Co-circulating genetically divergent A2 human metapneumovirus strains among children in southern Taiwan

An outbreak of human metapneumovirus (hMPV) among children in southern Taiwan in 2004 prompted the investigation of the molecular epidemiology of hMPV from September 2003 to August 2005. Respiratory specimens that were culture negative for a panel of respiratory viruses were examined for the presence of hMPV by RT-PCR. The results indicated that 59 out of 546 (10.8%) children were hMPV-positive. The majority of these hMPV-positive children were less than 2 years old (59.4%), females (61%), and inpatients (67.8%). Infections occurred throughout the year, but peaked during the spring and/or summer months. Sequence analysis of the fusion gene from the isolates revealed two phylogenetic groups with five possible lineages (A1, A2a/A2b, B1, and B2). Among these co-circulating strains, A2 strains were most frequently observed and demonstrated the greatest divergence. Deduced amino acid sequence analysis identified several variant amino acids specific to the A2 lineage. Lineage-specific amino acid substitutions were noted at aa233, aa286, aa312, aa348, and aa296. This study indicated that genetically divergent strains of hMPV which caused respiratory disease and hospitalization were circulating among children in Taiwan.     

Genetic heterogeneity of G and F protein genes from Argentinean human metapneumovirus strains

Human metapneumovirus (hMPV) is a newly identified paramixovirus, associated with respiratory illnesses in all age groups. Two genetic groups of hMPV have been described. The nucleotide sequences of the G and F genes from 11 Argentinean hMPV strains (1998-2003) were determined by RT-PCR and direct sequencing. Phylogenetic analysis showed that hMPV strains clustered into two main genetic lineages, A and B. Strains clustered into A group were split into two sublineages, A1 and A2. All strains belonging to group B clustered with representative strains from sublineage B1. No Argentinean strains belonged to sublineage B2. F sequences showed high percentage identities at nucleotide and amino acid levels. In contrast, G sequences showed high diversity between A and B groups. Most changes observed in the deduced G protein sequence were amino acid substitutions in the extracellular domain, and changes in stop codon usage leading to different lengths in the G proteins. High content of serine and threonine residues were also shown, suggesting that this protein would be highly glycosylated. The potential sites for N- and O-glycosylation seem to have a different conservation pattern between the two main groups. This is the first report on the genetic variability of the G and F protein genes of hMPV strains in South America. Two main genetic groups and at least three subgroups were revealed among Argentinean hMPV strains. The F protein seems to be highly conserved, whereas the G protein showed extensive diversity between groups A and B.     

Phylogenetic analysis of human metapneumovirus from New York State patients during February through April 2010

Background

Human metapneumovirus (hMPV) is the second leading cause of lower respiratory infection (LRI) in children around the world and has been linked to LRI in multiple studies. Currently, hMPV is classified into 2 major subtypes (A and B), each with 2 subgroups (1 and 2).

Objective

To determine which hMPV genotypes were present in NYS patients with influenza-like illness (ILI) from February through April 2010, during a period of unusually heightened activity.

Study design

Specimens were collected from February through April of 2010 from patients presenting with ILI who were previously confirmed as positive for hMPV by real-time RT-PCR. A 700 base pair region of the hMPV fusion (F) gene was amplified, sequenced and resulting sequences aligned. A phylogenic tree was constructed based on prototype strains, and the partial F gene sequences obtained in this study.

Results

Bi-directional sequence was obtained from 30 patient samples and included in the phylogenic analysis. Specimen sequences grouped into hMPV genotype A2a (16), A2b (9), B2 (4) and B1 (1). No A1 genotypes were found.

Conclusion

Previously, reports have demonstrated that genotypes A1, A2, B1 and B2 circulate every season, usually with one dominant strain. In contrast, late in the 2009-2010 respiratory season, 4 of the 5 recognized genotypes of hMPV were isolated from NYS ILI patients, and by sequencing a larger portion of the fusion gene, we were able to identify the A2a and A2b genotypes.     

北京地区人偏肺病毒融合蛋白F编码基因的序列分析

目的进一步了解北京地区人偏肺病毒（hMPV）编码基因的特征。方法在原先工作的基础上，从分属于hMPV的两个不同基因进化簇（即基因型）的两份临床标本BJ1816和BJ1887中扩增F蛋白全基因，克隆至pBS-T载体中并进行测序，与GenBank中hMPV的基因序列进行比较分析和种系进化分析。结果BJ1816和BJ1887的F基因全长均为1620个核苷酸，编码539个氨基酸。BJ1816和BJ1887F蛋白基因之间的核苷酸和推导的氨基酸同源性分别为83．8％和94．4％，与同一基因簇内的hMPV有相当高的氨基酸同源性（98．3％～99．6％）。BJ1816和BJ1887的F蛋白是典型的I型糖蛋白，具有与迄今已发现的hMPV相同的裂解位点（RQSR）。BJ1816和BJ1887之间F2亚单位的氨基酸同源性高于F1亚单位的同源性（96．9％vs93．8％）；除位于羧基末端的跨膜区和胞质尾区外，F1亚单位的其余部分较保守（95．4％）；糖基化位点保守。种系进化分析显示BJ1816和BJ1887属于不同的进化簇。结论F蛋白的序列分析进一步证明BJ1816和BJ1887分属于不同的基因型，其F蛋白的基因特征与其他国外文献中所报道的hMPV相似，是病毒的膜表面糖蛋白，提示其在病毒的感染与免疫中起到重要的作用。     

人偏肺病毒广州分离株全基因组序列测定及分析

目的 探讨广州地区儿童感染的偏肺病毒基因组分子结构特点和基因型类型.方法 参考GenBank上的偏肺病毒00-1株(AF371337)基因组设计分段扩增引物,进行RT-PCR分段扩增偏肺病毒基因组,克隆于T载体上,序列测定,用Clustal W/X、DNASTAR、MEGA4.1等软件分析基因组序列.结果 偏肺病毒hMPVgz01全基因组为13 327 bp,提交到GenBank上的序列号为GQ153651,有8个开放阅读框(open reading frames,ORFs),基因组结构为:3-N-P-M-F-M2-SH-G-L-5;将hMPVgz01株全基因组核苷酸序列与GenBank上的偏肺病毒全基因组序列进行Clustal W比较,发现与偏肺病毒的A组相似性较高,为92%～97%,与A2b组的BJ1887相似性为最高,而与B组相似性为81%,与C组的禽偏肺病毒为71%.将hMPVgz01株的N、F、G基因与偏肺病毒的A1、A2、B1、B2的相对应基因进行相似性比较,同样也是与A2b型的相似性为最高,因此确认广州地区的hMPVgz01株为A2b型.结论 广州地区儿童感染的偏肺病毒hMPVgz01株全基因组序列为13 327 bp,GenBank 序列号为GQ153651,hMPVgz01与偏肺病毒的A2b型相似性最高,确认hMPVgz01株属于偏肺病毒A2b型.     

Genotype variability of human metapneumovirus, South Korea

Human metapneumovirus (hMPV), a virus causing lower respiratory tract infections in children, is classified two major groups or genotypes of hMPV and recently existence of multiple lineages has been suggested. The purpose of this study was to examine the extent of genetic variation and circulation pattern of hMPV in Korea. Between January 2005 and April 2007, nasopharyngeal aspirates were collected from 1,214 children <16 years of age hospitalized with acute respiratory tract infection at Sanggyepaik Hospital. Nasopharyngeal aspirates were tested for common respiratory pathogens using immunofluorescence or multiplex RT-PCR. RT-PCR was used to detect hMPV. The PCR products were purified and subsequently sequenced directly on both strands. hMPV was detected in 8.4% (102/1,214) of nasopharyngeal aspirates from children with acute respiratory tract infection. The 102 hMPV strains detected in this study were classified into two distinct F lineages, 87 strains belonged to genogroup A2 (A2a in 42, A2b in 45) and 15 strains to genogroup B. All hMPV subtypes except A1 co-circulated in Korean population. Although alternating predominance of hMPV subtypes from year to year could not be found, the changing predominance of sublineage A2a and A2b was demonstrated.     

Serological recognition of feline infectious peritonitis virus spike gene regions expressed as synthetic peptides andE. coli fusion protein

Summary Cats exposed to feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV) cannot be differentiated by serological analysis. Three synthetic peptides and anE. coli recombinant fusion protein generated from FIPV 79-1146 spike gene sequence were produced. Coronavirus positive cat sera reacted to peptide aa 950–990 but were non-reactive to aa137–151 and aa 150–180 peptides as demonstrated by ELISA. Amino acid sequence 97–222 expressed as a galk fusion protein inE. coli was tested against coronavirus positive cat sera by western blot analysis. Only sera from cats exposed to the FIPV type-II strains DF-2 or 79–1146 that were exhibiting signs of FIP recognized the fusion protein. Sera from FECV exposed cats did not recognize the 97–222 fusion protein in western blot analysis.     

Variation in the fusion glycoprotein gene of human respiratory syncytial virus subgroup A

Six different genotypes (designated lineages SHL1–6) of human respiratory syncytial (RS) virus have been defined by partial nucleotide sequence analysis of the variable SH and the hypervariable G membrane protein genes, and by restriction fragment analysis of the conserved N protein gene of viruses isolated in south Birmingham. Viruses of very similar genotype appear to be present worldwide at the present time. We have determined the nucleotide sequences of the fusion protein genes of five viruses isolated in south Birmingham in the same year, but belonging to different lineages, and have compared them with the sequences of four subgroup A viruses isolated at earlier times from diverse localities. The sequence diversity of the F genes of these five viruses, as measured by nucleotide (94.5–98.5%) and inferred amino acid (97.0–99.3%) identities, is comparable with that of the nine subgroup A viruses considered as a whole. No sequence changes occur in any of the sites of known epitopes. Comparison of the nine subgroup A sequences with the published sequences of a subgroup B strain and three bovine RS viruses confirms that the F protein sequences are most divergent in the F₂ region.     

Immunofluorescence Assay for Detection of Human Metapneumovirus-Specific Antibodies by Use of Baculovirus-Expressed Fusion Protein

Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (κ = 0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.     

Prevalence of human metapneumovirus in hospitalized children with respiratory tract infections in Tianjin,China

Human metapneumovirus (hMPV) has recently been recognized as an important respiratory pathogen, especially in children. At present, our understanding of the characteristics of hMPV from China is very limited. Nasopharyngeal aspirates were taken from 310 hospitalized pediatric patients. Twenty (6.5%) of them were infected with hMPV, and they all developed pneumonia. Sixty five percent (13/20) of the cases were under 12 months. Phylogenetic analysis of F gene fragments indicated that three sub-genotypes of hMPV(A2a/A2b, B1,B2) circulated in Tianjin and A2b was the predominant subtype. The Vero-E6 cell line was better than LLC-MK2 for hMPV isolation. Three hMPV strains were successfully isolated using the Vero-E6 cell line.     

Seroepidemiology of human metapneumovirus (hMPV) on the basis of a novel enzyme-linked immunosorbent assay utilizing hMPV fusion protein expressed in recombinant vesicular stomatitis virus

Human metapneumovirus (hMPV) is a newly identified human respiratory virus now recognized as a major respiratory pathogen of infants and children. To define the seroepidemiology of hMPV, we developed a novel enzyme-linked immunosorbent assay (ELISA) based on expression of the fusion protein of hMPV (hMPV F) in recombinant vesicular stomatitis virus (VSV). Western blot analysis using an hMPV F-specific antiserum confirmed the expression of hMPV in recombinant VSV. The ELISA is specific for hMPV F; antibody specific for the most closely related human paramyxovirus, respiratory syncytial virus, does not bind to hMPV F. Overall, 216 serum specimens were tested. The percentages of seropositive individuals were 89.1% in children < or =5 months old, 55.0% in children 6 to 11 months old, 36.0% in children 12 to 23 months old, 45.0% in children 24 to 47 months old, 77.3% in children 48 to 59 months old, 91.3% in children 5 to 10 years old, and 95.5% for individuals 11 to 20 years old. This is the first seroepidemiological survey of hMPV in the United States and the first analysis to determine the prevalence of antibody to a specific hMPV protein. The data suggest that exposure to hMPV is common in childhood and that hMPV F is an antigenic determinant of hMPV.     

High genetic diversity of the attachment (G) protein of human metapneumovirus

Complete genes encoding the predicted nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), M2-1protein, M2-2protein, small hydrophobic protein (SH), and attachmentprotein (G) of seven newly isolated human metapneumoviruses (hMPVs) were analyzed and compared with previously published data for hMPV genes. Phylogenetic analysis of the nucleotide sequences indicated that there were two genetic groups, tentatively named groups 1 and 2, similar to the grouping of human respiratory syncytial virus. Although the predicted amino acid sequences of N, P, M, F, and M2 were highly conserved between the two groups (amino acid identities, 96% for N, 85% for P, 97% for M, 94% for F, 95% for M2-1, and 90% for M2-2), the amino acid identities of the SH and G proteins were low (SH, 58%; G, 33%). Furthermore, each group could be subdivided into two subgroups by phylogenetic analysis, tentatively named subgroups 1A and 1B and subgroups 2A and 2B. The predicted amino acid sequences of G within members of each subgroup were highly conserved (amino acid identities, 88% for group 1A, 93% for group 1B, and 96% for group 2B). The G of hMPV is thought to be the major antigenic determinant and to play an important role in the production of neutralizing antibodies. Clarification of the antigenic diversity of G is important for epidemiological analysis and for establishment of strategies to prevent hMPV infection.     

中国湖南地区人偏肺病毒的基因分型及其膜蛋白G基因特征的分析

目的 研究中国湖南地区人偏肺病毒(hMPV)的基因分型及其膜蛋白G序列的遗传学特征,比较分析其区域性流行特点.方法收集2005年中国湖南地区232份住院儿童鼻咽抽吸物(NPA)样本进行常见呼吸道病毒筛查,并扩增hMPV阳性样本的膜蛋白G全序列,与GenBank中已知hMPV参考株及其他地区公布的相关资料进行比对、进化等分子遗传学特征分析.结果从232份临床样本中共检测得到17份(7.3%)hMPV阳性样本,与其他病毒混合感染率达35%.扩增出其中13份hMPV样本的G蛋白全序列,分属于4种亚型(A1、A2、B1、B2).核酸长度类型有4种(711,675,660,696nt),N-连接糖基化位点数目和位置、半胱氨酸残基数目等特征与已报道的同期北京地区、日本、北美等地区hMPV调查分析结果不尽一致.结论中国湖南地区与其他地区同期hMPV调查分析结果各有特点,反映出hMPV变异显著,具有明显的地区流行特征.