人偏肺病毒感染住院儿童的临床特征及病毒基因特性分析

目的 了解急性呼吸道感染住院儿童中人偏肺病毒(hMPV)的感染情况、临床特征及人偏肺病毒基因特性.方法 对2006年2月至5月、2008年3月至4月及2008年9月至2009年2月收集的天津市儿童医院急性呼吸道感染住院儿童的310份鼻咽吸取物标本进行巢式PCR检测hMPV N基因片段,并对其中17株N基因片段进行序列测定,分析序列特征并绘制种系发生树.收集所有阳性患者的临床资料.同时采用多重PCR对N基因阳性标本进行其他常见呼吸道病毒的检测.结果 310份标本中共检出20份(6.5%)hMPV.hMPV阳性患儿的年龄中位数为15.0个月(16天～9岁),其中≤2岁患儿占90.0%(18/20).男性占60.0%(12/20).17株N基因种系发生树分析显示,11株为A2b亚型.20例hMPV阳性患儿的临床诊断均为支气管肺炎,占所有采集的肺炎患儿标本的7.1%(20/282),其临床症状以咳嗽、喘息、呼吸急促、发烧等常见.35%(7/20)需要重症监护,15%(3/20)需要吸氧治疗.平均住院时间为(11.9±4.8)d.A、B型间临床特征未见明显差别.hMPV无明显流行高峰.2例hMPV阳性患儿分别与腺病毒和鼻病毒混合感染.结论 hMPV可引起婴幼儿肺炎等严重疾病.天津地区流行的hMPV以A2b为优势流行型.     

2007—2011年长沙地区急性下呼吸道感染住院儿童人偏肺病毒的流行状况

目的了解人偏肺病毒（hMPV）在长沙地区急性下呼吸道感染住院患儿中的流行病学特点。方法收集2007年9月至2011年2月因急性下呼吸道感染在湖南省人民医院儿科医学中心住院儿童的鼻咽抽吸物（nasopharyngealaspirates,NPA）样本2613份,采用逆转录聚合酶链反应法（RT—PCR）检测hMPVM基因,将阳性PCR扩增产物测序并与GenBank中已知的hMPV参考株进行比对、分析。结果2613份标本中hMPV阳性检出数135例,检出率为5．2％,男女之间的检出率比较有统计学差异（x2=8．007,P=0．003）,hMPV阳性检出患儿的年龄以1岁以内多见（63．2％）。hMPV阳性检出率在春季呈现高峰,从检出季节分布显示A2b型主要在冬春季节流行,而B2型主要在春夏季流行。135例hMPV长沙株分为A型和B型两个主要的基因型,其中A2b亚型在2007--2008年为优势流行型别,2009--2010年A2b和B2型共同流行,B2亚型在2011年呈优势流行型别。135例hMPV检出阳性患儿中有66例（48．9％）存在混合病毒感染,其中与HBoV混合检出率最高。结论长沙地区部分儿童的急性下呼吸道感染与hMPV有关,且阳性检出患儿年龄主要集中在1岁以下,男多于女,主要流行季节在春季,A2b型和B2型优势基因型在长沙地区共同流行,与其他病毒混合检出率较高。     

Characterization of human metapneumovirus infections in Israel

Respiratory tract infections are a leading cause of morbidity and mortality worldwide. Even with the advancement of diagnostic tools, the causative agent of 20 to 30% of upper respiratory tract infections go undiagnosed. Recently, a newly identified human respiratory virus, human metapneumovirus (hMPV), was discovered in young children in The Netherlands. To study the prevalence of hMPV infections in Israeli children, respiratory specimens from 388 hospitalized children less than 5 years of age were evaluated for the presence of hMPV RNA, which was present in 42 (10.8%) of these samples. All hMPV-positive samples were negative for respiratory syncytial virus (RSV), influenza viruses (Flu) A and B, adenovirus, and parainfluenza viruses 1, 2, and 3. Conversely, hMPV RNA was not detected in 76 RSV-positive and 38 Flu A- or B-positive samples. Most hMPV activity was between the months February and April. Sequence analysis of 20 positive samples revealed that both of the hMPV genotypes (groups 1 and 2) have circulated in central Israel during the study period. Moreover, three of the four known hMPV subgroups (1A, 1B, and 2B) were detected among the tested samples. Seroprevalence of hMPV in 204 patients from the central part of Israel revealed that 100% of the children are hMPV seropositive by the age of 5 years old. We conclude that hMPV is a common respiratory pathogen in Israel, while mixed infections of hMPV with RSV or Flu in hospitalized children are apparently rare.     

南京地区儿童人偏肺病毒感染的流行病学及临床特征分析

目的了解南京地区儿童人偏肺病毒（hMPV）感染的流行病学特点及临床特征。方法收集2009年8月至2010年7月南京医科大学附属南京儿童医院住院及门诊呼吸道感染患儿的鼻咽抽吸物（NPA）及咽拭子（NPS）共642例,采用逆转录聚合酶链反应法（RT—PCR）检测hMPVM基因,将阳性PCR扩增产物进行测序、同源性和进化分析。结果642例标本中共检出hMPV阳性扩增产物35份,检出率为5．5％。系统进化分析显示南京地区hMPVB1型占51．4％,A2b型占31．4％。hMPV的发病高峰在4月份。其致呼吸道感染以1岁以内多见（71．4％）。35例hMPV感染患儿中有15例（42．8％）存在混合感染,其中与HRV的混合感染检出率最高。临床诊断以肺炎（17例,48．6％）最为常见。结论人偏肺病毒是南京地区儿童急性呼吸道感染的重要病原之一,该年度其优势流行型别为B1型,南京地区A、B两型hMPV感染患儿临床特征无明显差异。     

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.     

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.     

Epidemiological and clinical features of hMPV, RSV and RVs infections in young children.

BACKGROUND: Human metapneumovirus (hMPV) has recently been isolated from children with acute respiratory tract infections (RTIs). The epidemiological and clinical characteristics of hMPV infection need further investigation. OBJECTIVES: The purpose of this study was to compare the clinical features of hMPV, respiratory syncytial virus (RSV) and rhinoviruses (RV) infections in children less than 3 years of age presenting to an emergency department with acute respiratory illness. STUDY DESIGN: From December 2002 to April 2004, all children under age three (n=931) admitted for acute respiratory illness to Dijon Hospital, France, were investigated for respiratory viruses in nasal washes. RESULTS: hMPV was detected in 6% of children (in 10.1% (n=38) the first winter and in 3.3% (n=17) the second winter); RSV was detected in 28.5% of the children, while rhinoviruses were found in 18.3%. Five hMPV-infected children had evidence of dual infection, two with RSV and three others with RV. The median age of the patients with hMPV infection was 6 months, and the main clinical symptoms were rhinorrhea (74.5%) and cough (67%). A lower tract disease occurred in 66% of hMPV-positive patients. Gene sequencing of hMPV isolates revealed co-circulation of the two major groups of hMPV during the study period; no difference in pathogenicity was found. There was no difference in the prevalence of bronchiolitis where hMPV, RSV or rhinoviruses were present. Asthma was found more often in hospitalized children with hMPV and rhinoviruses than among those with RSV (p<0.001). CONCLUSIONS: These results provide further evidence of the importance of hMPV as a pathogen associated with respiratory tract infection in children.     

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.     

Usefulness of two new methods for diagnosing metapneumovirus infections in children

Human metapneumovirus (hMPV) is associated with acute respiratory tract infections, mainly in paediatric patients. The aim of this study was to evaluate the usefulness of two new commercial techniques available for the detection of hMPV in clinical samples from children: an enzyme immunoassay, hMPV EIA (Biotrin International Ltd), and a molecular assay, real-time RT-PCR (Pro hMPV Real Time Assay Kit; Prodesse). A total of 184 nasopharyngeal aspirate specimens from 173 children aged less than 5 years who were hospitalized with acute wheezing were analysed. Respiratory syncytial virus was detected in 27% of the samples, followed by influenza A virus (6%), parainfluenza virus (PIV)3 (2.2%), adenovirus (2%), PIV1 (1.1%), PIV2 (1.1%), and influenza B virus (0.5%). The presence of hMPV was tested in all samples, using the real-time RT-PCR and EIA. Real-time RT-PCR detected 13 hMPV-positive samples (8%), and EIA detected 17 (9.3%). When the EIA results were compared with those of real-time RT-PCR for the detection of hMPV, a good correlation was found (94%). A relatively low co-infection rate (15%) was observed in our patients. RT-PCR and EIA provide robust methods for the diagnosis of hMPV infection in children.     

Metapneumoviruses in birds and humans

Avian pneumovirus (APV, Turkey rhinotracheitis virus) and Human metapneumovirus (hMPV) are pathogens of birds and humans, respectively, that are associated with upper respiratory tract infections. Based on their different genomic organization and low level of nucleotide (nt) and amino acid (aa) identity with paramyxoviruses in the genus Pneumovirus, APV and hMPV have been classified into a new genus referred to as Metapneumovirus. First isolated in 1970s, APV strains have since been isolated in Europe, Africa, middle east, and United States (US) and classified in four subgroups, APV/A, APV/B, APV/C, and APV/D based on nt and predicted aa sequence identity. Although it was first isolated in 2001, serological evidence indicates that hMPV may have been present in human population from as early as the 1950s. There is only one subgroup of hMPV so far, whose nt and aa sequence identity indicates that it is more closely related to APV/C than to APV/A, APV/B, or APV/D.     

Co-circulation of genetically distinct human metapneumovirus and human bocavirus strains in young children with respiratory tract infections in Italy

The discovery of human Metapneumovirus (hMPV) and human Bocavirus (hBoV) identified the etiological causes of several cases of acute respiratory tract infections in children. This report describes the molecular epidemiology of hMPV and hBoV infections observed following viral surveillance of children hospitalized for acute respiratory tract infections in Milan, Italy. Pharyngeal swabs were collected from 240 children ≤3 years of age (130 males, 110 females; median age, 5.0 months; IQR, 2.0-12.5 months) and tested for respiratory viruses, including hMPV and hBoV, by molecular methods. hMPV-RNA and hBoV-DNA positive samples were characterized molecularly and a phylogenetical analysis was performed. PCR analysis identified 131/240 (54.6%) samples positive for at least one virus. The frequency of hMPV and hBoV infections was similar (8.3% and 12.1%, respectively). Both infections were associated with lower respiratory tract infections: hMPV was present as a single infectious agent in 7.2% of children with bronchiolitis, hBoV was associated with 18.5% of pediatric pneumonias and identified frequently as a single etiological agent. Genetically distinct hMPV and hBoV strains were identified in children examined with respiratory tract infections. Phylogenetic analysis showed an increased prevalence of hMPV genotype A (A2b sublineage) compared to genotype B (80% vs. 20%, respectively) and of the hBoV genotype St2 compared to genotype St1 (71.4% vs. 28.6%, respectively). Interestingly, a shift in hMPV infections resulting from A2 strains has been observed in recent years. In addition, the occurrence of recombination events between two hBoV strains with a breakpoint located in the VP1/VP2 region was identified.     

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.     

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.     

Diagnosis and epidemiological studies of human metapneumovirus using real-time PCR.

BACKGROUND: Human metapneumovirus (hMPV) is prevalent in children, the elderly and immunocompromised individuals, but available epidemiological data is limited. OBJECTIVES: (1) To develop and validate a real-time PCR method for hMPV diagnosis. (2) To determine the percentage of hMPV in respiratory specimens from the community and its association with outbreaks in our geographic area. (3) To provide epidemiological data in terms of age distribution, seasonality and co-infections. STUDY DESIGN: A real-time PCR assay was designed for detection of hMPV lineages A and B. Prospective testing for hMPV over a 22-month period was then undertaken. RESULTS: The real-time PCR was sensitive and specific for detection of both lineages of hMPV. hMPV was detected in 9.5% (n=8239) of the specimens and 25% of the outbreaks (n=100) tested. The hMPV-positive patients ranged in age from 18 days to 99 years with a median age of 24 months. The number of positive samples peaked during the winter months of December, January and February. A high rate of co-infections was noted in the samples tested. CONCLUSIONS: hMPV is common in the community and is associated with outbreaks. Including hMPV in routine testing improves etiological diagnosis of acute respiratory infections.