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 and antigenic variability of human respiratory syncytial virus (groups a and b) isolated over seven consecutive seasons in Argentina (1995 to 2001)

The genetic and antigenic variability of human respiratory syncytial virus (HRSV) strains isolated in Buenos Aires from 1995 to 2001 was evaluated by partial nucleotide sequencing of the G gene and enzyme-linked immunosorbent assay analysis with anti-G monoclonal antibodies. Phylogenetic analyses showed that 37 group A strains clustered into five genotypes, whereas 20 group B strains clustered into three genotypes. Group A showed more genetic variability than group B. A close correlation between genotypes and antigenic patterns was observed. Changes detected in the G protein of viruses from both groups included (i) amino acid substitutions and(ii) differences in protein length due to either changes in stop codon usage or sequence duplications. Three B strains from 1999 exhibited a duplication of 20 amino acids, while one B strain from 2001 had 2 amino acids duplicated. The comparison among Argentinean HRSV strains and viruses isolated in other geographical areas during different epidemics is discussed.     

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.     

Human metapneumovirus infections among children with acute respiratory infections seen in a large referral hospital in India.

BACKGROUND: Acute respiratory infections (ARI) are the leading cause of morbidity and mortality among children <5 years of age in developing countries. Human metapneumovirus (hMPV), a newly described respiratory pathogen, has been identified as an important cause of ARI in young children. OBJECTIVES: The objective was to describe the prevalence of hMPV in children who presented with ARI to a large referral hospital in Delhi, India and to genotype circulating strains on the basis of F gene nucleotide sequence analysis. STUDY DESIGN: We analyzed 97 samples from children <5 years of age with ARI seen at the All India Institute of Medical Sciences from June 2004 to March 2005. RT-PCR was performed for the N and F genes and partial F gene nucleotide sequences were used to characterize the viruses. RESULTS: hMPV was identified in 12% of children with ARI, including 13% of the children hospitalized with ARI. Most virus identification occurred in the winter. The Indian strains clustered in the A2 genetic sublineage. CONCLUSIONS: This report establishes hMPV as an important cause of ARI in children in India.     

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.     

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.     

Genetic variability of the fusion protein and circulation patterns of genotypes of the respiratory syncytial virus

Although antigenic and genetic variations were shown to occur both in the G and F protein of respiratory syncytial virus (RSV), few studies looked at the variation of F gene. The F genotypes were determined by the evaluation of clustering patterns, via the phylogenetic analysis of the nucleotide sequences of a variable region in the F gene. One hundred seventy-nine strains obtained from a children's hospital in Korea over nine consecutive epidemics were included. The relationship between the F and G genotypes was analyzed with the G genotypes previously published by the authors. The phylogenetic analysis of the variable region from the F gene revealed 9 genotypes among 129 group A RSVs and 4 genotypes among 50 group B RSVs. In each of the epidemics, the dominant genotypes were replaced with new genotypes in consecutive epidemics. In each of the epidemics of group B RSVs, the predominant genotype alternated between genotypes. Most of the strains which clustered to a particular F genotype were assigned to particular G genotype(s). By determining the nearly entire sequences of the F genes, we revealed the percentage of the nucleotide differences which resulted in amino acid coding changes was determined to be much great in two distinct variable regions of the F gene. Our results indicated that the F gene of the RSVs may be continuously evolving under selective pressure in a distinct pattern, and that the genetic variability of the F protein is associated with that of the G protein.     

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.     

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.     

Sequence and phylogenetic analysis of SH, G, and F genes and proteins of Human respiratory syncytial virus isolates from Singapore

To study the genetic variability and molecular epidemiology of Human respiratory syncytial virus (HRSV) occurring in Singapore, nucleotide sequencing of three membrane-associated genes (SH, G and F) of four local isolates was performed. Comparison of their nucleotide and amino acid sequences with those of the prototype strains A2 (subgroup A) and CH-18537 (subgroup B) indicated that the Singapore isolates belong to the subgroup A. Comparison of the Singapore isolates with the reference strain A2 showed that whereas the G protein was the most divergent with up to 15% difference, the F and SH proteins showed less diversity of only up to 4%. Each gene exhibited its distinct variable and conserved regions. The N- and O-glycosylation sites within the G protein of the isolates were analyzed to ascertain their potential implications on the antigenicity of the viral glycoprotein. Based on the second variable region of the G protein, phylogenetic analysis of the Singapore isolates with 91 previously identified genotypes of subgroup A revealed that more than one genotype (GA2 and GA5) may circulate in the local population at a given time. This epidemiological study reflects the pattern of genetic relationships between the HRSV isolates from Singapore to those from other parts of the world.     

Antigenic and genetic variability of human respiratory syncytial viruses (group A) isolated in Uruguay and Argentina: 1993-2001

The antigenic and genetic diversity of G glycoprotein from 25 human respiratory viruses (group A) isolated during nine consecutive epidemics (1993-2001) in Montevideo, Uruguay, and 7 strains isolated in Buenos Aires, Argentina, in the same period were analyzed. Genetic variability was evaluated by partial sequence of the G protein gene. Phylogenetic analysis indicated that most Uruguayan and Argentinean group A isolates clustered into three genotypes: GA5, GA2, and GA1. Some strains clustered into the GA3 genotype characterized previously. The antigenic analysis was carried out with a panel of anti-G monoclonal antibodies that recognized conserved and strain-specific epitopes. A close correlation between the antigenic and genetic relatedness of the strains analyzed was observed.     

Molecular diversity of the aminoterminal region of the G protein gene of human respiratory syncytial virus subgroup B

Two major antigenic subgroups (designated A and B) have been described for human respiratory syncytial virus (HRSV). Between and within the two main subgroups, there is antigenic variation in the attachment protein G. The variability of the G protein is known to be located in two hypervariable regions of the ectodomain. Most investigators have studied the gene segment coding the C-terminal end of the protein, and little is known about the N-terminal variable region. In the present study, the genetic variability of HRSV subgroup B was evaluated by nucleotide sequencing of the N-terminal region of the G gene of 52 Tunisian isolates. Tunisian subgroup B isolates clustered into two main lineages designated arbitrarily as Tu-GB1 and Tu-GB2. Three distinct subtypes were identified within genotype Tu-GB2. The inter- and intragenotype nucleotide variability ranged from 4 to 8% and from 0 to 4%, respectively. Overall divergence values of the G sequences were inferior or equal to 15% at the aminoacid level. Comparison of sequences among Tunisian HRSV strains and viruses isolated in other geographical areas during different epidemics demonstrated close similarity to strains from Kenya, Belgium, the UK, Qatar, Canada and South Korea.     

Detection of genetic lineages of human metapneumovirus in Croatia during the winter season 2005/2006

Human metapneumovirus (HMPV) is an important respiratory pathogen, especially among young children. The genetic characteristics of HMPV circulating in Croatia have not been studied so far. The aim of this study was to determine the incidence of HMPV infection in hospitalized children with acute respiratory tract infection (ARTI) in the season 2005/2006 in Croatia, as well as to perform the genotypic analysis of detected HMPV strains. From December 1 to March 31 nasopharyngeal secretions (NPSs) were collected from 402 inpatients up to 5 years of age with ARTI. NPSs were tested by real-time RT-PCR assay targeting the nucleoprotein (N) gene of HMPV. HMPV infection was detected in 33 patients (8.2%). To perform the phylogenetic study, partial nucleotide sequences were obtained for HMPV fusion (F) gene of 30 HMPV positive samples. Phylogenetic analysis showed the circulation of two main genetic lineages (A and B), with B lineages being prevalent. It also showed the existence of two sublineages within the group B (B1 and B2) and three subclusters within lineage A (A1, A2a and A2b). Further molecular analysis revealed point mutations in HMPV strains of sublineage B1.     

Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses

Rotavirus groups A to E are mainly defined by antibody reactivity to the capsid protein VP6. Additionally, two putative rotavirus groups (F and G) have been identified in birds. Here, the first nucleotide sequences of the VP6-encoding genome segment of group F (strain 03V0568) and group G (strain 03V0567) rotaviruses, both derived from chickens, are presented. The group F rotavirus is most closely related to avian group A and D rotaviruses, with 49.9-52.3% nucleotide and 36.5-39.0% amino acid sequence identity. The group G rotavirus is most closely related to mammalian group B rotaviruses, with 55.3-57.5% nucleotide and 48.2-49-9% amino acid sequence identity. The terminal sequences of the genome segment were similar in groups A, D and F, and in groups B and G. The findings indicate a long-term evolution of rotavirus groups in two separated clades and support the development of a sequence-based classification system for rotavirus groups.     

Classification of duck hepatitis virus into three genotypes based on molecular evolutionary analysis

The nucleotide sequences of the complete VP1, VP0, VP3, and partial 3D regions of seven duck hepatitis virus (DHV) serotype 1 (DHV-1) strains isolated in China between 2001 and 2007 and one DHV-1 strain originally obtained from ATCC were determined and compared with previously available DHV sequences in GenBank. Phylogenetic analysis on the basis of VP1 sequences demonstrated three distinct genetic groups. There was an excellent concordance among the genetic groups assigned based on the complete VP0, VP3, and the partial 3D regions. In view of the growing importance of molecular techniques in diagnosis, we propose that the three genetic groups should be termed DHV types A, B, and C. All DHV-1 strains grouped in genotype A, whereas the new serotype strains isolated in Taiwan and the new serotype strains isolated in South Korea clustered into genotypes B and C, respectively, suggesting a potential genetic correlates of serotype. In pairwise comparisons of complete VP1, VP0, and VP3 nucleotide and amino acid sequences and the partial 3D nucleotide sequence, DHVs of the same genotype were clearly distinguished from those of heterologous genotypes. Analysis of the amino acid sequences of the three capsid proteins demonstrated the presence of conserved elements that form the eight-stranded beta-barrel structures, as well as intervening domains that vary in sequence between strains of different genotypes as seen in other picorna viruses.     

Antigenic and genetic characterization of the fusion (F) protein of mumps virus strains

Summary.  Twelve strains of mumps virus, belonging to the A, C and D genotypes of the small hydrophobic (SH) protein gene, were investigated by nucleotide sequencing of the fusion protein gene. The nucleotide sequences and deduced amino acid sequences were aligned and compared with previously reported sequences of the gene. In addition an antigenic comparison between the F protein of different strains of the A, C and D genotypes was performed with ten monoclonal antibodies directed against the F protein of genotype A. Phylogenetic analysis of the coding region of the F gene showed the expected clustering of the different genotypes, as previously determined from the SH protein gene. Comparison of the 538 long amino acid sequence of the protein showed that only a small number of amino acids differed between the viral strains. The A genotype differed from B, C and D whereas the latter showed fewer consistent amino acid differences between themselves. Nine of the ten monoclonal antibodies reacted with the C and D genotypes and one failed to react with these genotypes. It is concluded that the structure and antigenicity of the F protein is well conserved both intra- and intergenotypically over long periods of time. Received September 7, 1999 Accepted November 17, 1999     

Comparison of the RNA polymerase genes of marine birnavirus strains and other birnaviruses

Summary.  The cDNA nucleotide sequence of the genome segment B encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp), was determined for 5 marine birnavirus (MABV) strains from different host or geographic origins and 1 infectious pancreatic necrosis virus (IPNV) strain AM-98. Segment B of the IPNV AM-98 strain and 4 MABV strains, Y-6, YT-01A, H1 and NC1, contained a 2535 bp ORF, which encoded a protein of 845 amino acid residues with a predicted MW of 94.4 kDa. Only the MABV AY-98 RdRp had 1 amino acid shorter RdRp. Pairwise comparisons were made among our data and 4 other known IPNV sequences. The nucleotide sequences of the 5 MABV strains were very similar each other, with identities of 98.3–99.7%. The highest divergence of the nucleotide level was between MABV strains and IPNV SP strain (serotype A2), with 20.4–20.8% divergences in the coding region, which gave 10.1–11.3% divergence in the amino acid level. The aquabirnavirus RdRp was noticeably conserved in amino acid sequences. Though the identities of the nucleotide sequences of encoding region were 85.1–85.9% between MABV strains and IPNV serotype A1 strains, they shared as high as 95.1–95.9% identities in amino acid level. A phylogenetic tree was constructed based on the amino acid sequences of the RdRp gene from different birnaviruses including avibirnavirus and entomobirnavirus. Ten aquabirnavirus strains were clustered into 3 Genogroups. The Genogroup I consisted of four IPNV A1 serotype strains. All MABV strains were clustered into Genogroup II. Only IPNV SP strain was clustered into an independent Genogroup III. Received August 19, 2002; accepted October 30, 2002     

Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus

The envelope glycoproteins of Newcastle disease virus (NDV), hemagglutinin-neuraminidase (HN) and fusion (F) proteins, play important roles in determining the host immune response and the virulence of that particular virus strain. The complete nucleotide sequence of the HN and F genes of a highly neurovirulent strain of NDV (Texas G. B., 1948) was determined in an effort to study the molecular basis of this strain's neurotropic properties. Comparison of the predicted amino acid sequences for the HN and F among the American NDV strains revealed that the Texas G. B. and Beaudette C envelope genes are closely related to each other and are less closely related to the avirulent B1 Hitchner strain. We have found 11 amino acid changes in the predicted HN protein between the Beaudette C and Texas G. B. strain but only 2 conservative amino acid changes (amino acids 11 and 197) in the F protein between these two strains. Although the virulence of NDV strains has been related to sequences at the cleavage site of F0, the property of neurovirulence cannot depend solely upon these sequences because there are no sequence differences between the Beaudette C and Texas G. B. strains. We suggest that the neurovirulence phenotype could be due to the molecular properties of the HN protein; however, we cannot exclude the possibility that the two conservative amino acid differences between the two F proteins could also play a role in determining the phenotypic differences between these two virus strains.