Human respiratory syncytial virus (HRSV) is the most common cause of serious acute lower respiratory tract disease among infants and young children, and is found mainly in late fall, winter, and spring in temperate zones of the world (6). Some 50% to 70% of infants experience infection in the first year of life, and virtually all are infected by 2 years of age (3). In a population-based birth cohort study, 1.1% of the cohort were admitted to the hospital within 12 months of birth with HRSV-induced bronchiolitis (14). The consequences of HRSV infection in children with underlying conditions, such as prematurity, cardiac and pulmonary disease, or immunosuppression, may include prolonged substantial illness and even death (18,38). Reinfections are very common throughout life (7,10,11,13).HRSV belongs to the family Paramyxoviridae and has a nonsegmented, negative-sense RNA genome of approximately 15,200 nucleotides (3). HRSV has been classified into antigenic subgroups A and B (HRSV-A and HRSV-B, respectively), initially on the basis of the reactivity of the virus with monoclonal antibodies directed against the attachment glycoprotein (G protein) (1,5,15,21) and now by genetic analyses (9,19,(31)(32)(33).The G protein is the most variable HRSV protein, with two hypervariable regions. Its C-terminal region (the second hypervariable region) accounts for strain-specific epitopes (2-4, 9, 16, 27, 29, 31). The molecular epidemiology and evolutionary patterns of the G protein have provided important information about the epidemiological features of HRSV. Some studies showed that several different genotypes cocirculated and some predominated in a community every year (23, 27). However, the relationship between strain diversity and the clinical and epidemiological features of HRSV has yet to be elucidated in detail.The subgroups have been subdivided further, into genotypes, by genetic analyses. HRSV-A is divided into seven genotypes (GA1 to -7) and HRSV-B into four genotypes (GB1 to -4) (3,8). An additional HRSV-A genotype, SAA1, has been proposed, as well as the new HRSV-B genotypes SAB1 to -3 (37). Another HRSV-B genotype includes the Buenos Aires (BA) type strain, which has a 60-nucleotide insertion in the second hypervariable region of the G protein and has been reported in Buenos Aires in 1999, as well as in other areas of the world (31,35,40). BA strains are further subdivided into six clusters (BA-I to BA-VI) (36).National surveillance of HRSV infection based on weekly reports from sentinel pediatric clinics throughout Japan began

Phylogenetic analysis of respiratory syncytial virus (RSV) group B genotype BA strains from the 2002-2003 to 2009-2010 seasons collected in Niigata, Japan, revealed four distinct clusters, designated new BA genotypes BA7, BA8, BA9, and BA10. These new genotypes were not associated with large outbreaks in the community.

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

bAfter receptor binding and internalization during influenza virus entry, the hemagglutinin (HA) protein is triggered by low pH to undergo irreversible conformational changes that mediate membrane fusion. To investigate how mutations that alter the activation pH of the HA protein influence the fitness of an avian H5N1 influenza virus in a mammalian model, we infected C57BL/6J or DBA/2J mice and compared the replication and virulence of recombinant A/chicken/Vietnam/C58/04 (H5N1) HA-Y23 1 H mutant, wild-type, and HA-H24 1 Q and HA-K58 2 I mutant viruses that have HA activation pH values of 6.3, 5.9, 5.6, and 5.4, respectively. The HA-Y23 1 H mutant virus was highly susceptible to acid inactivation in vitro and was attenuated for growth and virulence in mice, suggesting that an H5N1 HA protein triggered at pH 6.3 is too unstable for the virus to remain fit. Wild-type and HA-H24 1 Q viruses were similar in pathogenicity and grew to similar levels in mice, ducks, and cell cultures derived from both avian and mammalian tissues, suggesting that H5N1 HA proteins triggered at pH values in the range of 5.9 to 5.6 broadly support replication. The HA-K58 2 I mutant virus had greater growth and virulence in DBA/2J mice than the wild type did, although the mutant virus was highly attenuated in ducks. The data suggest that adaptation of avian H5N1 influenza virus for infection in mammals is supported by a decrease in the HA activation pH to 5.4. Identification of the HA activation pH as a host-specific infectivity factor is expected to aid in the surveillance and risk assessment of currently circulating H5N1 influenza viruses.

bInfluenza virus entry is mediated by the acidic-pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in the HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) (VN1203) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (K to I at position 58) (activation pH 5.5). The VN1203-HA2-K58I virus had replication kinetics similar to those of wild-type VN1203 in MDCK and normal human bronchial epithelial cells and yet had reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar levels of morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to naive contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract, and yet it was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer ␣(2,6) receptor binding specificity and remove a critical N-linked glycosylation site. The information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals in the background of a non-laboratory-adapted virus provides essential information for the surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses.