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。     

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.     

北京地区2株人偏肺病毒的P和M蛋白基因序列分析

目的进一步了解北京地区人偏肺病毒(hMPV)编码基因的特征。方法从分属于hMPV的两个不同基因进化簇(即基因型)的2份临床标本BJ1816和BJ1887中扩增P和M全基因，克隆至pBS-T载体中并进行测序，与GenBank中的hMPV的基因序列进行比较分析和种系进化分析。结果经扩增BJ1816和BJ1887的P基因全长为885个核苷酸，编码294个氨基酸；M基因全长765个核苷酸，编码254个氨基酸。BJ1816和BJ1887的P和M蛋白基因与同一基因簇内的hMPV有相当高的氨基酸同源性(91．9％-100％)。BJ1816和BJ1887间P基因的氨基酸同源性仅为85．8％，而M基因的氨基酸同源性为96．9％。结论BJ1816和BJ1887的P和M蛋白基因特征与其他国外文献中所报道的hMPV相似，不同型的hMPV的P基因之间比M基因显示了更强的基因多样性。     

Evidence of human metapneumovirus in children in Argentina

Human metapneumovirus (hMPV) is a virus, which was first associated with acute lower respiratory infection in children but is detected currently in all age groups. Clinical symptoms are similar to those described for respiratory syncytial virus (RSV) infections, ranging from mild respiratory illness to severe bronchiolitis and pneumonia in children. To date, no cases of hMPV have been reported in Argentina. In this study, 440 respiratory samples obtained during the period 1998-2002 from children under 5 years old with acute respiratory infection were evaluated. Routine detection for RSV, adenovirus, influenza, and parainfluenza was undertaken by immunofluorescent assay. Of the samples negative for these viruses, only 100 were available. All these samples were tested for hMPV by RT-PCR using primers for the L gene. Eleven out of 100 (11%) respiratory samples were positive for hMPV by RT-PCR. A higher frequency of detection was observed in spring. hMPV was detected in all the years studied, except in 2001. Ten out of 11 children positive for hMPV were hospitalized. Median age was 5 months. Of seven patients, five (71%) required oxygen supplementation. The most frequent diagnosis was bronchiolitis (86%), sometimes accompanied by conjunctivitis and otitis media. The present study showed that hMPV was associated with acute lower respiratory infections in children in Buenos Aires, Argentina. This evidence strongly suggests that hMPV is a common pathogen with a wide geographical distribution, which should be included in the routine diagnosis of respiratory viruses in young children.     

Comparison of the seroprevalence of human metapneumovirus and human respiratory syncytial virus

Human metapneumovirus (hMPV) is a virus that induces human respiratory syncytial virus (hRSV)-like illnesses, ranging from upper respiratory tract infection to severe bronchiolitis and pneumonia. The 100 serum samples from children aged 1 month to 5 years were tested for the presence of hMPV and hRSV antibodies using an indirect immunofluorescence assay and a neutralizing-antibody assay, respectively. The seroprevalence of hMPV was significantly lower than that of hRSV in children over 4-months-old (43% vs. 60%, P < 0.025), and the difference was particularly notable between the ages of 4 months and 1 year (11% vs. 48%, P = 0.006). The results suggest that primary infection with hMPV occurs somewhat later than that with hRSV.     

Seroprevalence of human metapneumovirus in Japan

A new human pneumovirus, provisionally designated human metapneumovirus, was discovered by Dutch researchers. We examined 142 serum samples from the general population aged from 1 month to 35 years in Japan for human metapneumovirus antibody by indirect immunofluorescence assays using human metapneumovirus-infected monkey kidney cells. The overall prevalence of human metapneumovirus infection was 72.5%. The seropositive rate was lowest in the age group of 6 months to 1 year and gradually increased with age. All of the children had been exposed to human metapneumovirus by the age of 10 years. The results show that human metapneumovirus is circulating in the Japanese population and is a ubiquitous virus acquired early in life.     

北京新发现的人偏肺病毒N蛋白编码基因的序列分析

目的　了解新近从北京地区发现的人类偏肺病毒 (humanmetapneumovirus ,HMPV)N蛋白编码基因的特征。方法　从经RT PCR检测HMPV阳性的临床鼻咽洗液标本中进行HMPVN蛋白全基因扩增、克隆至pUCm T载体中并进行测序 ,与GenBank中的多株HMPVN蛋白基因序列进行比较和进化树分析。结果　经扩增并测序 ,标本BJ1816和BJ1887N蛋白基因全长为 1185bp ,编码 394个氨基酸。BJ1816N蛋白基因与国际上第一株HMPV分离株NDL0 0 1和GenBank中其它的多株HMPV的核苷酸和氨基酸同源性分别为 86 .2 %～ 99.0 %和 96 .2 %～ 99.7%。BJ1887N蛋白基因与以上进行比较的各株HMPV的核苷酸和氨基酸同源性分别为 86 .6 %～ 97.0 %和 96 .4 %～ 99.5 %。BJ1816和BJ1887之间N基因的核苷酸和氨基酸同源性分别为 86 .6 %和 96 .4 %。提示BJ1816和BJ1887分属于HMPV的两个不同的基因进化簇。结论　从基因水平进一步证明新近发现的婴幼儿肺炎的新病原确为HMPV ,提示北京地区婴幼儿中同时存在两个不同进化簇 (即基因型 )的HMPV感染。     

Limited Inter- and Intra-patient Sequence Diversity of the Genetic Lineage A human metapneumovirus fusion gene

Human metapneumovirus (hMPV) is associated with respiratory tract illness especially in young children. Two hMPV genetic lineages, A and B, and four sublineages A1, A2 and B1, B2 have been defined. Infection with hMPV occurs through membrane fusion mediated by the hMPV fusion (F) protein. In this study, the inter- and intra-patient genetic diversity of the lineage A hMPV F gene was investigated. Ten isolates were collected from 10 hMPV infected children. Viral RNA was isolated and amplified, and approximately 10 clones from each isolate were sequenced. Altogether 108 clones were successfully sequenced. The average interpatient sequence diversity was 1.68% and 1.64% at nucleotide and amino acid levels, respectively. The samples were divisible into two groups on the basis of intrapatient sequence diversity. In group 1 (4 children) the intra-patient sequence diversity was low (nt: 0.26–0.39%, aa: 0.51–0.94%) whereas group 2 (6 children) had a higher intra-patient sequence diversity (nt: 0.85–1.98%, aa: 1.08–2.22%). Phylogenetic analyses showed that the group 1 children harboured sublineage Al only, but interestingly group 2 children harboured both sublineages Al and A2, indicating they had been infected with at least two viruses. Several independent viruses contained premature stop codons in exactly identical positions resulting in truncated fusion proteins. Possibly this is a mechanism for immune system evasion. The F protein is a major antigenic determinant, and the limited sequence diversity observed lay emphasis on the hMPV F gene as a putative target for future vaccine development.     

The role of lipid rafts in cell entry of human metapneumovirus

Human metapneumovirus (hMPV) is a crucial pathogen in children. A cell entry is the first step for infection. Our previous study indicated that there was an endocytosis pathway for hMPV cell entry. Lipid raft is a specific structure at the cell surface and it has been demonstrated to play an important role in endocytosis process of many viruses. In this study, we investigated whether and how lipid raft can take part in the hMPV entry. The confocal microscope was used to detect colocalization of hMPV and lipid raft marker. We demonstrated that colocalizations were increased along with the viral infection and hMPV particles transferred to the perinuclear region with lipid raft. When specific lipid raft inhibitors: methyl-β cyclodextrin and nystatin were used, hMPV cell entry was inhibited and viral titer decreased dramatically. With the replenishment of exogenous cholesterol, hMPV recovered quickly. These data suggest that lipid raft plays an important role in hMPV endocytosis and maybe one of the pathways for hMPV cell entry.     

北京地区人偏肺病毒核蛋白基因的原核表达及抗原活性分析

目的用大肠杆菌表达获得人偏肺病毒主要结构蛋白N蛋白，为下一步的深入研究奠定基础。方法从重组质粒pUCm-N1816中PCR扩增获得N基因，用BamHⅠ和EcoRⅠ双酶切后插入到原核表达载体pET30a（＋）中，得到重组表达质粒pE130a-N1816，转化大肠杆菌BL21（DE3），IPIG诱导培养。SDS-PAGE和Western blot检测目的蛋白的表达和抗原性。结果经双酶切和测序证明1．2kb N1816。基因正确插入pET30a中，并具有正确的读码框架，得到预期的pET30a-N1816重组原核表达质粒。37℃，1mmol／L IFIG诱导培养6h后产生大量带6个组氨酸标记的重组N蛋白，主要以包涵体形式存在，占细胞总蛋白的20％左右。N蛋白粗提物经Co^2＋亲和层析获得较理想纯化。Western blot结果显示，体外所表达的蛋白能和特异性抗血清及人血清反应。结论本研究成功构建了重组pET30a-N1816原核表达质粒，N蛋白获得了高效表达，并且具有特异抗原活性，可用于人偏肺病毒的深入研究。     

Seroepidemiological study of human metapneumovirus in New Delhi, India

Purpose: There are a few seroepidemiological studies reported on human metapneumovirus (hMPV) as hMPV was only discovered in the year 2001. This respiratory virus has been reported to be ubiquitous and associated with respiratory tract infections in all age groups. The present study aimed at determining the prevalence of antibodies to hMPV in children and adults of 1 month to 55 years of age. Materials and Methods: Serum samples from 100 study subjects were tested for hMPV antibody by an in-house ELISA system that used hMPV-infected cell lysate antigen. Result: The prevalence of antibody to hMPV was lowest in children less than 5 years of age (60%) and increased throughout age to > 80%. Similarly, geometric mean titres were 1:180 in children less than 5 years of age and reached a peak of 1:419 in adults over 35 years of age. Conclusion: The results show that hMPV infection is acquired early in life and re-infection in later life may maintain the seroprevalence and antibody levels in adult population.     

绿色荧光蛋白标记重组人偏肺病毒的制备

目的 人偏肺病毒(human metapneumovirus,hMPV)可致人上、下呼吸道感染.研究利用反向遗传学技术,以带绿色荧光蛋白(GFP)标记的hMPV NL/1/00全长cDNA质粒及4种辅助质粒pCITE-N、pCITE-P、pCITE-M2.1和pClTE-L为基础,在体外制备GFP标记的重组hMPV病毒(命名为rhMPV NL/1/00 GFP).方法 采用LipofectAMINE 2000将带GFP标记的hMPV NL/1/00全长cDNA质粒以及蛋白表达质粒pcITE-N、pCITE-P、pCITE-M2.1和pcITE-L共转染BSR-T7细胞,3 d后取BSR-T7细胞上清液感染Vero-E6细胞,1-4 d后观察Veto-E6细胞出现明显细胞病变(CPE)和绿色荧光,持续观察至第10天.收集病毒上清用于病毒滴度检测.提取培养上清的病毒RNA并通过RT-PCR方法扩增病毒N基因、F基因和G基因验证所获重组病毒.结果 Vero-E6细胞接种1～4 d后观察到明显CPE和绿色荧光,随后CPE加重,荧光信号加强,持续至感染后10 d;第1、5、10、15和20代病毒的滴度波动于105.0～106.5TCID50/ml;RT-PCR检测出符合预期大小的910 bp(N)、450 bp(F)和980 bp(G)条带,经卜述片段cDNA序列测定和比对表明获得的重组病毒与hMPV NL/1/00原型病毒序列一致.rhMPV NL/1/00 GFP重组病毒在体外传代20代后,遗传信号和荧光信号均稳定.结论 采用反向遗传学技术成功拯救了具有感染性的重组hMPV病毒,为hMPV感染发病机制及疫苗研究奠定了基础.     

Clinical impact of human metapneumovirus genotypes and genotype-specific seroprevalence in Yamagata, Japan

The clinical impact of human metapneumovirus (hMPV) genotypes and the relation between the hMPV genotype in circulation and genotype-specific seroprevalence are yet to be clarified. We determined the genotypes of 93 hMPV strains that were isolated between 2004 and 2006 in Yamagata, Japan, and identified 35 genotype A2, 14 genotype B1, and 44 genotype B2 isolates. Children infected with genotype A2 hMPV were significantly older than those infected with genotype B1 hMPV. Diagnosis of laryngitis was more common in children with genotype B1 hMPV infection and wheezing was more prevalent in children with genotype B1 and B2 hMPV infection than in those with genotype A2 hMPV infection. We then examined genotype-specific seroprevalence by neutralization assay. The higher seropositive rate for the B2 genotype among the children aged 1-2 years is likely to reflect the outbreak of B2 genotype strains in the previous year in this community. The low seropositive rate for the B1 genotype among children aged 1-2 years appears to be associated with a finding that more than 70% of children infected with the B1 genotype were less than 3 years old. In conclusion, we found that the different clinical characteristics of hMPV infection may be associated with hMPV genotype, and the predominant genotype during a season and the affecting age may be closely related to genotype-specific immune status within a community.     

Detection of antibodies against human metapneumovirus by Western blot using recombinant nucleocapsid and matrix proteins

Detection of antibodies against individual proteins of human metapneumovirus (hMPV) is important in the analysis of immune responses to hMPV. Specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 serum samples were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. The results were compared with those of immunofluorescence assays (IFAs) based on hMPV-infected LLC-MK2 cells, which expressed the whole hMPV proteins. Thirty (61.2%) and 31 (63.3%) of 49 serum samples with titers of > or = 1:160 by IFA reacted with N and M proteins, respectively. Only 2 (4.2%) of 11 serum samples with titers of 1:80 by IFA reacted with N and M proteins. Antibodies against N and M proteins were not detected in 37 serum samples with titers of < 1:40 by IFA. These results indicate that the antibodies against N and M proteins are highly specific (100%) but less sensitive (42.1%, N protein; 40.8%, M protein) than those against whole proteins of hMPV detected by IFA. The reactivity of sera with the recombinant N protein and that with the recombinant M protein correlated well (correlation coefficient of 0.79), and the concordance of reactivities was 91% (kappa = 0.79). In summary, both recombinant N and M proteins of hMPV were antigenic, and the responses to N and M protein varied among patients. Therefore, Western blot using N and M proteins provide a useful tool for analysis of immune responses to hMPV.