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.     

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.     

RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses

The sequence of the RNA genome of bovine ephemeral fever virus (BEFV) was determined from the start of the L (polymerase) gene to the end of the untranslated 5′ trailer sequence, completing the sequence of the 14 900 nucleotide (nt) genome. The 6470 nt L gene encodes a single long ORF of 2144 amino acids with a deduced molecular weight of 249 766 Da. The 70 nt BEFV 5′ trailer region displays partial terminal complementarity with the 3′ leader sequence and contains a 26 nt direct repeat of the U-rich domain of the 3′ leader region. The 47 nt 5′ trailer region of Adelaide River virus (ARV) displays terminal sequence similarity to the BEFV trailer and partial terminal complementarity with the ARV 3′ leader sequence, but does not contain the direct repeat sequence. The BEFV L protein contains all characteristic sequence motifs of amino acid blocks I–VI, conserved among RNA polymerase proteins of single-stranded (−) RNA viruses, separated by regions of lower homology. Phylogenetic analysis using the complete BEFV L protein sequence indicated a closer relationship to vesicular stomatitis virus than to rabies virus. Sequence comparison of two conserved central domains encompassing blocks II and III and block VI of the BEFV and ARV L proteins indicated they are closely related. An extended phylogenetic analysis using the block III sequence, confirmed the relationship of these ephemeroviruses to vesiculo- and lyssaviruses and to other single-stranded (−) RNA viruses.     

Complete genome sequence of rabies virus CVS-24 from China

The entire genome of the mouse-adapted rabies virus strain CVS-24 (challenge virus standard 24), was sequenced. The overall length of the genome was 11,927 nucleotide (nt), comprising a leader sequence of 58 nt, a nucleoprotein (N) gene of 1353 nt, phosphoprotein (P) gene of 894 nt, a matrix protein (M) gene of 609 nt, a glycoprotein (G) gene of 1575 nt, an RNA-dependent RNA polymerase (RdRp, L) gene of 6384 nt and a trailer region of 70 nt. There was a TGAAAAAAA (TG₇) consensus sequence at the end of each gene, except the G gene which had an AGAAAAAAA sequence at the end, and the L/trailer region had the sequence CGAAAAAAA. Three were AACAYYYCT consensus start signals close to TG₇. The five cistrons were separated by intergenic regions (IGRS) of 2, 5, 5, 24 nt, respectively. Residue 333 of the mature G protein, which is considered to be associated with pathogenicity, was Ala in CVS-24. The topology of the phylogenetic trees generated using N protein sequences suggested that CVS-11 and CVS-N₂C have a close relationship to CVS-24.     

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.     

Complete genome sequence of highly virulent neurotropic Newcastle disease virus strain Texas GB

Newcastle disease virus (NDV) strain Texas GB is a highly virulent neurotropic virus that is used as a standard vaccine challenge virus in the U.S. In this study, the complete genome sequence of strain Texas GB was determined and compared with the complete genome sequences of other NDV strains. The genome is 15,186 nucleotides (nt) long and consists of six genes in the order of 3′leader-N-P-M-F-HN-L-5′trailer. The genome contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. The intergenic sequences are 2, 1, 1, 31, and 47 nt between N/P, P/M, M/F, F/HN, and HN/L genes, respectively. The putative cleavage site of fusion protein showed amino acid sequence of R-R-Q-K-R↓F in position 112 to 117, which corresponds to those of virulent NDV strains. The phylogenetic analysis showed that strain Texas GB is closely related to the neurovirulent mesogenic strain Beaudette C (BC) and to NDV viruses isolated in China and Egypt than to other strains of NDV.     

Whole-genome analysis of a human rabies virus from Sri Lanka

The complete genome sequence of a human rabies virus, strain H-08-1320, from Sri Lanka was determined and compared with other rabies viruses. The size of the genome was 11,926 nt, and it was composed of a 58-nucleotide 3?? leader, five protein genes ?C N (1353 nt), P (894 nt), M (609 nt), G (1575 nt), and L (6387 nt) ?C and a 70-nt 5?? trailer. The intergenic region G?CL contained 515 nt. The sizes of the nucleoprotein, phosphoprotein, matrix-protein, glycoprotein and large-protein was 450, 296, 202, 524 and 2,128 residues, respectively. The phosphoprotein and large protein were one amino acid shorter and longer, respectively, than those of most rabies viruses. The glycoprotein of H-08-1320 had a unique amino acid substitution at antigenic site I. Whole-genome phylogenetic analysis showed that strain H-08-1320 formed an independent lineage and did not cluster with rabies viruses from other countries.     

Determination of the complete genomic sequence and analysis of the gene products of the virus of Spring Viremia of Carp, a fish rhabdovirus

The complete genome of spring viremia of carp virus (SVCV) was cloned and the sequence of 11019 nucleotides was determined. It contains five open reading frames (ORF's) encoding for the nucleoprotein N; phosphoprotein P; matrix protein M; glycoprotein G; and the viral RNA dependent RNA polymerase L. Genes are organised in the order typical for rhabdoviruses: 3'-N-P-M-G-L-5'. The short leader and trailer regions of SVCV exhibit inverse complementarity and are similar to the respective 3' and 5' ends of the genome of vesicular stomatitis virus. To verify the predicted open reading frames proteins were expressed in bacteria and analysed with a polyclonal anti-SVCV serum. Furthermore, monospecific antisera against the distinct viral proteins were generated. Comparison of genome and protein confirm the assignment of SVCV to the genus Vesiculovirus.     

Beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded RNA viruses

During a subtraction study on gene expression in human kidney mesangial cells (HMCs), cDNA clones with sequence homology to paramyxovirus P, M and F genes were isolated. Subsequent investigation revealed that this particular HMC line was infected with a previously unknown paramyxovirus. Here, we report the isolation and genome characterization of this new virus, now named Beilong virus (BeV). The genome of BeV is 19,212 nucleotides (nt) in length and is the largest among all known members of the order Mononegavirales. The BeV genome contains eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. The SH and TM genes code for a small hydrophobic protein of 76 aa and a transmembrane protein of 254 aa, respectively. The BeV G gene, at 4527 nt, codes for an attachment protein of 734 aa and contains two additional open reading frames (ORFs) in the 3' half of the gene, coding for putative proteins of 299 and 394 aa in length. Although the exact origin of BeV is presently unknown, we provide evidence indicating that BeV was present in a rat mesangial cell line used in the same laboratory prior to the acquisition of the HMC line, suggesting a potential rodent origin for BeV.     

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.     

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.     

Characterization of human rabies virus vaccine strain in China

Human rabies virus vaccine strain CTN181 from China was sequenced. The overall length of the genome was 11,923 nucleotides (nt), comprising a leader sequence of 58 nt, nucleoprotein (N) gene of 1353 nt, phosphoprotein (P) gene of 894 nt, matrix protein (M) gene of 609 nt, glycoprotein (G) gene of 1575 nt, RNA-dependent RNA polymerase (RdRp, L) gene of 6387 nt, and a trailer region of 70 nt. The five monocistrons are separated by intergenic regions (IGRs) of 2, 5, 5 and 24 nucleotides (nt), respectively. Two obvious differences between CTN181 and the other rabies virus vaccine strains were (1) the putative stop/polyadenylation signal of the G gene has only one poly (A) tract for CTN181, and (2) the start of the open reading frame for L has two repeats of ATG for CTN181. Both were similar to the SHBRV-18 (silver-haired bat-associated RV strain 18) strain. In addition, some mutations and new functional regions were discovered that are presumed crucial to the function of leader region and L protein. There is an equal role for all five genes in the phylogenetics of rabies virus.     

Complete genome sequence of a virulent Newcastle disease virus isolated from an outbreak in chickens in Egypt

The complete genome sequence of a virulent Newcastle disease virus (NDV) isolated from chickens in Egypt was determined and compared to the sequence of NDV strains isolated from different parts of the world. The genome is 15,186 nucleotides (nt) long and consists of 6 genes in the order of 3′-N-P-M-F-HN-L-5′. The genome contains a 55-nt leader region at the 3′ end and a 114-nt trailer region at the 5′ end. Interestingly, the phylogenetic analysis showed that strain Egypt is closely related with the NDV strains isolated in China. In addition, the sequence of the fusion protein cleavage site of strain Egypt was identical to that of the NDV strain recently isolated in Mali. Determination of complete genome sequences of additional NDV strains from Africa is necessary to understand the epidemiology of currently circulating viruses in Africa.     

Complete nucleotide sequence of Northern cereal mosaic virus and its genome organization

Summary.  We determined the complete nucleotide sequence, 13, 222 nucleotides (nts) of the Northern cereal mosaic virus (NCMV). The genome had 273 nt 5′ trailer sequence and 90 nts 3′leader sequence. It formed a terminal complementarity in 25 nts of both terminal sequences. A characteristic intergenic sequence (consensus) separating genes, 3′-AUUCUUUUUGACUCUAGU-5′ was presented. The genome had nine open reading frames (ORFs) on the viral complementary sequence. Five putative proteins of NCMV were postulated by its molecular weight or comparison of the similarities to other rhabdovirus proteins: nucleocapsid (N), non-structural protein or phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). A series of four small ORFs (genes 3 to 6) were also presented between P and M gene sequences. The proposed NCMV genome organization was 3′leader-N-P-3-4-5-6-M-G-L-5′trailer. N and L proteins of NCMV had low but distinct similarities to those of lettuce necrotic yellows virus and Sonchus yellow net virus, respectively. Received September 29, 1999 Accepted January 26, 2000     

Molecular characterization of Sunflower chlorotic mottle virus: a member of a distinct species in the genus Potyvirus

The complete nucleotide (nt) and deduced amino acid (aa) sequences of the C (common) and CRS (chlorotic ringspot) Argentine strains of SuCMoV have been determined. The SuCMoV-C RNA genome consists of 9,965 nt, whereas indels within the P1 coding region of SuCMoV-CRS make its genomic length 15 nt shorter. Nucleotide and aa sequence identities between the polyproteins of the C and CRS strains of SuCMoV were 92.3 and 95.6%, respectively. Pairwise comparisons between the polyproteins of the C and CRS strains of SuCMoV and the viruses of the Potato virus Y (PVY) subgroup revealed identities of 66.5–66.9% at the nt level and 69.7–69.8% at the aa level. These results and phylogenetic analyses show that although SuCMoV strains cluster together with the potyviruses belonging to the PVY subgroup, SuCMoV should be considered a member of a distinct species in the genus Potyvirus.