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.     

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.     

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.     

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.     

Complete sequence and phylogenetic analysis of a porcine bocavirus strain swBoV CH437

Porcine bocavirus (PBoV), a member of genus Bocavirus, family Parvoviridae, was first identified in 2009 in Swedish swine herds suffering from postweaning multisystemic wasting syndrome. Up to date, the different species of PBoVs have been reported in different countries. Especially, the virus isolated in China was complicated. In this study, we detected a novel PBoV strain swBoV CH437 from clinical samples collected in Gansu Province, Northwest China. The complete genome of swBoV CH437 was 5,275 nucleotides (nt) in length and contains three ORFs: ORF1 encodes NS1 (2,004 nt, 667 aa), ORF3 encodes NP1 (681 nt, 226 aa), and ORF2 encodes VP1 (2,049 nt, 682 aa) and VP2 (1,641 nt, 546 aa). Sequence analysis demonstrated that the NS1 gene shared 24.2–88.6 % nucleotide sequence identity, the NP1 shared 21.3–89.9 %, less than 95 % nucleotide sequence identity with other PBoV strains. Therefore, we propose that swBoV CH437 should be classified as a novel PBoV species.     

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.     

Comparative sequence analysis of envelope protein genes of Indian buffalopox virus isolates

Summary. Buffalopox virus (BPXV) is considered to be a close variant of vaccinia virus (VACV), the prototype member of the genus Orthopoxvirus. In the present study, we have analyzed the sequences of H3L, A27L, and D8L gene-homologues of VACV in BPXV to elucidate its genetic relationship to VACV and other orthopoxviruses (OPVs). Products of these genes have been shown to be important in attachment of VACV to host cell surface receptors during viral entry. Additionally, the A27L gene is also responsible for cell fusion during infection, while the H3L gene is required for synthesis of the highly immunogenic major envelope protein p35. Full-length nucleotide sequences of H3L, A27L, and D8L genes of three BPXV isolates were determined by PCR amplification, cloning, and sequencing. The nucleotide (nt) sequence and the deduced amino acid (aa) sequences were compared with published sequences from other members of the genus Orthopoxvirus. Comparative sequence analysis of all the three genes revealed high sequence identity of BPXV isolates with VACV (close to 99% sequence identity) at both the nt and aa level. Phylogenetic analysis based on the deduced aa sequences of the H3L, A27L, and D8L genes also showed that BPXVs are more closely related to VACV than to any of the other OPVs.     

Complete nucleotide sequence and genome organisation of grapevine Bulgarian latent virus

The complete genome sequence of grapevine Bulgarian latent virus (GBLV) has been determined. RNA-1 (7,452 nt in length) contains a single ORF of 6,285 nt, encoding a polyprotein with conserved motifs characteristic of the viral protease cofactor (Prot-cofact), the NTP-binding protein (NTP), the cysteine-like protease (Cyst-Prot) and the RNA-dependent RNA polymerase (RdRp) of members of the order Picornavirales and show high aa sequence identity with blackcurrant reversion virus (BRV, 64%). RNA-2 (5,821 nt) contains a single ORF of 4,500 nt, encoding a polyprotein in which the conserved motifs of the movement protein (MP) and coat protein (CP) have been identified. The GBLV CP aa sequence shows highest homology with that of blueberry leaf mottle virus (BLMoV, 68%). Both RNAs have a poly(A) tail and a NCR at the 3' and 5' termini, respectively. The results of this study confirm the classification of GBLV as a member of a distinct species in subgroup C of the genus Nepovirus.     

Sequence analysis of the nucleocapsid and phosphoprotein genes of avian pneumoviruses circulating in the US

Avian pneumovirus (APV) has recently been described as the cause of a new respiratory syndrome in turkey flocks in the United States. We here describe the complete sequence of the nucleocapsid (N) and phosphoprotein (P) genes of this emerging APV (APV/US). Our results show 59 and 61% nucleotide sequence identity of the APV/US N gene with N genes of previously described European APV subgroups A and B, respectively. The P gene of APV/US showed only 53% nucleotide sequence identity with the ortholog from APV subgroup A. Phylogenetic analyses of both N and P genes clearly demonstrate that the APV/US lineage is evolutionarily related but distinct from European APVs. Moreover, sequence analysis of the N and P genes from two laboratory adapted isolates of APV/US (APV/MN-1a and APV/MN-1b) and from ten clinical samples from APV-infected turkeys suggests only modest level of amino acid divergence in the N (0-0.3%) and P (0-1.4%) proteins. Taken together, the results of this study indicate support that APV/US represents a new subgroup (subgroup C) of APV and show that there is limited heterogeneity in the N and P genes of APV/US isolates.     

Molecular characterization of the full-length L and M RNAs of Tomato yellow ring virus, a member of the genus Tospovirus

Tomato yellow ring virus (TYRV), first isolated from tomato in Iran, was classified as a non-approved species of the genus Tospovirus based on the characterization of its genomic S RNA. In the current study, the complete sequences of the genomic L and M RNAs of TYRV were determined and analyzed. The L RNA has 8,877 nucleotides (nt) and codes in the viral complementary (vc) strand for the putative RNA-dependent RNA polymerase (RdRp) of 2,873 amino acids (aa) (331 kDa). The RdRp of TYRV shares the highest aa sequence identity (88.7 %) with that of Iris yellow spot virus (IYSV), and contains conserved motifs shared with those of the animal-infecting bunyaviruses. The M RNA contains 4,786 nt and codes in ambisense arrangement for the NSm protein of 308 aa (34.5 kDa) in viral sense, and the Gn/Gc glycoprotein precursor (GP) of 1,310 aa (128 kDa) in vc-sense. Phylogenetic analyses indicated that TYRV is closely clustered with IYSV and Polygonum ringspot virus (PolRSV). The NSm and GP of TYRV share the highest aa sequence identity with those of IYSV and PolRSV (89.9 and 80.2–86.5 %, respectively). Moreover, the GPs of TYRV, IYSV, and PolRSV share highly similar characteristics, among which an identical deduced N-terminal protease cleavage site that is distinct from all tospoviral GPs analyzed thus far. Taken together, the elucidation of the complete genome sequence and biological features of TYRV support a close ancestral relationship with IYSV and PolRSV.     

Sequence analysis of the large polymerase (L) protein of the US strain of avian metapneumovirus indicates a close resemblance to that of the human metapneumovirus

The complete nucleotide sequence of the large polymerase (L) protein of the avian metapneumovirus subgroup C strain Colorado was determined. The L protein gene of avian pneumovirus Colorado isolate (APV-C) was 6173 nucleotides in length from the gene-start to the gene-end and encoded a polypeptide of 2005 amino acids in length. The length of the L protein of APV-C was exactly the same as that of human metapneumovirus (hMPV) and one amino acid longer than the L protein of APV subgroup A. The L protein of APV-C showed 80% amino acid identity with the L protein of hMPV, but only 64% amino acid identity with the L protein of APV-A. The nucleotide and deduced amino acid sequences were compared with the corresponding sequences of eleven other paramyxoviruses. All six domains characteristic of paramyxovirus L proteins were also observed in the L protein of APV-C. All the polymerase core motifs in domain III were conserved to nearly 100% in the metapneumoviruses. Similarly, the putative ATP-binding motif in domain VI was completely conserved among the metapneumoviruses and differed in length, by one intermediate residue, from other paramyxoviruses. Phylogenetic analysis of the different L proteins also revealed a closer relationship between APV-C and hMPV.     

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.     

Complete genome sequence of Brugmansia suaveolens mottle virus, a potyvirus from an ornamental shrub

Brugmansia suaveolens mottle virus (BsMoV) was the first potyvirus isolated from “angel trumpet” (Brugmansia suaveolens), described in Brazil. In this study, the complete nucleotide (nt) genome sequence of BsMoV was determined, and the deduced amino acid (aa) sequence was analyzed. The BsMoV RNA genome consists of 9870 nt without a poly-A tail, encoding a putative typical potyviral polyprotein of 3090 aa. Pairwise comparisons of the complete BsMoV genome with those of the most closely related potyviruses revealed a maximum nucleotide identity of 63.7% with pepper mottle virus. These results and phylogenetic analyses based on the complete genome sequence of the most closely related potyviruses confirmed that BsMoV should be considered a member of a distinct species of the genus Potyvirus.     

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.     

Molecular characterization of G11P[25] and G3P[3] human rotavirus strains associated with asymptomatic infection in South India

Rotaviruses are the major etiological agents of diarrhea in children less than 5 years of age. Two unusual rotavirus strains not previously reported in India, G11P[25] (CRI 10795) and G3P[3] (CRI 33594) were isolated from faecal samples of asymptomatic children in India. The strains were characterized by sequence analysis of the genes encoding the VP7, VP4, VP6, and NSP4. The G11P[25] strain was closely related to the human G11P[25] strains from Bangladesh (with 98% identity at the nucleotide [nt] level and the amino acid [aa] level for the VP7 gene and 96% identity at the nt and 98% at the aa level for the VP4 gene). The G3P[3] strain was found to be related to a G3P[3] strain isolated in Thailand (CMH222; 88% identity at the nt level and 97% at aa level for the VP7 gene and 84% identity at the nt level and 90% at the aa level for the VP4 gene). Phylogenetic analysis of the VP6 and the NSP4 genes revealed that the Vellore G11P[25] strain was of VP6 subgroup II and NSP4 genotype B. The G3P[3] strain was identified as NSP4 genotype C and the VP6 gene showed 97% identity at the deduced amino acid level with strain CMH222 (Thailand) strain but did not cluster with sequences of SGI, SGII, SGI+II or SG-nonI/nonII. Both strains had gene segments of animal rotavirus origin suggesting inter-species transmission of rotavirus, and in the case of G11P[25] possibly underwent reassortment subsequently with human strains resulting in an animal-human hybrid strain.     

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.     

Identification and molecular characterization of a new potyvirus from Panax notoginseng

A potyvirus causing distortion and mosaic symptoms in the herbal plant Sanqi (Panax notoginseng) was isolated from Yunnan province, China, and the complete nucleotide sequence of one isolate and the partial sequences of two other isolates were determined. The viral RNA genome comprised 9,750 nt excluding the 3′-terminal poly(A) tail, with the capacity to encode a single polyprotein of 3,089 amino acids. Phylogenetic analysis with other completely sequenced potyviruses revealed that the virus in this study was most closely related to plum pox virus, with 56.3% nt identity in the genomic RNA sequence and 53.3% aa identity in the polyprotein. However, the most closely related 3′-terminal sequences were from four partially sequenced potyviruses infecting plants of the family Apiaceae (67.7–75.3% nt identity and 73.8–76.7% aa identity in their coat protein cistrons), especially Angelica virus Y. These results suggest that this virus isolate should be designated a member of a new species in the genus Potyvirus, which is tentatively named Panax virus Y (PanVY).     

Grapevine deformation virus: completion of the sequence and evidence on its origin from recombination events between Grapevine fanleaf virus and Arabis mosaic virus

The complete nucleotide (nt) sequence of Grapevine deformation virus (GDefV) RNA-1 has been determined. It consists of 7386 nt, excluding the poly(A) tail, and contains a single open reading frame (ORF) encoding a polyprotein (p1) of 252 kDa. P1 comprises the 1A(Pro-cof) proteinase cofactor, the 1B(Hel) NTP-binding protein, the 1C(VPg) viral protein genome-linked, the 1D(Prot) proteinase and the 1E(Pol) RNA-dependent RNA polymerase, all of which are conserved domains in polyproteins of different members of the order Picornavirales. The amino acid (aa) sequence of GDefV RNA1 p1 has the highest identity with the homologous products of Grapevine fanleaf virus (GFLV, 86-88%) and Arabis mosaic virus (ArMV, 73-74%), two nepoviruses of subgroup A. Four cleavage sites for proteins processing were predicted (C/A, C/S, G/E and R/G) and found similar to those of GFLV RNA1. Phylogenetic trees constructed with the complete aa sequences of protein p1 and the RNA2-encoded protein p2 of GDeFV, GFLV and ArMV, showed an incongruent allocation of GDefV in these trees. Pairwise alignment and prediction of recombination sites of both RNA segments showed that GDefV RNA2 has a mosaic structure resulting from recombination events between GFLV and ArMV at the level of the 2A(HP) (homing protein), 2B(MP) (movement protein), 2C(CP) (capsid protein) and the 3'NCR (non coding region). This strongly suggests that GDefV originated from the interspecific recombination between isolates of GFLV and ArMV.