重庆地区急性呼吸道感染患儿呼吸道合胞病毒流行特征及其G基因序列分析

目的 分析重庆地区2008-2009年度急性呼吸道感染住院患儿呼吸道合胞病毒(respiratory syncytial virus,RSV)的亚型流行情况,并了解优势流行株BA株的G蛋白基因特征.方法 采集2008年4月-2009年3月全年于重庆医科大学附属儿童医院因急性呼吸道感染住院的508例患儿鼻咽深部分泌物,用RT-PCR方法检测RSV并进行亚型鉴定,选取29例B亚型和10例A亚型RSV阳性标本,用RT-PCR的方法扩增全长G蛋白并测序.结果 在508例标本中,RSV阳性126例(24.8%),其中检测出A亚型43例(34.1%),B亚型80例(63.5%),A、B亚型混合感染3例(2.4%).所测的10株A亚型的G基因与标准株A2的核苷酸同源性为91.4%～92.0%,均属GA2基因型;29株B亚型的G基因与标准株CH18537的核苷酸同源性为92.0%～93.0%,其中19株均为具有60个高度重复核苷酸插入的BA株.B亚型流行株与CH18537标准株相比,G基因有多种核苷酸变异如缺失、插入等,尤其在G蛋白近C端1/3处的高变区.结论 2008-2009年RSV仍是重庆地区儿童急性呼吸道感染的主要病原,与既往两年A亚型优势流行不同,2008-2009年度B亚型毒株流行占优;近年新发现的BA株可能已成为本地区优势流行株,BA株G基因变异是否导致G蛋白功能增强,进而促进其优势流行尚有待研究.     

Prevailing genotype distribution and characteristics of human respiratory syncytial virus in northeastern China

Although human respiratory syncytial virus (RSV) is one of the most common viruses inducing respiratory tract infections in young children and the elderly, the genotype distribution and characteristics of RSV in northeastern China have not been investigated. Here, we identified 25 RSV‐A and 8 RSV‐B strains from 80 samples of patients with respiratory infections between February 2015 and May 2015. All 25 RSV‐A viruses were classified as the ON1 genotype, which rapidly spread and became the dominant genotype in the world since being identified in Ontario (Canada) in December 2010. All eight RSV‐B viruses belonged to the BA genotype with a 60‐nucleotide duplication, seven of which formed two new genotypes, BA‐CCA and BA‐CCB. The remaining RSV‐B virus clustered with one of the Hangzhou strains belonging to genotype BA11. Construction of a phylogenetic tree and amino acid substitution analysis showed that Changchun ON1 viruses exclusively constituted Lineages 3, 5 and 6, and contained several unique and newly identified amino acid substitutions, including E224G, R244K, L289I, Y297H, and L298P. Selective pressure was also evaluated, and various N and O‐glycosylation sites were predicted. This study provides the first genetic analysis of RSV in northeastern China and may facilitate a better understanding of the evolution of this virus locally and globally. J. Med. Virol. 89:222–233, 2017 . © 2016 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.

     

Nosocomial outbreak of respiratory syncytial virus subgroup B variants with the 60 nucleotides-duplicated G protein gene

In January 2001, 20 children among 40 residents under 2 years old at a nursery home in Sapporo, Japan had respiratory symptoms and were confirmed as having respiratory syncytial virus (RSV) infection by a conventional diagnostic kit. Nasopharyngeal aspirates were collected from four RSV-positive patients and total RNA was extracted directly from the specimens for the analysis of RSV grouping and genotyping. All four RSV strains had the same G protein gene sequence of subgroup B and were assigned to identical strains. Interestingly, the G protein gene had a duplication of 60 nucleotides at the C-terminal third of the G protein gene in which three nucleotides differed each other. The predicted polypeptide is lengthened by 20 amino acids. The clinical picture of these cases was not different from those of patients with other RSV strains. These novel mutations were thought to be introduced in vivo.     

Circulating Respiratory Syncytial Virus Genotypes and Genetic Variability of the G Gene during 2017 and 2018/2019 Seasonal Epidemics Isolated from Children with Lower Respiratory Tract Infections in Daejeon,Korea

BackgroundRespiratory syncytial virus (RSV) is a major pathogen causing respiratory tract infections in infants and young children. The aim of this study was to confirm the genetic evolution of RSV causing respiratory infections in children at Daejeon in Korea, through G gene analysis of RSV-A and RSV-B strains that were prevalent from 2017 to 2019.MethodsPediatric patients admitted for lower respiratory tract infections at The Catholic University of Korea Daejeon St. Mary''s Hospital in the 2017 and 2018/2019 RSV seasonal epidemics, who had RSV detected via multiplex polymerase chain reaction (PCR) were included. The nucleic acid containing RSV-RNA isolated from each of the patients'' nasal discharge during standard multiplex PCR testing was stored. The G gene was sequenced and phylogenetic analysis was performed using MEGA X program and the genotype was confirmed.ResultsA total of 155 specimens including 49 specimens from 2017 and 106 specimens from 2018-2019 were tested. The genotype was confirmed in 18 specimens (RSV-A:RSV-B = 4:14) from 2017 and 8 specimens (RSV-A:RSV-B = 7:1) from 2018/2019. In the phylogenetic analysis, all RSV-A type showed ON1 genotype and RSV-B showed BA9 genotype.ConclusionRSV-B belonging to BA9 in 2017, and RSV-A belonging to ON1 genotype in 2018/2019 was the most prevalent circulating genotypes during the two RSV seasons in Daejeon, Korea.     

Genetic diversity among respiratory syncytial viruses that have caused repeated infections in children from rural India

Respiratory syncytial virus (RSV) causes repeat infections throughout life. Antigenic variability in the RSV G protein may play a significant role in reinfections. A variable region of the RSV G gene was analyzed for 14 viruses from seven children who experienced initial and repeat infections. Eleven group A strains were in clades GA2 and GA5 and the three group B viruses were in the newly identified BA clade. In five children reinfections were caused by a heterologous group or genotype of RSV. Two children experienced infection and reinfection by viruses of the same clade, these virus pairs differed by only two to three amino acids in the region compared. This is the first report of RSV nucleotide sequence analysis from India and one of the few molecular characterizations of paired RSV from reinfections. Determining the molecular basis of reinfections may have important implications for RSV vaccine development.     

Chimeric subgroup A respiratory syncytial virus with the glycoproteins substituted by those of subgroup B and RSV without the M2-2 gene are attenuated in African green monkeys

Using the existing reverse genetics system developed for the subgroup A respiratory syncytial virus (RSV), a chimeric virus (designated rA-G(B)F(B)) that expresses subgroup B-specific antigens was constructed by replacing the G and F genes of the A2 strain with those of the 9320 strain of subgroup B RSV. rA-G(B)F(B) grew well in tissue culture, but it was attenuated in the respiratory tracts of cotton rats and African green monkeys. To further attenuate this chimeric RSV, the M2-2 open reading frame was removed from rA-G(B)F(B). rA-G(B)F(B)DeltaM2-2 was highly attenuated in replication in the respiratory tracts of the infected monkeys, but it provided complete protection against wild-type subgroup B RSV challenge following two doses of infection. In this study, rA2DeltaM2-2 (a recombinant A2 RSV that lacks the M2-2 gene) was also evaluated in African green monkeys. The replication of rA2DeltaM2-2 was highly restricted in both the upper and lower respiratory tracts of the infected monkeys and it induced titers of serum anti-RSV neutralizing antibody that were slightly lower than those induced by wild-type rA2. When rA2DeltaM2-2-infected monkeys were challenged with wild-type A2 virus, the replication of the challenge virus was reduced by approximately 100-fold in the upper respiratory tract and 45,000-fold in the lower respiratory tracts. rA2DeltaM2-2 and rA-G(B)F(B)DeltaM2-2 could represent a bivalent RSV vaccine composition for protection against multiple strains from the two RSV subgroups.     

Distinct patterns of evolution between respiratory syncytial virus subgroups A and B from New Zealand isolates collected over thirty-seven years

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract infections in infants and children worldwide. In New Zealand, infants with RSV disease are hospitalized at a higher rate than other industrialized countries, without a proportionate increase in known risk factors. The molecular epidemiology of RSV in New Zealand has never been described. Therefore, we analyzed viral attachment glycoprotein (G) gene sequences from 106 RSV subgroup A isolates collected in New Zealand between 1967 and 2003, and 38 subgroup B viruses collected between 1984 and 2004. Subgroup A and B sequences were aligned separately, and compared to sequences of viruses isolated from other countries during a similar period. Genotyping and clustering analyses showed RSV in New Zealand is similar and temporally related to viruses found in other countries. By quantifying temporal clustering, we found subgroup B viruses clustered more strongly than subgroup A viruses. RSV B sequences displayed more variability in stop codon usage and predicted protein length, and had a higher degree of predicted O-glycosylation site changes than RSV A. The mutation rate calculated for the RSV B G gene was significantly higher than for RSV A. Together, these data reveal that RSV subgroups exhibit different patterns of evolution, with subgroup B viruses evolving faster than A.     

Identification of a variant subgroup A strain of respiratory syncytial virus.

During epidemiologic surveillance of children with respiratory syncytial virus (RSV) disease in Huntington, W.Va., we identified seven strains of a new variant subgroup A RSV (subgroup A-Var) by their reactions in an enzyme immunoassay with two anti-F monoclonal antibodies (MAbs) specific for two epitopes, F1 and F4, generated against the subgroup B RSV. The prototype strain of subgroup A and all other subgroup A field strains from that epidemiologic year failed to react with these two subgroup B MAbs. Additional enzyme immunoassays with 18 subgroup B anti-F MAbs specific for 14 epitopes showed that subgroup A-Var strains also reacted with a MAb specific for the subgroup B F2 epitope. In a radioimmune precipitation assay, the molecular size of the subgroup A-Var F2 subunit of the fusion (F) protein clearly differed from those of both prototype strains of subgroup A and subgroup B RSV. The molecular size of the F2 subunit of subgroup A-Var (24 kDa) was intermediate between the size of the F2 subunit of subgroup A (25 kDa) and that of subgroup B (23 kDa). However, the molecular sizes of the F1 subunits of both subgroup A and subgroup A-Var were identical (54 kDa) and slightly larger than those of the F1 subunits of both subgroups B1 and B2 (53 kDa). These data suggest that subgroup A-Var may represent a distinct RSV A subgroup, analogous to subgroup B1 and B2 RSV, and it is the first-identified naturally occurring subgroup A RSV with an F protein different from that of the prototype A RSV.     

Molecular epidemiology and genetic variability of respiratory syncytial virus (RSV) in Stockholm, 2002-2003

The epidemiology and genetic variability of circulating respiratory syncytial virus (RSV) strains in Stockholm during the season 2002-2003 were studied in consecutive RSV isolates derived from respiratory samples and diagnosed in the laboratory. Two hundred thirty-four viruses were sequenced. The samples were mainly from children under 1 year old (79%). The phylogeny of the N-terminal part of the G gene was studied after amplification and sequencing. One hundred fifty-two viruses belonged to subgroup B and 82 to subgroup A. The subgroup A viruses could be further divided into genotypes GA2 (25) and GA5 (57) and the subgroup B viruses into GB3 (137) and SAB1 (15) strains. These strains clustered with subgroup A and subgroup B strains from Kenya from the same period, as well as with strains from Great Britain from 1995 to 1998. The dominance of subgroup B strains in Stockholm during 2002-2003 is in agreement with findings from other parts of the world during the same years. Only two genotypes of subgroup A, GA2 and GA5, were circulating during this time, and GA2 has been circulating in Sweden for more than 20 years. Consecutive strains from the same individual displayed no variability in the sequenced region, which was also true of strains that had been passaged in cell cultures.     

Occurrence of respiratory syncytial virus subgroup A and B strains in Japan, 1980 to 1987.

The subgroup characteristics of 71 strains of respiratory syncytial virus (RSV) isolated in Sapporo, Japan, during 5 epidemic years from 1980 to 1987 were determined by the use of 17 monoclonal antibodies (MAbs) raised against the RSV Long strain, which is now recognized as the prototype subgroup A strain. Nine of these MAbs immunoprecipitated the fusion protein (F), five immunoprecipitated the large glycoprotein (G), two immunoprecipitated the nucleoprotein (NP), and one immunoprecipitated the phosphoprotein (P). Based on the pattern of reaction of these MAbs to RSV isolates in an indirect immunofluorescence assay, we were able to distinguish two different subgroups. Subgroup A strains reacted to all 17 MAbs. Subgroup B strains reacted to none of the anti-G MAbs, eight of the nine anti-F MAbs, and all anti-NP and anti-P MAbs. Subgroup A included 38 (53.5%) isolates from every epidemic year. Subgroup B included 32 (45.1%) strains isolated in the last 4 epidemic years. One virus strain with an intermediate character of reactivity was isolated in 1983. From the first epidemic year, six subgroup A strains and no B strains were isolated. During the next three annual epidemics, subgroup B strains were predominantly isolated, i.e., 8 of 13, 10 of 13, and 13 of 17 strains. However, in the last epidemic year only one strain of 22 isolates belonged to subgroup B, and the remainder belonged to subgroup A. This variability of dominance in the occurrence of different RSV subgroup strains may suggest a possible role of the subgroup-specific immune response in RSV epidemics.     

Molecular characterization of human respiratory syncytial virus, 2010-2011: identification of genotype ON1 and a new subgroup B genotype in Thailand

This study reports the molecular epidemiology and genetic characterization of human respiratory syncytial virus (RSV) samples collected in Thailand from January 2010 to December 2011. In total, 1,315 clinical samples were collected from Bangkok and Khon Kaen provinces and were screened by semi-nested PCR for RSV infection. We found 74 samples (27.7 %) and 71 samples (6.8 %) to be RSV positive for Bangkok and Khon Kaen, respectively, and we sequenced 122 of these samples. Phylogenetic analysis revealed that 100 of the RSV-A-positive samples clustered into either genotype NA1 or the recently discovered genotype ON1 strain, which has a 72-nucleotide duplication in the second variable region of its G protein. Moreover, 22 of the RSV-B-positive samples clustered into four genotypes; BA4, BA9, BA10 and genotype THB, first described here. The NA1 genotype was found to be the predominant strain in 2010 and 2011. The ON1 strain detected in this study first emerged in 2011 and is genetically similar to ON1 strains characterized in other counties. We also describe the THB genotype, which was first identified in 2005 and is genetically similar to the GB2 genotype. In conclusion, this study indicates the importance of molecular epidemiology and characterization of RSV in Thailand in order to better understand this virus. Further studies should be conducted to bolster the development of antiviral agents and a vaccine.     

Antigenic diversity of respiratory syncytial virus subgroup B strains circulating during a community outbreak of infection

The epidemiological characteristics and relationship between respiratory syncytial virus (RSV) subgroup and virulence during an outbreak of RSV infection occurring in Southeast Texas in the winter season 1991/92 are described. Fiftytwo infants and children were diagnosed with RSV infection by rapid viral antigen detection and/or viral isolation. Subgrouping of the isolates was carried out using 11-monoclonal antibodies. Ten isolates were found to be subgroup B, and 8 isolates were subgroup A. The subgroup B strains showed 3 different patterns of reaction with monoclonal antibodies; one of these subgroups was examined further by restriction analysis of parts of its nucleocapsid and attachment protein genes. The peak of RSV outbreak was in December 1991. Both subtypes A and B circulated simultaneously in the same territory, and caused lower respiratory tract infections in similar proportions. The more frequent occurrence of the B subgroup and the diversity of its simultaneously circulated RSV strains have made this outbreak unusual. © 1994 Wiley-Liss, Inc.     

Evaluation of subgroup-specific peptides of the G protein of respiratory syncytial virus for characterization of the immune response

Two synthetic peptides, designated peptides 12G(A) and 12G(B), representing amino acids 174–188 of the G glycoprotein of respiratory syncytial virus (RSV) subgroup A (strain A2) and subgroup B (strain CH18537) were evaluated for their properties as subgroup-specific antigens for enzyme immunoassay (ELISA). These peptides were used to characterize the immune response of children with naturally occurring RSV infection during six annual epidemics in the Huntington area, West Virginia, USA; viz. 1978–1979, 1979–1980, 198G1981, 1983–1984, 1989–1990, and 1990–1991. The study group comprised 43 paired sera from 42 infants and children, who ranged in age between 1 month and 5.5 years of age (median age 16 months). The inclusion criteria were subgroup identification of RSV, respiratory tract illness requiring admission to hospital, and the availability of paired sera. Five of 30 children with subgroup A and 3 of 13 children with subgroup B infections developed homologous or dual fourfold or greater antibody responses to peptides 12G(A) and 12G(B) during convalescence; six of these eight children also developed antibody rises to whole virus antigens. Twenty children (14 subgroup A and 6 subgroup B) developed such responses in antibody only to whole virus (not to the peptides), and 15 children (11 subgroup A and 4 subgroup B) failed to develop a rise in antibody. Children who developed rises in antibody to the peptides were usually less than 9 months of age, suggesting that a response to peptides was more likely to occur during primary infection. Peptides 12G(A) and 12G(B) of RSV G protein lacked sufficient sensitivity and specificity to serve as antigens for ELISA for characterizing the subgroup-specific immune responses to RSV infection in infants and children. © 1995 WiIey-Liss, Inc.     

Molecular epidemiology of human respiratory syncytial virus subgroups A and B identified in adults with hematological malignancy attending an Irish hospital between 2004 and 2009

Human respiratory syncytial virus (HRSV) is an important cause of respiratory infection in patients with hematological malignancy, particularly hematopoietic stem cell transplant recipients. This study investigated the genetic variability of the attachment (G) protein gene among HRSV isolates collected from adult patients with hematological malignancy. Between December 2004 and March 2009, 60 samples collected from 58 adults attending an Irish hospital were positive for HRSV by direct immunofluorescence. Nucleotide sequence analysis of the G gene showed a slightly higher frequency of HRSV subgroup A (52%) than HRSV subgroup B (48%). Genetic variability was higher among subgroup A viruses (up to 13% at nucleotide level) than among subgroup B viruses (up to 4%). Phylogenetic analysis revealed two genotypes of HRSV subgroup A, GA2 and GA5, which cocirculated between 2004/2005 and 2007/2008, although GA2 alone was identified in season 2008/2009. Genotype BA was the only genotype of HRSV subgroup B identified. Genotype-specific amino acid substitutions were identified, with two and seven changes for GA2 and GA5, respectively. Furthermore, one to four potential N-glycosylation sites were found among HRSV subgroup A isolates while two to three were identified in HRSV B isolates. Predicted O-glycosylation sites included 25-34 and 40-43 in HRSV subgroups A and B, respectively. The average synonymous mutation-to-non-synonymous mutation ratios (dS/dN) implied neutral selection pressure on both HRSV subgroup isolates. This study provides data for the first time on the molecular epidemiology of HRSV isolates over five successive epidemic seasons among patients attending an Irish hospital.     

RSV molecular characterization and specific antibody response in young children with acute lower respiratory infection.

The presence of respiratory syncytial virus (RSV) in nasopharyngeal aspirates (NPA) were studied in 254 hospitalized Argentinean children with acute lower respiratory infection tract (ALRI). The specific humoral immune response and partial sequences of the G protein gene were studied in a subset of 22 children with RSV confirmed infection. The RSV IgM detection and the RSV IgG titration were made by immunofluorescence assay (IFA) in pairs of sera. The partial RSV G gene sequences were obtained by an RT-PCR amplification directly from de NPAs. RSV was present in 44.5% of the children. The RSV IgM was detected in 22.7 and 68.8% of the first and second sera, respectively. The IgG geometric mean titers of the acute and convalescent sera were 8 and 589. The RSV IgG titration was able to define 86.4% of the RSV confirmed cases. The percentage of coincidence between RSV IgM detection in the second sera and diagnosis by RSV IgG titration was 72.7% and no significant differences were observed. The nucleotide sequence of one group A and three group B viruses were identified. The first one was related with circulating viruses in Madrid, Montevideo and Mozambique during 1992, 1989 and 1999, respectively. The three sequences identified as group B viruses were closely related with circulating viruses in 1998 from South Africa and Canada during 1999 and 2000. The data obtained in our study provide the first approach at the molecular level (nucleotide) of the RSV circulating strains in Argentina and the lack of genotype patterns previously determined make necessary a continuous molecular surveillance in order to contribute to the understanding of the behavior of this virus in our community.     

Prevalence and genetic characterization of respiratory syncytial virus (RSV) in hospitalized children in Korea

Human respiratory syncytial virus (HRSV) is the most common respiratory pathogen among infants and young children. To investigate the prevalence and genetic characteristics of HRSVs circulating in South Korea, we analyzed medical records of patients and performed molecular analysis of the G-protein gene of viruses detected from nasopharyngeal aspirates (NPA) of admitted patients at the Pediatrics Department of Chungbuk National University Hospital from April 2008 to April 2010. Epidemiological data revealed that the prevalence of HRSV infection was high during both winter seasons (October 2008 to February 2009 and November 2009 to February 2010). Of the 297 positive NPA specimens from infants or children tested, 67% were identified as HRSV-A while 33% were HRSV-B. The HRSV subgroup B was the most dominant in December 2008, but its dominance was dramatically replaced by HRSV subgroup A strains by February 2009. Phylogenetic analysis of the G protein sequences of HRSVs revealed novel genotypes within the HRSV-A (genotype CB-A) and B (genotypes BA11 and CB-B) subgroups in South Korea in addition to other strains identified in other countries. Molecular analysis also revealed genetic variability at the C-terminal end of the G proteins of the two HRSV subgroups, suggesting selection pressure in this region, which may potentially impact immune recognition. This is the first report of these HRSV variants in South Korea, indicating active genetic evolution of HRSV strains. Therefore, this study provides information on the molecular epidemiology of current HRSVs in the country and presents data for comparative analysis with other HRSV strains circulating worldwide.