Analysis of the genomic sequence of a human metapneumovirus

We recently described the isolation of a novel paramyxovirus from children with respiratory tract disease in The Netherlands. Based on biological properties and limited sequence information the virus was provisionally classified as the first nonavian member of the Metapneumovirus genus and named human metapneumovirus (hMPV). This report describes the analysis of the sequences of all hMPV open reading frames (ORFs) and intergenic sequences as well as partial sequences of the genomic termini. The overall percentage of amino acid sequence identity between APV and hMPV N, P, M, F, M2-1, M2-2, and L ORFs was 56 to 88%. Some nucleotide sequence identity was also found between the noncoding regions of the APV and hMPV genomes. Although no discernible amino acid sequence identity was found between two of the ORFs of hMPV and ORFs of other paramyxoviruses, the amino acid content, hydrophilicity profiles, and location of these ORFs in the viral genome suggest that they represent SH and G proteins. The high percentage of sequence identity between APV and hMPV, their similar genomic organization (3'-N-P-M-F-M2-SH-G-L-5'), and phylogenetic analyses provide evidence for the proposed classification of hMPV as the first mammalian metapneumovirus.     

Subtype B avian metapneumovirus resembles subtype A more closely than subtype C or human metapneumovirus with respect to the phosphoprotein,and second matrix and small hydrophobic proteins

Avian metapneumovirus (aMPV) subtype B (aMPV/B) nucleotide sequences were obtained for the phosphoprotein (P), second matrix protein (M2), and small hydrophobic protein (SH) genes. By comparison with sequences from other metapneumoviruses, aMPV/B was most similar to subtype A aMPV (aMPV/A) relative to the US subtype C isolates (aMPV/C) and human metapneumovirus (hMPV). Strictly conserved residues common to all members of the Pneumovirinae were identified in the predicted amino acid sequences of the P and M2 protein-predicted amino acid sequences. The Cys(3)-His(1) motif, thought to be important for binding zinc, was also present in the aMPV M2 predicted protein sequences. For both the P and M2-1 protein-predicted amino acid sequences, aMPV/B was most similar to aMPV/A (72 and 89% identity, respectively), having only approximately 52 and 70% identity, respectively, relative to aMPV/C and hMPV. Differences were more marked in the M2-2 proteins, subtype B having 64% identity with subtype A but < or = 25% identity with subtype C and hMPV. The A and B subtypes of aMPV had predicted amino acid sequence identities for the SH protein of 47%, and less than 20% with that of hMPV. An SH gene was not detected in the aMPV/C. Phylogenetically, aMPV/B clustered with aMPV/A, while aMPV/C grouped with hMPV.     

Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3' end and a 45 nt trailer at its 5' end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.     

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.     

Topology and cellular localization of the small hydrophobic protein of avian metapneumovirus

The small hydrophobic protein (SH) is a type II integral membrane protein that is packaged into virions and is only present in certain paramyxoviruses including metapneumovirus. In addition to a highly divergent primary sequence, SH proteins vary significantly in size amongst the different viruses. Human respiratory syncytial virus (HRSV) encodes the smallest SH protein consisting of only 64 amino acids, while metapneumoviruses have the longest SH protein ranging from 174 to 179 amino acids in length. Little is currently known about the cellular localization and topology of the metapneumovirus SH protein. Here we characterize for the first time metapneumovirus SH protein with respect to topology, subcellular localization, and transport using avian metapneumovirus subgroup C (AMPV-C) as a model system. We show that AMPV-C SH is an integral membrane protein with N(in)C(out) orientation located in both the plasma membrane as well as within intracellular compartments, which is similar to what has been described previously for SH proteins of other paramyxoviruses. Furthermore, we demonstrate that AMPV-C SH protein localizes in the endoplasmic reticulum (ER), Golgi, and cell surface, and is transported through ER-Golgi secretory pathway.     

Expression of Recombinant Small Hydrophobic Protein for Serospecific Detection of Avian Pneumovirus Subgroup C

The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.     

Expression of recombinant small hydrophobic protein for serospecific detection of avian pneumovirus subgroup C

The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.     

Genetic variability of the G glycoprotein gene of human metapneumovirus

Human metapneumovirus (hMPV) has been associated with respiratory illnesses like those caused by human respiratory syncytial virus (HRSV) infection. Similar to other pneumoviruses, genetic diversity has been reported for hMPV. Little information is currently available on the genetic variability of the G glycoprotein (G), which is the most variable gene in RSV and avian pneumovirus. The complete nucleotide sequences of the G open reading frame (ORF) of 24 Canadian hMPV isolates were determined. Phylogenetic analysis showed the existence of two major groups or clusters (1 and 2). All but one of the hMPV isolates that we examined belonged to cluster 1. Additional genetic variability was observed in cluster 1, which separated into two genetic subclusters. Within cluster 1 the nucleotide sequence identity for the G ORF was 74.2 to 100%, and the identity for the predicted amino acid sequence was 61.4 to 100%. The G genes of cluster 1 isolates were more divergent from the cluster 2 isolates, with 45.6 to 50.5% and 34.2 to 37.2% identity levels for the nucleotide and amino acid sequences, respectively. Sequence analysis also revealed changes in stop codon usage, resulting in G proteins of different lengths (217, 219, 228, and 236 residues). Western blot analysis with the use of hMPV-specific polyclonal antisera to each hMPV cluster showed significant antigenic divergence between the G proteins of clusters 1 and 2. These results suggest that the G protein of hMPV is continuously evolving and that the genetic diversity observed for the hMPV genes is reflected in the antigenic variability, similar to HRSV.     

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.     

Sequence analysis of the N,P, M and F genes of Canadian human metapneumovirus strains

The complete nucleotide sequences of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) genes of 15 Canadian human metapneumovirus (hMPV) isolates were determined. Phylogenetic analysis revealed two distinct genetic clusters, or groups for each gene with additional sequence variability within the individual groups. Comparison of the deduced amino acid sequences for the N, M and F genes of the different isolates revealed that all three genes were well conserved with 94.1-97.6% identity between the two distinct clusters The P gene showed more diversity with 81.6-85.7% amino acid identity for isolates between the two clusters, and 94.6-100% for isolates within the same cluster.     

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。     

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.     

European and American Subgroup C Isolates of Avian Metapneumovirus belong to Different Genetic Lineages

The gene encoding the attachment glycoprotein (G) was sequenced in three French isolates of-subgroup C avian metapneumovirus (APV-C) from ducks. With 1771 nt, this gene proved as long as recently published for North-American APV-C isolates from turkeys. The nt sequences of the duck viruses shared 99% identity but proved only 75-83% identical with their North-American counterparts, viruses of both origins encoding 585 amino acid (aa)-long G proteins. Alignments revealed more homogeneity within the European and North-American groups (at least 98 and 79% aa identity, respectively) than between European and North-American viruses (at best 70% a identity), and confirmed the presence of an extracellular divergent domain (positions 302-484) in APV-C G. A phylogenetic analysis demonstrated that North-American and French isolates of APV-C belonged to significantly different genetic lineages, in agreement with the different geographical origin and host species of these viruses.     

Molecular epidemiology of subgroup C avian pneumoviruses isolated in the United States and comparison with subgroup a and B viruses

The avian pneumovirus (APV) outbreak in the United States is concentrated in the north-central region, particularly in Minnesota, where more outbreaks in commercial turkeys occur in the spring (April to May) and autumn (October to December). Comparison of the nucleotide and amino acid sequences of nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), and second matrix (M2) genes of 15 U.S. APV strains isolated between 1996 and 1999 revealed between 89 and 94% nucleotide sequence identity and 81 to 95% amino acid sequence identity. In contrast, genes from U.S. viruses had 41 to 77% nucleotide sequence identity and 52 to 78% predicted amino acid sequence identity with European subgroup A or B viruses, confirming that U.S. viruses belonged to a separate subgroup. Of the five proteins analyzed in U.S. viruses, P was the most variable (81% amino acid sequence identity) and N was the most conserved (95% amino acid sequence identity). Phylogenetic comparison of subgroups A, B, and C viruses indicated that A and B viruses were more closely related to each other than either A or B viruses were to C viruses.     

Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses

A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted M(r) of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States.     

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.     

Positive natural selection in the evolution of human metapneumovirus attachment glycoprotein

Human metapneumovirus (hMPV), a newly discovered virus of the family Paramyxoviridae, has been associated with upper and lower respiratory tract infections in different age groups in many countries. The putative attachment (G) glycoprotein of this virus was previously reported to have shown more extensive nucleotide and deduced amino acid sequence polymorphism than any other genomic regions of this virus, leading to four sub-lineages. Using a maximum likelihood-based codon substitution model of sequence evolution, here we report that sequences of extracellular domain of 8 amino acid sites in lineage 1a, and 3 amino acid sites each in lineage 1b, 2a, and 2b have a higher rate of nonsynonymous substitutions (d(N)) than the synonymous substitutions (d(S)) with a posterior probability above 0.95, thus suggesting the evidence of adaptive evolution driven by Darwinian selection. Although it is unclear whether these amino acid adaptations are driven by differential immune pressure or some other factors, identification of these positively selected amino acid sites would help in better screening using epitope mapping technology to identify and localize the sites that can be recognized by the immune system. We also observed surprisingly higher nucleotide substitution rates per site, per year for each lineage of hMPV than the rates that were previously reported for the human respiratory syncytial virus, suggesting rapid evolutionary dynamics of hMPV.     

Fusion protein predicted amino acid sequence of the first US avian pneumovirus isolate and lack of heterogeneity among other US isolates

Avian pneumovirus (APV) was first isolated from turkeys in the west-central US following emergence of turkey rhinotracheitis (TRT) during 1996. Subsequently, several APV isolates were obtained from the north-central US. Matrix (M) and fusion (F) protein genes of these isolates were examined for sequence heterogeneity and compared with European APV subtypes A and B. Among US isolates the M gene shared greater than 98% nucleotide sequence identity with only one nonsynonymous change occurring in a single US isolate. Although the F gene among US APV isolates shared 98% nucleotide sequence identity, nine conserved substitutions were detected in the predicted amino acid sequence. The predicted amino acid sequence of the US APV isolate's F protein had 72% sequence identity to the F protein of APV subtype A and 71% sequence identity to the F protein of APV subtype B. This compares with 83% sequence identity between the APV subtype A and B predicted amino acid sequences of the F protein. The US isolates were phylogenetically distinguishable from their European counterparts based on F gene nucleotide or predicted amino acid sequences. Lack of sequence heterogeneity among US APV subtypes indicates these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the F protein among APV isolates supports classification of US isolates as a new APV subtype C.