (HI), NAI and neutralisation assays. Most paired sera from naturally infected patients exhibited marked elevation in the NAI activity, and seroconversion rates (SCR) among HI-LRs and HI-responders (HI-Rs) were 60% and 87%, respectively; however, those from vaccinees displayed low increase in the NAI activity, and the SCR among HI-LRs and HI-Rs were 0% and 12%, respectively. In both HI-LRs and HI-Rs, vaccination with the inactivated, monovalent, split vaccine failed to elicit the NAI activity efficiently in the sera of the naive population, compared with the natural infection. Hence, the improvement of influenza vaccines is warranted to elicit not only HI but also NAI antibodies., Competing Interests: The authors have declared that no competing interests exist.

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.

Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production. IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines., (Copyright © 2017 American Society for Microbiology.)

In 2013, the first case of human infection with an avian influenza A virus (H7N9) was reported in China, and the human infection with this virus has continued as of 2016. At the request of the WHO, we have successfully developed candidate reassortant vaccine virus using A/Anhui/1/2013 and the high egg-growth master virus A/PR/8/1934. Recent plans regarding influenza vaccine production include using cell-cultured systems in Japan and several other countries. However, egg-based vaccine viruses are not always suitable for cell-cultured vaccine production due to potential issues with growth, protein yield and antigenic stability. Therefore, in this study, we have developed a high-growth master virus (hg-PR8) adapted to qualified NIID-MDCK cells that are competent for vaccine production. The virus hg-PR8 was obtained after 20 serial passages of A/Puerto Rico/8/1934 (PR8) in NIID-MDCK cells. The viral titer of hg-PR8 was 108.6 plaque-forming units per milliliter (PFU/mL). Seven amino acid substitutions were identified in the amino acid sequences of PB2, PB1, PA, NA, M and NS of hg-PR8 compared to the sequence of the original PR8 (org-PR8) strain. The growth capacities of the reassortant viruses, which possess heterologous internal genes from hg-PR8 or org-PR8, indicated that the amino acid changes in PB2 and NS2 similarly affected growth capacity in NIID-MDCK cells. To assess the suitability of hg-PR8 as a master virus, we generated 6:2 reassortant viruses possessing the HA and NA segments from A/Anhui/1/2013 (H7N9) and the remaining segments from hg-PR8. The virus titers of the reassortant strains were 107-108 PFU/mL. The antigenicity of the viruses was stable during ten passages of the viruses in NIID-MDCK cells. In comparison with the egg-based reassortant vaccine viruses with identical HA and NA segments, the hg-PR8-based viruses showed 1.5- to 2-fold higher protein yields in NIID-MDCK cells.

In April 2013, the first three fatal cases of human infection with an avian influenza A virus (H7N9) were reported in China. Because of a pandemic threat by this virus, we have commenced to develop candidate vaccine viruses (CVVs). Three 6:2 genetic reassortant viruses with different hemagglutinin (HA) sequences, NIIDRG-10, -10.1 and -10.2, were generated by a reverse genetics technique between the high egg-growth master virus, A/Puerto Rico/8/34 (H1N1) and A/Anhui/1/2013 (H7N9), kindly provided by the Chinese Center for Disease Control and Prevention. The different HA gene sequences of the three CVVs were derived from the original virus stock. NIIDRG-10 possesses HA, whose sequence is identical to that of the original A/Anhui/1/2013 (H7N9) in the Global Initiative on Sharing Avian Influenza Data (EPI439507), while NIIDRG-10.1 and -10.2 possess amino acid differences, A125T and N123D/N149D, respectively, compared with NIIDRG-10. NIIDRG-10 replicated in embryonated chicken eggs with low hemagglutination titer 128, whereas NIIDRG-10.1 and -10.2 grew well with hemagglutination titer 1024. These viruses reacted well with a ferret antiserum raised against the original A/Anhui/1/2013 virus. Ferret antiserum against NIIDRG-10.1 reacted well with A/Anhui/1/2013 similar to the homologous virus NIIDRG-10.1. These results indicated that NIIDRG-10.1 passed the two-way test of antigenic identity. In contrast, the ferret antiserum against NIIDRG-10.2 reacted with A/Anhui/1/2013 at an 8-fold lower hemagglutination inhibition titer than with the homologous virus NIIDRG-10.2, indicating an antigenic change. The total and HA protein yields of NIIDRG-10.1 were 14.7 and 6.9 μg/ml, respectively, similar to those levels of high-yield seed viruses of seasonal influenza vaccines. NIIDRG-10.1 was approved as one of the CVVs for H7N9 viruses by the WHO in 2013. The candidate vaccine derived from NIIDRG-10.1 is currently being evaluated in a phase II clinical study in Japan., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A/Narita/1/2009 (A/N) was the first H1N1 virus from the 2009 pandemic (H1pdm) to be isolated in Japan. To better understand and predict the possible development of this virus strain, the effect of passaging A/N was investigated in Madin-Darby canine kidney cells, chicken eggs and mice. A/N that had been continuously passaged in cells, eggs, or mice obtained the ability to grow efficiently in each host. Moreover, A/N grown in mice had both a high level of pathogenicity in mice and an increased growth rate in cells and eggs. Changes in growth and pathogenicity were accompanied by amino acid substitutions in viral hemagglutinin (HA) and PB2. In addition, the adapted viruses exhibited a reduced ability to react with ferret antisera against A/N. In conclusion, prolonged passaging allowed influenza A/N to adapt to different hosts, as indicated by a high increase in proliferative capacity that was accompanied by an antigenic alteration leading to amino acid substitutions.

Unlabelled: We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain., Importance: The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2+/+ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with ≥1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo., Importance: Influenza A virus (IAV) is a leading pathogen that infects and kills many humans every year. We clarified that the infectivity and pathogenicity of IAVs, including a recently emerged H7N9 subtype, are determined primarily by a host protease, TMPRSS2. Our data showed that TMPRSS2 is the key host protease that activates IAVs in vivo through proteolytic cleavage of their HA proteins. Hence, TMPRSS2 is a good target for the development of anti-IAV drugs. Such drugs could also be effective for many other respiratory viruses, including the recently emerged Middle East respiratory syndrome (MERS) coronavirus, because they are also activated by TMPRSS2 in vitro. Consequently, the present paper could have a large impact on the battle against respiratory virus infections and contribute greatly to human health.

To improve the hemagglutinin (HA) antigen yield of influenza A(H1N1)pdm09 candidate vaccine viruses, we generated 7:1, 6:2, and 5:3 genetic reassortant viruses between wild-type (H1N1)pdm09 (A/California/7/2009) (Cal7) and a high-yielding master virus, A/Puerto Rico/8/34 (PR8). These viruses contained the HA; HA and neuraminidase (NA); and HA, NA, and M genes, respectively, derived from Cal7, on a PR8 backbone. The influence of the amino acid residue at position 223 in Cal7 HA on virus growth and HA antigen yield differed between these reassortant viruses. NIIDRG-7, a 7:1 virus possessing arginine at position 223, exhibited a 10-fold higher 50% egg infectious dose (EID(50)) (10.0 log(10)EID(50)/ml) than the 5:3 and 6:2 viruses. It also had 1.5- to 3-fold higher protein (13.8 μg/ml of allantoic fluids) and HA antigen (4.1 μg/ml of allantoic fluids) yields than the 5:3 and 6:2 viruses, which possessed identical Cal7 HA proteins. However, the HA antigen yield of the other 7:1 virus, which possessed glutamine at position 223 was 60% of that of NIIDRG-7. In addition, a novel 6:2 virus possessing Cal7 HA and the NA of A/Wisconsin/10/98 (a triple reassortant swine-like H1N1 virus), produced 107% of the HA yield of NIIDRG-7. In this study, we showed that the balance between HA and NA in the influenza A(H1N1)pdm09 virus affects its protein and antigen yield.

Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 10(6)EID(50) of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds., (Copyright © 2012 Elsevier B.V. All rights reserved.)

How the antibodies of individual convalescent human sera bind to each amino acid residue at the antigenic sites of hemagglutinin (HA) of influenza viruses, and how the antigenic drift strains of influenza viruses are selected by human sera, is not well understood. In our previous study, it was found by a binding assay with a chimeric HA between A/Kamata/14/91 (Ka/91) and A/Aichi/2/68 that convalescent human sera, following Ka/91 like (H3N2) virus infection, bind to antigenic site A of Ka/91 HA. Here using chimeric HAs possessing single amino acid substitutions at site A, it was determined how those human sera recognize each amino acid residue at antigenic site A. It was found that the capacity of human sera to recognize amino acid substitutions at site A differs from one person to another and that some amino acid substitutions result in all convalescent human sera losing their binding capacity. Among these amino acid substitutions, certain ones might be selected by chance, thus creating successive antigenic drift. Phylogenetic analysis of the drift strains of Ka/91 showed amino acid substitutions at positions 133, 135 and 145 were on the main stream of the phylogenetic tree. Indeed, all of the investigated convalescent sera failed to recognize one of them., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

Ab initio electronic-state calculations for influenza virus hemagglutinin (HA) trimer complexed with Fab antibody were performed on the basis of the fragment molecular orbital (FMO) method at the second and third-order Møller-Plesset (MP2 and MP3) perturbation levels. For the protein complex containing 2351 residues and 36,160 atoms, the inter-fragment interaction energies (IFIEs) were evaluated to illustrate the effective interactions between all the pairs of amino acid residues. By analyzing the calculated data on the IFIEs, we first discussed the interactions and their fluctuations between multiple domains contained in the trimer complex. Next, by combining the IFIE data between the Fab antibody and each residue in the HA antigen with experimental data on the hemadsorption activity of HA mutants, we proposed a protocol to predict probable mutations in HA. The proposed protocol based on the FMO-MP2.5 calculation can explain the historical facts concerning the actual mutations after the emergence of A/Hong Kong/1/68 influenza virus with subtype H3N2, and thus provides a useful methodology to enumerate those residue sites likely to mutate in the future., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Quantum mechanical fragment molecular orbital calculations have been performed for receptor binding of the hemagglutinin protein of the recently pandemic influenza 2009 H1N1, A/swine/Iowa/1930, and A/Puerto Rico/8/1934 viruses to α2-6 linked sialyloligosaccharides, as analogs of human receptors. The strongest receptor binding affinity was observed for the 2009/H1N1pdm. The inter-fragment interaction energy analysis revealed that the amino acid mutation of 2009/H1N1pdm, Ser145Lys, was a major cause of such strong binding affinity. Strong ionic pair interaction between the sialic acid and Lys145 was observed only in the 2009/H1N1pdm, in addition to the hydrogen bond between the sialic acid and Gln226 observed in all the HAs. Therefore, pandemic 2009/H1N1pdm has been found to recognize the α2-6 receptor much stronger than the 1930-swine and 1934-human.

In order to clarify the effect of amino acid substitutions on the structure and function of the neuraminidase (NA) protein of influenza A virus, we introduced single-point amino acid substitutions into the NA protein of the A/Tokyo/3/67 (H2N2) strain using PCR-based random mutation. The rate of tolerant random one amino acid substitutions in the NA protein was 47%. Rates of tolerant substitutions for the stalk and for the surface and inner portion of the head region of the NA protein were 79, 54, and 19%, respectively. Deleterious changes, such as those causing the NA protein to stop at the Golgi/endoplasmic reticulum, were scattered throughout the protein. On the other hand, the ratio of mutations with which the NA protein lost neuraminidase activity, but was transported to the cell surface, decreased in proportion to the distance from the structural center of enzyme active site. In order to investigate the effect of accumulated amino acid substitutions on the structural character of the N2NA protein during evolution, the same amino acid substitutions were introduced by site-directed mutagenesis at 23 homologous positions on N2 proteins of A/Tokyo/3/67, A/Bangkok/15/85 (H3N2), and A/Mie/1/2004 (H3N2). The results showed a shift, or discordance, in tolerance at some of the positions. An increase in discordance was correlated with the interval in years between virus strains, and the discordance rate was estimated to be 0.6-0.7% per year.

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.

We introduced 38 single-point amino acid changes into the hemagglutinin (HA) protein of the reassortmented A/Duck/Mongolia/54/01 (H5N2) strain by a PCR random mutation method. The percentage of amino acid changes on the HA domain that did not abrogate hemadsorption activity was calculated to be 89%. Changes in the amino acids of the HA2 domain were observed to be about half of those in the HA1 domain of these mutants. We assumed that amino acid changes in the HA1 domain afforded more flexibility in maintaining the functions of the HA protein than did those in the HA2 domain. Changes at two positions allowed the mutants to have same characteristics with respect to HA function despite the difference in the substituted amino acid. The results suggested that the effect on hemadsorption activity of an amino acid change on the HA protein primarily depends on the position rather than the species of substituted amino acid. An amino acid change at residue 122 from Trp to Arg and 179 from His to Arg resulted in the loss of hemadsorption activity of the HA protein. Site 122 is near the antibody binding site A, and site 179 is in the receptor binding domain (RBD) of HSHA. So that we suggest residue position 179 or 122 is very important to maintain the structure of RBD or antigenic site of H5HA. Position 4 in HA1 changed from Cys to Arg and position 148 in HA2 changed from Cys to Tyr also resulted in the loss of hemadsorption activity of the HA protein. Cys plays an important role in maintaining the structure of HA protein by means of S-S bonds. 3 potential glycosylation sites (Asn-X-Ser/Thr) were lost in our experiment that did not lose the hemadsorption activity of HA. Some interesting positions need to be analyzed more finely. Some amino acid changes identified in vitro experiment may serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203. HA protein with substitutions of some amino acid residues created by site-directed mutagenesis in the escape mutants retained the ability to bind to mAb203. Changes in the amino acid character affecting charge or hydrophobicity accounted for the binding capacity to the antibody of the HA with most of the substitutions in the escape mutants and binding-positive mutants. However, the effect of some amino acid substitutions remained unexplained. A three-dimensional model of the 1991 HA was constructed and used to analyze substituted amino acids in these mutants for the accessible surface hydrophobic and hydrophilic characters. One amino acid substitution in an escape mutant and another amino acid substitution in a binding-positive mutant seemed to be explained by the changes noted on this model.

During protein evolution the amino acid substitutions accumulate with time. However, the effect of accumulation of the amino acid substitutions to structural changes has not been estimated well. We will propose that the discordance of amino acid substitution on the HA protein of influenza A virus is useful for the assessment of structural changes during evolution. Discordance value can be obtained from the experimental data of tolerance or intolerance by introducing site directed mutagenesis at the homologous positions of two HA proteins holding the same amino acid residues. The value of discordance correlated to the number of amino acid differences among proteins. In the H3HA discordance rate was calculated to be 0.45% per one amino acid change. Furthermore, discordance of amino acid substitutions suggests that tolerable amino acid substitutions in different order have a probability of promoting irreversible divergence of the HA protein to different subtypes.

The C-terminal sequence of the cytoplasmic tail (CT) of influenza B haemagglutinin (BHA) consists of strictly conserved, hydrophobic amino acids, and the endmost C-terminal amino acid of the CT is Leu. To elucidate the role of this amino acid in the fusion activity of BHA (B/Kanagawa/73), site-specific mutant HAs were created by replacing Leu at this position with Arg, Lys, Ser, Try, Val or Ile or by the deletion of Leu altogether. All mutants were expressed at the cell surface, bound to red blood cells, were cleaved properly into two subunits and could be acylated like the wild-type (wt) HA. The membrane-fusion ability of these mutants was examined with a lipid (R18) and aqueous (calcein) dye-transfer assay and quantified with a syncytium-formation assay. All mutant HAs showed no measurable effect on lipid mixing or fusion-pore formation. However, mutant HAs with a hydrophobic value of the C-terminal amino acid lower than that of Leu had a reduced ability to form syncytia, whereas mutants with a more hydrophobic amino acid (Val or Ile) promoted fusion to the extent of the wt HA. On the other hand, the mutant HA with the deletion of Leu supported full fusion. These results demonstrate that Leu at the endmost portion of the C terminus of the BHA-CT is not essential for BHA-mediated fusion, but that the hydrophobicity of the single amino acid at this position plays an important role in syncytium formation.

Human influenza A viruses evolve more rapidly than influenza B viruses. To clarify the cause of this difference, we have evaluated the mutation rate of the nonstructural gene as revealed by the genetic diversity observed during the growth of individual plaques in MDCK cells. Six plaques were studied, representing two strains each of type A and B viruses. A total of 813,663 nucleotides were sequenced, giving rates of 2.0 x 10(-6) and 0.6 x 10(-6) mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates.

In order to clarify the effect of an accumulation of amino acid substitutions on the hemadsorption character of the influenza AH3 virus hemagglutinin (HA) protein, we introduced single-point amino acid changes into the HA1 domain of the HA proteins of influenza viruses isolated in 1968 (A/Aichi/2/68) and 1997 (A/Sydney/5/97) by using PCR-based random mutation or site-directed mutagenesis. These substitutions were classified as positive or negative according to their effects on the hemadsorption activity. The rate of positive substitutions was about 50% for both strains. Of 44 amino acid changes that were identical in the two strains with regard to both the substituted amino acids and their positions in the HA1 domain, 22% of the changes that were positive in A/Aichi/2/68 were negative in A/Sydney/5/97 and 27% of the changes that were negative in A/Aichi/2/68 were positive in A/Sydney/5/97. A similar discordance rate was also seen for the antigenic sites. These results suggest that the accumulation of amino acid substitutions in the HA protein during evolution promoted irreversible structural changes and therefore that antigenic changes in the H3HA protein may not be limited.

We studied the effects of an increase in the hydrophobicity of the transmembrane domain (TM) and cytoplasmic tail (CT) of influenza B virus hemagglutinin (BHA) on fusion activities. For this purpose, we created mutant HAs with novel acylation site(s) in the TM and/or CT. All mutants were able to induce hemifusion and to form fusion pores as well as could wild type (wt) BHA. However, the ability of these mutants to form syncytia was impaired, indicating that the increase in the hydrophobicity of these domains (especially the CT) affected fusion pore dilation.

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.

Sera from 27 children and eight older persons, which had been collected in 1998 and 1999 and showed haemagglutination-inhibition (HI) activity against influenza A/Sydney/5/97 (H3N2) strain, were characterized with a binding assay using chimeric haemagglutinin (HA) proteins between A/Aichi/2/68 (A/AI/68) and A/Sydney/5/97 (A/SD/97) strains. Sera from the young children had a tendency to recognize only the antigenic site B1 of the HA1 region. On the other hand, sera of the older individuals were fully reactive to all antigenic sites of HA1 except antigenic site D. Recent epidemic strains, A/Panama/2007/99 (A/PM/99)-like viruses have differences in amino acids in antigenic sites A, C, and B2 but not B1. However, human antisera obtained even from young children had HI activity to Panama-like viruses. The limited epidemic of A/PM/99-like viruses may have been due to the existence of antibody against B1, which had been produced in response to infection by the A/SD/97-like viruses.

We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations.

In an effort to find efficient DNA vaccine candidates, cDNA of influenza A virus hemagglutinin (HA) gene and several derived mutants were injected into mice using a gene gun. Mice immunized with HA1 DNA, with or without a membrane domain, showed a humoral immune response and the survival rate against homologous virus challenge was comparable to that of mice injected with HA DNA. In order to analyze epitopes recognized by antibodies induced by gene gun immunization, we used a binding assay employing the chimeric HA protein method. Serum antibodies of mice immunized with HA DNA recognized the HA1 domain but not the HA2 domain. In addition, antisera obtained from mice immunized with HA1 DNA reacted with each of the known antigenic sites on the HA1 domain, similar to the results obtained with HA DNA immunization.

It was reported previously that haemadsorption by the haemagglutinin (HA) protein of influenza B virus required that the protein must undergo desialidation. When MDCK and COS cells were infected with influenza B/Kanagawa/73 virus in the presence of a neuraminidase (NA) inhibitor, Zanamivir, haemadsorption on MDCK cells was inhibited but that on COS cells was not. The activity of the NA protein of the two types of infected cells was similar and both were inhibited by Zanamivir in a dose-dependent manner. A comparison of the desialidation of the HA protein was made on MDCK and COS cells in the presence of bacterial NA and both cells were found to have similar sensitivity. On the accumulation of the HA and NA proteins in the trans-Golgi network of MDCK cells by means of low-temperature treatment, desialidation of the HA protein in the presence of Zanamivir was detected by two-dimensional gel electrophoresis. Because this agent was reported to be unable to penetrate cells, these data suggest that, in MDCK cells, desialidation of the HA protein occurs on the cell surface but, in COS cells, the HA and NA proteins might accumulate in the trans-Golgi network, thus allowing NA desialidation before their migration to the cell surface.

Human H3N2 influenza A viruses were known to preferentially bind to sialic acid (SA) in alpha2,6Gal linkage on red blood cells (RBC). However, H3N2 viruses isolated in MDCK cells after 1992 did not agglutinate chicken RBC (CRBC). Experiments with point-mutated hemagglutinin (HA) of A/Aichi/51/92, one of these viruses, revealed that an amino acid change from Glu to Asp at position 190 (E190D) was responsible for the loss of ability to bind to CRBC. A/Aichi/51/92 did not agglutinate CRBC treated with alpha2, 3-sialidase, suggesting that SAalpha2,3Gal on CRBC might not inhibit the binding of the virus to SAalpha2,6Gal on CRBC. However, the virus agglutinated derivatized CRBC resialylated with SAalpha2, 6Galbeta1,4GlcNAc. These findings suggested that the E190D change might have rendered the HA able to distinguish sialyloligosaccharides on the derivatized CRBC containing the SAalpha2,6Galbeta1,4GlcNAc sequence from those on the native CRBC., (Copyright 2000 Academic Press.)

Eight convalescent human sera obtained from patients aged 3 to 14 years old, who were infected with influenza A(H3N2) virus during the 1990/1991 influenza season, were characterized by a binding assay with chimeric hemagglutinin (HA) proteins between influenza virus A/Aichi/2/68 and A/Kamata/14/91(H3N2) strains. These sera did not recognize the HA protein of the A/Aichi/2/68 strain but recognized that of the A/Kamata/14/91 strain. The binding assay revealed that these sera recognized only the HA1 domain of A/Kamata/14/91 HA protein. A further assay of the binding of these sera to the chimeric proteins of the HA1 domain revealed that three sera (A-1, A-2, and A-3) from very young patients bound only to region 150-170 (site B1) and one serum (Y-1) bound to regions 96-150 (site A) and 96-170 (sites A and B1). These four sera showed reduced hemagglutination inhibition (HI) activity with the 203v2 strain, a monoclonal variant of the A/Kamata/14/91 strain with two amino acid changes in the HA protein at antigenic sites A and B1. The other four sera (Y-2, G-1, G-2, and A-4) bound to regions 1-96 (site C/E), 96-150 (site A), 96-170 (sites A and B1), and 170-200 (site B2), two of which further bound to region 240-306 (site C); these sera were all fully reactive with the 203v2 strain. All eight sera showed reduced HI reactivity to a drift strain A/Aichi/4/93. Amino acid changes of the A/Aichi/4/93 strain from the A/Kamata/14/91 strain were located at antigenic sites A, B1, B2, and C. We propose a possible model for the emergence of a drift strain A/Aichi/4/93 from an A/Kamata/14/91-like strain by sequential changes during reinfections of individuals starting from A-1-like, next to Y-1-like, and then to Y-2-like populations., (Copyright 2000 Academic Press.)

In order to conduct a survey of influenza viruses entering Japan via travellers arriving by airplanes, gargle solutions were collected from passengers who reported to the quarantine station of Nagoya International Airport complaining of respiratory symptoms. From 504 samples collected between August 1996 and March 1999, 30 influenza virus strains were isolated. Twenty-eight of the isolates were influenza A (H3N2) viruses and two were influenza B viruses. No H1N1 virus was isolated. Among 28 isolates of H3N2 virus, 3 strains were obtained outside the influenza season. Nucleotide sequences of the haemagglutinin (HA) genes of these isolates along with those from domestic patients were analysed in order to determine the influence of imported influenza viruses by travellers on epidemics in Japan. From the phylogenetic and chronological aspects, the possibility was suggested in one case in 1997/8 and two in the 1998/9 season that imported virus by travellers may have influenced the domestic influenza epidemics.

Cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for the pathogenicity and even for the neurovirulence of influenza A viruses. WSN, a neurovirulent virus, adapted to mouse brain, grew in vitro in several types of cells including neuroblastoma cells in the absence of trypsin. When mice were intracerebrally inoculated with WSN, the viral antigen was found in the substantia nigra zona compacta and hippocampus. The mice inoculated with viruses isolated from children with acute encephalopathy associated with an influenza virus infection, on the other hand, showed no neurological symptoms. Furthermore, these viruses did not grow in the human neuroblastoma and glioblastoma cells. Since 1991, most of the human influenza A viruses have not agglutinated chicken erythrocytes. Whether this altered receptor binding specificity is related to the occurrence of influenza encephalitis and encephalopathy is now under investigation.

We analysed the role of neuraminidase (NA) on haemadsorption by the haemagglutinin (HA) protein of influenza B virus. The influenza B virus mutant ts-7 has a temperature-sensitive mutation in the NA protein. At high temperature, cells infected with this virus did not exhibit haemadsorption activity, but the addition of bacterial neuraminidase (bNA) restored haemadsorption activity. COS cells transfected with HA cDNAs of B/Kanagawa/73 or B/Lee/40 virus showed no evidence of haemadsorption. However, with the addition of bNA or co- transfection with NA cDNA of the B/Lee/40 virus, haemadsorption was observed. Experiments with point-mutated HA cDNAs of B/Lee/40 virus showed that two N-acetyl glycosylation sites at amino acid residues 160 and 217 were responsible for the inability of the HA protein to adsorb to erythrocytes. These results indicated that haemadsorption by the HA protein of influenza B virus required the involvement of NA. Because the NA inhibitor Zanamivir was reported not to penetrate cells, we investigated the action of this inhibitor and found that Zanamivir inhibited haemadsorption on MDCK cells infected with B/Kanagawa/73 or B/Lee/40 virus. After removing Zanamivir by washing, the addition of bNA restored the haemadsorption activity on the infected cells. Scanning electron microscopy indicated that at 0.4 microM Zanamivir, HA protein did not adsorb to erythrocytes but retained the ability to aggregate virions. However, at 4 microM Zanamivir, distinct virion formation could not be observed.

Phylogenetic analysis was carried out for genes encoding hemagglutinin (HA) (24 new and 25 previously reported sequences) and nonstructural proteins (NS) (22 new and 14 previously reported sequences) of influenza B virus isolates obtained from 1940 to 1999. Two antigenically and genetically distinct HA lineages are presently known to exist. Divergence into these two lineages was estimated to have occurred around 1969. Phylogenetic analysis of NS genes revealed that their phylogenetic relationships were not linked to the two HA lineages but suggested that reassortment of viral genes between the viruses of two HA lineages had occurred. In addition two distinct NS lineages which were not linked to the two HA lineages were observed. Viruses isolated after 1997 formed their own lineage in combination with B/Houston/84 while other virus isolates obtained from 1973 to 1995 comprised the other NS lineage.

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.

From the reassortment experiments between A/Aichi/4/92 and A/WSN/33 (WSN) (H1N1) viruses, two different phenotype viruses which contained the haemagglutinin (HA) gene from A/Aichi/4/92 virus and the neuraminidase (NA) gene from WSN virus were obtained. PW13 and PW15 viruses agglutinated chicken red blood cells (CRBC), while PW10 and PW70 viruses did not. However, the expressed HA proteins of these viruses did not adsorb CRBC. The difference in gene constellation between PW13, PW15 and PW10, PW70 viruses was the membrane protein (M) gene. The former two had the M gene from A/Aichi/4/92 virus and the latter two had that from WSN virus. In PW15-infected cells, haemadsorption of CRBC was observed 30 min later than that of goose red blood cells and the M1 protein migrated from the nucleus to the cytoplasm 30 min earlier than adsorption of CRBC was observed. On the other hand, in PW10-infected cells, haemadsorption of CRBC was not observed through the virus replication and the M1 protein stayed in the nucleus after HA and NA activities reached maximum levels. Co-expression of the M and the HA proteins of A/Aichi/4/92 virus did not help the HA protein gain the ability to adsorb CRBC. However, neuraminidase treatment of COS cells expressing the HA protein of A/Aichi/4/92 virus or MDCK cells infected by PW10 virus restored the ability to adsorb CRBC. We discussed the possibility that the M1 protein helped the NA protein in its role to modify the HA protein on the cell surface.

Two phenotypes of human influenza A(H1N1) virus are currently circulating in Japan. One (group 1) agglutinates both chicken and goose red blood cells (CRBC and GRBC), the other (group 2) agglutinates GRBC but not CRBC. In the 1995/6 season, group 2 viruses accounted for 70% of the H1N1 viruses isolated in MDCK cells. The 1995/6 viruses were located on two branches of the genetic tree. One branch contained both group 1 and group 2 viruses and the other branch contained only group 2 viruses. Group 2 viruses had aspartic acid at residue 225 in the haemagglutinin (HA) protein, the key amino acid residue for group 2 phenotype. The HA protein of group 1 viruses had a change from aspartic acid to asparagine at residue 225 and the expressed HA protein of these viruses adsorbed CRBC. Serial passage of group 2 viruses in MDCK cells or embryonated chicken eggs caused these viruses to gain the ability to agglutinate CRBC. MDCK-adapted viruses had the same amino acid sequences of HA polypeptide as the original ones, but egg-adapted viruses had changed amino acid sequences. The expressed HA protein from one egg-adapted virus that originally belonged to group 2 adsorbed CRBC.

Influenza A virus is unique among human viruses in its capacity to alter the antigenic phenotype with relative ease and evade neutralizing antibodies. This property is ascribed to the accumulation of a series of amino acid changes in the antigenic regions of hemagglutinin (HA) molecule. Neutralizing antibodies against HA prevent the early stage of infection, whereas neuraminidase (NA) antibodies mediate the antiviral effect by restricting spread of viruses in the host cells after infection. Thus the understanding of antigenic structure on those protective antigen is significant. Sequence analysis of natural variants and escape mutants selected by monoclonal antibodies allowed us to assign each antigenic sites and epitopes to a particular region on the three dimensional structure of HA and NA molecules.

The hemagglutinin(HA) of influenza virus is a major glycoprotein and plays a crucial role in the early stage of virus infection: HA is responsible for binding of the virus to cell surface receptors, and it mediates liberation of the viral genome into the cytoplasm through membrane fusion. The essential component of the receptor for influenza viruses has been considered to be the sialic acid. Influenza A and B viruses recognize N-acetylneuraminic acid, whereas influenza C virus specifically recognizes N-acetyl-9-O-acetylneuraminic acid as the receptor. Influenza A viruses are subdivided into 15 subtypes by their antigenic differences, but several amino acid residues composing functional domains (receptor binding site and fusion peptide) are shown to be conserved among HAs.

We previously reported that the neurovirulent influenza A virus strain, AIWSN/33 has strong affinity for the substantia nigra (SN) in mouse brain. In this study, we used a neuron-glia co-culture system in order to observe the cellular responses to viral infection. Co-cultured cells were infected with the A/WSN/33 strain and were examined immunohistochemically. Viral antigens were strictly confined to the neuronal cell bodies and their neurites. More than 90% of tyrosine hydroxylase (TH)-positive neurons in the SN were also positive to anti-WSN antibody. There was no increase in class I major histocompatibility complex (MHC) expression in virus antigen positive cells. Thus, we can conclude that the virus preferentially infects neurons, mainly TH-positive neurons in the SN. An antigen presenting response mediated by class I MHC was not observed in infected neurons.

Influenza A viruses are suitable for the analysis of virus evolution because the genes of the viruses are well analyzed. The origin of the present human influenza A viruses are deduced to be direct descendent of the viruses which caused Spanish flu in 1918. The analysis of NS gene shows the branching point between avian and human viruses are early 1900. By comparison of the amino acid sequences, HA serotypes could be divided into two groups, i.e., an H1 and an H3 groups. The branching between subtypes H1 and H2 occurred fairly recently. The HA genes of influenza A viruses evolve causing two kinds of antigenic variation, saift and drift, which are caused by different mechanisms.

Recent strains of influenza A but not B viruses have lost the ability to agglutinate chicken red blood cells (CRBC). The H1N1 viruses isolated in Japan during the 1991/92 season could be divided into two groups. Group 1 viruses (A/Aichi/4/92 and A/Aichi/7/92) agglutinated goose red blood cells (GRBC) and CRBC, while group 2 viruses (A/Aichi/24/92 and A/Aichi/26/92) did not agglutinate CRBC. There were no amino acid differences between them in the haemagglutinin (HA) polypeptide. Reassortment experiments between a group 1 virus (A/Aichi/4/92) or a group 2 virus (A/Aichi/24/92) and the A/WSN/33 influenza A (H1N1) virus strain suggested that the HA gene products of the viruses of both groups had lost the capacity to agglutinate CRBC. The HA proteins expressed on Cos cells by transfecting the cDNAs of the virus HA gene of A/Aichi/4/92 and A/Aichi/24/92 agglutinated GRBC but not CRBC. These experiments indicated that the HA proteins of H1N1 viruses of both groups isolated in 1992 had lost the ability to agglutinate CRBC even though the group 1 virions showed haemagglutinating capacity with CRBC. By using the cDNAs of the HA gene of seven natural isolates obtained from 1977 to 1992, it was found that the expressed HA proteins of influenza A (H1N1) viruses isolated since 1988 had lost the ability to agglutinate CRBC. Experiments with chimeric and point-mutated HA cDNAs of A/Aichi/24/92 showed that an amino acid change at residue 225, which occurred after 1986, and a cluster of amino acid changes at residues 193, 196 and 197, which occurred before 1986, were responsible for loss of the ability to agglutinate CRBC. Egg-adapted virus derived from A/Aichi/24/92 had one amino acid change at residue 225 compared to the parental virus.