Genotyping and phylogenetic analysis of the major genes in respiratory syncytial virus isolated from infants with bronchiolitis

We performed the genotyping and phylogenetic analysis of respiratory syncytial virus (RSV) isolated from 17 infants with bronchiolitis in Kanagawa Prefecture, Japan in 2005 and 2006. The major genes in these samples (attachment [G] glycoprotein gene, fusion [F] protein gene, and nucleoprotein [N] gene) were sequenced and analyzed genetically. Phylogenetic analysis of these genes revealed that 7 and 10 strains could be classified into subgroups A and B, respectively. Phylogenetic analysis of the G gene revealed that the subgroup A and B strains were unique genotypes GA2 and BA, respectively. Moreover, the amino acid sequences for these genotypes suggested a relatively high frequency of amino acid substitutions in the G and F proteins in these strains, whereas the N protein was highly homologous. These results suggest that RSV genotypes GA2 and BA may be associated with bronchiolitis in the cases studied here.     

Respiratory Syncytial Virus G Protein Sequence Variability among Isolates from St. Petersburg,Russia, during the 2013–2014 Epidemic Season

Human respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. It is actively evolving under environmental and herd immunity influences. This work presents, for the first time, sequence variability analysis of RSV G gene and G protein using St. Petersburg (Russia) isolates. Viruses were isolated in a cell culture from the clinical samples of 61 children hospitalized (January–April 2014) with laboratory-confirmed RSV infection. Real-time RT-PCR data showed that 56 isolates (91.8%) belonged to RSV-A and 5 isolates (8.2%) belonged to RSV-B. The G genes were sequenced for 27 RSV-A isolates and all of them belonged to genotype ON1/GA2. Of these RSV-A, 77.8% belonged to the ON1(1.1) genetic sub-cluster, and 14.8% belonged to the ON1(1.2) sub-cluster. The ON1(1.3) sub-cluster constituted a minor group (3.7%). Many single-amino acid substitutions were identified in the G proteins of St. Petersburg isolates, compared with the Canadian ON1/GA2 reference virus (ON67-1210A). Most of the amino acid replacements were found in immunodominant B- and T-cell antigenic determinants of G protein. These may affect the antigenic characteristics of RSV and influence the host antiviral immune response to currently circulating viruses.     

Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it.

We have sequenced the virulent Asibi strain of yellow fever virus and compared this sequence to that of the 17D vaccine strain, which was derived from it. These two strains of viruses differ by more than 240 passages. We found that the two RNAs, 10,862 nucleotides long, differ at 68 nucleotide positions; these changes result in 32 amino acid differences. Overall, this corresponds to 0.63% nucleotide sequence divergence, and the changes are scattered throughout the genome. The overall divergence at the level of amino acid substitution is 0.94%, but these changes are not randomly distributed among the virus protein. The capsid protein is unchanged, while proteins NS1, NS3, and NS5 contain 0.5% amino acid substitutions, and proteins ns4a and ns4b average 0.8% substitutions. In contrast, proteins ns2a and ns2b have 3.0 and 2.3% amino acid divergence, respectively. The envelope protein also has a relatively high rate of amino acid change of 2.4% (a total of 12 amino acid substitutions). The large number of changes in ns2a and ns2b, which are largely conservative in nature, may result from lowered selective pressure against alteration in this region; among flaviviruses, these polypeptides are much less highly conserved than NS1, NS3, and NS5. However, many of the amino acid substitutions in the E protein are not conservative. It seems likely that at least some of the difference in virulence between the two strains of yellow fever virus results from changes in the envelope protein that affect virus binding to host receptors. Such differences in receptor binding could result in the reduced neurotropism and vicerotropism exhibited by the vaccine strain.     

中国12株狂犬病街毒株G基因序列测定与分析

目的了解中国狂犬病毒的流行情况以及街毒株与中国人用、兽用狂犬病疫苗株在G基因核苷酸和氨基酸水平的差异,为有效控制狂犬病疫情提供初步科学依据。方法对12株街毒株G基因进行了全基因测序,与其它36株中国狂犬病街毒株,以及中国的疫苗株和其它国家毒株的G基因序列进行了综合分析。结果序列分析表明来源于中国的50株狂犬病毒均为基因Ⅰ型狂犬病毒;其中具有代表性的6株病毒与我国现在使用的各种疫苗株在G基因的核苷酸和氨基酸水平上均存在不同程度的差异,与我国人用疫苗株CTN同源性较高;进化分析表明,我国主要流行狂犬病毒与泰国、印度尼西亚、马来西亚等东南亚狂犬病毒株处于同一分支。中国毒株之间G基因核苷酸同源性分别≥82.3%;氨基酸同源性分别≥92.1%;中国街毒株与疫苗株相比较核苷酸的同源性为79.3%～94.2%,氨基酸的同源性为87.8%～97.9%。结论我国的狂犬病毒为基因Ⅰ型狂犬病毒,可以明确分为6个进化群。无论在核苷酸还是氨基酸水平上,中国多数街毒株与疫苗株CTN之间的同源性要高于与其它疫苗株。     

Molecular cloning of bullfrog saxiphilin: a unique relative of the transferrin family that binds saxitoxin.

Plasma and tissue of certain vertebrates contain a protein called saxiphilin that specifically binds the neurotoxin saxitoxin with nanomolar affinity. We describe the isolation of a cDNA clone of saxiphilin from liver of the North American bullfrog (Rana catesbeiana). The cDNA sequence encodes a protein that is evolutionarily related to members of the transferrin family of Fe(3+)-binding proteins. Pairwise sequence alignment of saxiphilin with various transferrins reveals amino acid identity as high as 51% and predicts 14 disulfide bonds that are highly conserved. The larger size of saxiphilin (91 kDa) versus serum transferrin (approximately 78 kDa) is primarily due to a unique insertion of 144 residues. This insertion contains a 49-residue domain classified as a type 1 repetitive element of thyroglobulin, which is shared by a variety of membrane, secreted, and extracellular matrix proteins. Saxiphilin also differs from transferrins in 9 of 10 highly conserved amino acids in the two homologous Fe3+/HCO3-binding sites of transferrin. Identification of saxiphilin implies that transferrin-like proteins comprise a diverse superfamily with functions other than iron binding.     

Characterisation of RSV Fusion Proteins from South African Patients with RSV Disease, 2019 to 2020

Respiratory syncytial virus (RSV) is classified into RSV-A and RSV-B, which are further classified into genotypes based on variability in the G gene. The fusion (F) protein is highly conserved; however, variability within antigenic sites has been reported. This study aimed to characterise F proteins from RSV strains detected in South Africa from 2019 to 2020. Patients of all ages, from whom respiratory samples were submitted to the National Health Laboratory Service at Charlotte Maxeke Johannesburg Academic Hospital, South Africa during 2019 to 2020, were included. Complete RSV F genes were amplified for next-generation sequencing. MEGA X software was used for phylogenetic analysis. The overall prevalence of RSV was 5.8% (101/1734). Among 101 RSV positive samples only 69.3% (70/101) were available for characterization of the RSV F protein gene. Among cases included for F gene characterisation, viral co-infections were observed in 50% (35/70) and 25.7% (18/70) were admitted to intensive care units (ICU). About 74.2% (23/31) of F gene sequences cluster with other African NA1/ON1 genotypes. At antigenic site I, the V384I mutation was replaced by V384T in South African strains. The S275F mutation was seen in a single South African strain. The N120 N-linked glycosylation site was present in 25.8% (8/31) of RSV-A F proteins described in this study. For the first time, we detected the rare S275F mutation that is associated with palivizumab resistance.     

Clinical characteristics of respiratory syncytial virus (RSV) subgroup infections in Japan.

The subgroup characteristics of 130 strains of respiratory syncytial virus (RSV) isolated in Sapporo during 9 epidemic years 1980-1989 were determined. Monoclonal antibodies raised against the RSV Long strains were used. Subgroup A included 77 (59.2%) isolates and subgroup B 52 (40.0%) strains, while 1 strain was considered to be a variant of a subgroup A strain. The distribution by age of infants and children was different for the 2 subgroups: less than 1 year of age infants with subgroup A infection dominated, greater than 1 year of age subgroup A infections were less common than subgroup B infections. These was no difference in type of illness between the subgroups. Bronchiolitis was the dominant diagnosis in all patients.     

Prediction of protein antigenic determinants from amino acid sequences.

A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant. It was found that the prediction success rate depended on averaging group length, with hexapeptide averages yielding optimal results. The method was developed using 12 proteins for which extensive immunochemical analysis has been carried out and subsequently was used to predict antigenic determinants for the following proteins: hepatitis B surface antigen, influenza hemagglutinins, fowl plague virus hemagglutinin, human histocompatibility antigen HLA-B7, human interferons, Escherichia coli and cholera enterotoxins, ragweed allergens Ra3 and Ra5, and streptococcal M protein. The hepatitis B surface antigen sequence was synthesized by chemical means and was shown to have antigenic activity by radioimmunoassay.     

Sequence relationships among the hemagglutinin genes of 12 subtypes of influenza A virus.

Nucleotide sequences of the 3' 20% of the hemagglutinin gene of 32 influenza A virus strains from the 12 known hemagglutinin subtypes have been determined. Although the sequences of hemagglutinin genes and proteins of different subtypes differ greatly, cysteine and some other amino acid residues are totally conserved, presumably reflecting evolution of the 12 different hemagglutinins from a single gene. When viruses of one subtype, isolated over a period of time, are compared, the hemagglutinin gene and protein sequences show a slow accumulation of nucleotide changes and some amino acid changes. Since sequence data from the genes coding for the matrix and nonstructural proteins also show an accumulation of changes with time, it seems that antigenic selection (of the surface antigens) does not contribute significantly to the rate of change on influenza gene sequences. Although the rate of nucleotide change during drift is more than sufficient to account for the amino acid sequence differences observed in the 12 subtypes, there is a clear distinction, by antigenic as well as sequence analyses, between viruses of one subtype (0-9% amino acid variation) and viruses of other subtypes (20-74% amino acid variation). No virus has yet been found that is intermediate between subtypes.     

Genomic divergence of HIV-2 from Ghana

Genetic variability in human immunodeficiency virus type 1 (HIV-1) has been studied extensively, but the total nucleotide sequence of the HIV-2 genome has been reported only in two strains. For phylogenetic analyses of HIV, the genetic variability of HIV-2 should be investigated. This paper reports the complete nucleotide sequence of an HIV-2 isolate from Ghana, HIV-2[GH-1]. This virus showed approximately 85% homology in overall nucleotide sequence with HIV-2ROD. The amino acid sequence of the gag and pol proteins of HIV-2[GH-1] showed 90% homology with those of HIV-2ROD, but its env gene and central regions were highly variable (more than 20% divergence in amino acids), indicating the presence of extensive genetic heterogeneity in HIV-2. However, the sequences with specific functions were relatively well conserved in these HIV-2 isolates.     

Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus: comparison of the individual contributions of the F and G glycoproteins to host immunity.

A cDNA clone representing the mRNA coding sequence of the fusion glycoprotein (F) gene of human respiratory syncytial virus (RSV) was constructed and inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of a vaccinia virus promoter. The resulting recombinant vaccinia virus, vaccinia F, expressed the F1 and F2 cleavage products (48 and 20 kDa, respectively) of the F glycoprotein in cell culture. F1 and F2 were indistinguishable from their authentic RSV counterparts with respect to glycosylation, disulfide linkage, electrophoretic mobility, cell-surface expression, and antigenic specificity. Cotton rats infected intradermally with vaccinia F developed a high titer of serum F-specific antibodies, which neutralized infectivity of RSV. This neutralizing antibody response exceeded that induced by infection of the respiratory tract with RSV and was 6-fold higher than that induced by vaccinia G, a recombinant vaccinia virus that expressed the RSV G glycoprotein gene. Immunization with vaccinia F stimulated almost complete resistance to replication of RSV in the lower respiratory tract as well as significant resistance in the upper respiratory tract. The degree of resistance conferred by vaccinia F exceeded that induced by vaccinia G.     

Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan, 2008–2017

In this study, we investigated the molecular evolution and phylodynamics of respiratory syncytial virus (RSV) over 10 consecutive seasons (2008–2017) and the genetic variability of the RSV genotypes ON1 and BA in central Taiwan. The ectodomain region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the second hypervariable region of the G protein in RSV ON1 and BA were analyzed. A total of 132 RSV-A and 81 RSV-B isolates were obtained. Phylogenetic analysis revealed that the NA1, ON1, and BA9 genotypes were responsible for the RSV epidemics in central Taiwan in the study period. For RSV-A, the NA1 genotype predominated during the 2008–2011 seasons. The ON1 genotype was first detected in 2011 and replaced NA1 after 2012. For RSV-B, the BA9 and BA10 genotypes cocirculated from 2008 to 2010, but the BA9 genotype has predominated since 2012. Amino acid sequence alignments revealed the continuous evolution of the G gene in the ectodomain region. The predicted N-glycosylation sites were relatively conserved in the ON1 (site 237 and 318) and BA9 (site 296 and 310) genotype strains. Our results contribute to the understanding and prediction of the temporal evolution of RSV at the local level.     

In silico identification and characterization of common epitope-based peptide vaccine for Nipah and Hendra viruses

Objective: To explore a common B-and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus(He V) and Nipah virus(Ni V). Methods: Membrane proteins F, G and M of He V and Ni V were retrieved from the protein database and subjected to different bioinformatics tools to predict antigenic B-cell epitopes. Best B-cell epitopes were then analyzed to predict their T-cell antigenic potentiality. Antigenic B-and T-cell epitopes that shared maximum identity with He V and Ni V were selected. Stability of the selected epitopes was predicted. Finally, the selected epitopes were subjected to molecular docking simulation with HLA-DR to confirm their antigenic potentiality in silico. Results: One epitope from G proteins, one from M proteins and none from F proteins were selected based on their antigenic potentiality. The epitope from the G proteins was stable whereas that from M was unstable. The M-epitope was made stable by adding flanking dipeptides. The 15-mer G-epitope(VDPLRVQWRNNSVIS) showed at least 66% identity with all Ni V and He V G protein sequences, while the 15-mer M-epitope(GKLEFRRNNAIAFKG) with the dipeptide flanking residues showed 73% identity with all Ni V and He V M protein sequences available in the database. Molecular docking simulation with most frequent MHC class-II(MHC II) and class-I(MHC I) molecules showed that these epitopes could bind within HLA binding grooves to elicit an immune response. Conclusions: Data in our present study revealed the notion that the epitopes from G and M proteins might be the target for peptide-based subunit vaccine design against He V and Ni V. However, the biochemical analysis is necessary to experimentally validate the interaction of epitopes individually with the MHC molecules through elucidation of immunity induction.     

广西新分离流行性乙型脑炎病毒GP0722株全基因组分子特性研究

目的 对广西新分离乙脑病毒GP0722株进行全基因序列测定和分析,了解其基因组结构及毒力特征。方法 应用乙脑病毒全基因组扩增引物进行RT-PCR扩增,PCR产物直接测序,拼接后得到全基因序列。应用Clustal X(1.8)、DNASTAR、Mega 4. 1等生物软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析。结果 广西新分离乙脑病毒GP0722全基因长10 965个核苷酸,从97到10 395位编码一个开放阅读框,编码3 432个氨基酸,与目前使用的减毒活疫苗株SA-14-14-2株比较,只有88.9%的核苷酸同源性,97.6%的氨基酸同源性,全基因组共存在1 222个核苷酸差异,83个氨基酸差异。与GenBank中选择的21株乙脑病毒全基因序列比较发现,其核苷酸总体差异率为0.9%~18.8%,氨基酸总体差异率为0.1%~5.2%。通过PrM/C区段、E区段、3′NTR区段和全基因序列进行系统进化分析显示该毒株属于基因1型乙脑病毒。结论 新分离的乙脑病毒GP0722株属于基因1型,与JEV/sw/Mie/40/2004进化关系最近,与疫苗株SA-14-14-2相比关键位点氨基酸未见变异,现行使用的疫苗仍能保护GP0722引起的感染。     

cDNAs encoding the large subunit of human replication factor C.

Replication factor C (RFC) is a multisubunit, DNA polymerase accessory protein required for the coordinated synthesis of both DNA strands during simian virus 40 DNA replication in vitro. Previous studies have shown that RFC is a DNA-dependent ATPase that binds in a structure-specific manner to the 3' end of a primer hybridized to a template DNA, an activity thought intrinsic to the 140-kDa component of this multisubunit complex. Here, the isolation and analysis of cDNAs encoding this subunit is described. Analysis of the full-length coding sequence revealed an open reading frame of 3.4 kb, encoding an 1148-amino acid protein with a predicted molecular mass of 130 kDa. A putative ATP-binding motif was observed that is similar to a motif in several of the smaller subunits of RFC and in functionally homologous replication factors of bacterial and viral origin. A "DEAD" box is also conserved among these proteins. The predicted protein shows significant identity with a DNA-binding protein of murine origin (B. Luckow, P. Lichter, and G. Schütz, personal communication). Regions of similarity were also seen between the amino acid sequences of the 140-kDa subunit of RFC, poly(ADP-ribose) polymerase, and bacterial DNA ligases--possibly representing a conserved structural feature of these proteins that bind similar DNA substrates.     

Complete nucleotide sequence of the neuraminidase gene of influenza B virus.

The complete nucleotide sequence of the neuraminidase gene of influenza virus B/Lee/40 was derived from a cloned cDNA copy of virion RNA segment 6 and its corresponding mRNA. The RNA segment contains 1,557 virus-specific nucleotides, and the protein encoded by the longest open reading frame has a total of 466 amino acids with a molecular weight of 51,721. As is the case with the influenza A virus neuraminidases, the deduced amino acid sequence of the influenza B protein includes a single hydrophobic region near the amino terminus which would be capable of spanning the lipid bilayer of the viral or cell membrane. There are four potential glycosylation sites in the protein, two of which are near the amino-terminal hydrophobic region. Comparisons of the nucleotide and amino acid sequences with those of influenza A virus neuraminidases revealed seven regions of extensive homology within the central portion of the molecules, including 12 conserved cysteine residues. Five other cysteine residues in the terminal portions were also conserved.     

Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria.

Three antigenic determinants of Borrelia burgdorferi that were shared with other spirochetes and gram-negative bacterial species, as measured by Western blot, ELISA, or both, were identified and characterized using monoclonal antibodies (MAbs). Two were associated with immunogenic integral membrane lipoproteins of 19- and 22-kDa, respectively, by [3H]palmitate incorporation and Triton X-114 phase partitioning. A third antigenic determinant was shown to be associated with a 72-kDa heat shock protein that was also immunogenic in human patients. MAb agglutination assays with B. burgdorferi and treatment of the spirochete with proteases indicated that none of the antigenic determinants were surface exposed. NH2-terminal sequence analysis revealed the 72-kDa protein to have 100% identity with the first 13 amino acid residues of the Escherichia coli dnaK heat shock protein. The presence of these and other shared antigenic determinants in ELISA antigen preparations could explain the high degree of serologic cross-reactivity in current diagnostic procedures.     

Complete nucleotide sequence of an Indian strain of Japanese encephalitis virus: sequence comparison with other strains and phylogenetic analysis.

The RNA genome of an Indian strain of Japanese encephalitis virus (JEV), GP78, was reverse transcribed and the cDNA fragments were cloned in bacterial plasmids. Nucleotide sequencing of the cDNA clones covering the entire genome of the virus established that the GP78 genome was 10,976 nucleotides long. An open reading frame of 10,296 bases, capable of coding for a 3,432 amino acid polyprotein, was flanked by 95- and 585-base long 5'- and 3'-non-coding regions, respectively. When compared with the nucleotide sequence of the JaOArS982 strain, the JEV GP78 genome had a number of nucleotide substitutions that were scattered throughout the genome except for the 5'-noncoding region, the sequence of which was fully conserved. Comparison of the complete genome sequences of different JEV isolates showed a 1.3-4.1% nucleotide sequence divergence among them, which resulted in 0.6-1.8% amino acid sequence divergence. Analysis based on the complete genome sequences of different JEV isolates showed that the GP78 isolate from India was phylogenetically closer to the Chinese SA14 isolate.