Sequencing And Bioinformatics Analysis Of The Complete Genome Of Paramyxovirus Tianjin Strain

Members of the family Paramyxoviridae are pleomorphic enveloped virusespossessing a nonsegmented negative-strand RNA genome, such as Nipah virus,Hendra virus, and Menangle virus, which can cause fatal disease outbreaks in bothanimals and humans. In 1999, a strain of virus was isolated from the lungs ofcommon cotton-eared marmoset that died during an outbreak of disease in an animallaboratory. Virological and morphological analysis and sequence determination ofpart of the HN gene indicated that the virus responsible for the outbreak belonged tothe family Paramyxoviridae, designated temporarily paramyxovirus Tianjin strain.In our previous work, a fragment of 375 nt was amplified from viral RNA byRT-PCR and sequenced, then sequence similarity searches were conducted using theBLAST service at the National Center for Biotechnology Information (NCBI). Theresults showed that the fragment had higher homology with partial HN gene ofSendai viruses. But the deduced protein sequence showed high divergence amongthem. More importantly, Sendai virus usually causes outbreaks of lethal pneumonia inmouse colonies, whereas the experimental mice had not been suffering fromrespiratory disease during the epidemic in the same animal laboratory. In addition,ELISA tests demonstrated that the faculty in the animal laboratory had antibodyspecific to this virus, and 14 of 40 people who had never contacted with themarmosets had antibody to the virus. We also detected the sera of young childrenwith acute respiratory tract infection, and found that the positive rate of IgM to thevirus was 19.28%. These results suggested that the virus had a close relation tohuman, and may be a common respiratory virus in human and marmoset. In order tounderstand the genomic structure and taxonomic position of the strain, as well as therelationship with Sendai viruses, we determined the complete genome sequence ofTianjin strain and compared it with those of other paramyxoviruses withbioinformatics methods. In the first work of our study, the complete genome sequence of theparamyxovirus Tianjin strain had been determined. A total of 13 overlapping cDNAclones, covering the entire genome of Tianjin strain, were obtained by primer walkingRT-PCR. The sequences of the 3' and 5' termini of the viral genome were amplifiedby 3' and 5' RACE. Sequences compiled from these clones show that Tianjin strain is15,384 nt long. The genome size is identical to that of Sendai virus. The number15,384 is a multiple of 6, therefore, Tianjin strain conforms to the 'rule of six', whichplays an important role in the replication of paramyxoviruses. The genome of Tianjinstrain consists of six genes in the order 3'-NP-P/C-M-F-HN-L-5', coding for thenucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large (L) proteins, respectively. The P gene containsanother ORF, which is predicted to encode C protein. A putative RNA editing site isfound 942 nt downstream of the initiation codon (ATG) of the P protein (nt2785-2793). Sequence of the editing site of Tianjin strain, 3'-UUUUUCCC-5',conform well to the conserved sequence, UU(U/C)UCCC, found in all members ofthe subfamily Paramyxovirinae. The gene junctions contain highly conservedtranscription start and stop signal sequences and trinucleotide intergenic regionssimilar to those of Sendai viruses. The genomic structure of Tianjin strain divergesslightly from other Sendai viruses. Two significant nucleotide substitutions are found.One is in the gene start signal sequence between the HN and L ORFs and the other isin the stop codon of L protein, which results in the extended L gene mRNA. Theseunique substitutions indicate that Tianjin strain maybe has some novel features.In the second work, we analyzed the complete genome sequence and thededuced protein sequence of Tianjin strain with bioinformatics methods. Phylogeneticanalysis of genome sequences among Tianjin strain, and other members of the familyParamyxoviridae demonstrate that Tianjin strain, SeV, hPIV-1, hPIV-3 and bPIV-3are in one lineage, and Tianjin strain is most closely akin to Sendai virus. This resultsuggests that Tianjin strain should be assigned to the genus respirovirus within the subfamily Paramyxovirinae and is most likely a new genotype of Sendai virus.Consequently, the complete genome sequence of Tianjin strain was compared withthose of Sendai viruses from GenBank. Phylogenetic tree shows that Tianjin strainlocates a new branch and is more closely related to BB1 strain with 94.9% nucleotidehomology, which was submitted by Institute of Viral Disease Control and Prevention,Beijing in China.Sequence comparisons based on the predicted protein sequences indicate that Lprotein is the most conserved, having 96.0%～98.0% amino acid identity with otherSeVs. As the reported for other paramyxovirus P proteins, Tianjin strain P protein ispoorly conserved, sharing only 78.7%～91.9% amino acid identity with the knownSendai viruses. In addition, there are 29 unique substitutions and 35 commonsubstitutions with BB1 strain in P protein sequence of Tianjin strain. These resultsindicate P protein is maybe the specific protein of Sendai viruses. All of the treesbased on protein sequences are similar to that of complete genome sequence exceptM protein. In M phylogenetic tree, Tianjin strain is more closely related to the branchrepresented by Ohita and Hamamatsu strains than to BB1 strain.The 3' and 5' ends of Tianjin strain genome comprise the leader and trailerregions. The leader sequence is 55 nt long and the trailer sequence is 57 nt long. Theyare exactly complementary for the first 12 nt, which is similar to known Sendaiviruses. The nucleotide sequence comparisons among Tianjin strain and Sendaiviruses showed that the homologies of the leader and the trailer were 89.1%～98.2%,93.0%～96.5%, respectively. The result suggests that the leader and the trailersequences are highly conserved among Sendai viruses, which had been proved to beassociated with transcription and replication of the viral genome.In addition, we analyzed the distribution of the mutations in the genomesequence of Tianjin strain and the significance of amino acid substitutions. Tianjinstrain possesses 444 unique nucleotide variations and 546 common nucleotidevariations with BB1 strain in the entire genome sequence. These variations mainly distribute in the coding region of predicted protein, and result in a lot of amino acidsubstitutions in the protein sequences, which suggests that Tianjin strain maybe has asignificant difference in biological, pathological, immunological, or epidemiologicalcharacteristics from other Sendai viruses. To prove the relationship between thesubstitutions and viral biological properties or pathogenicity, generation ofrecombinant SeVs carrying the mutation by reverse genetic technology would benecessary.In our study, we showed the complete genome sequence of Tianjin strain anddemonstrated the taxonomic position of Tianjin strain and the relationship withSendai viruses, as well as the significance of amino acid substitutions in the predictedprotein sequences. These results might lay the foundation for the further research ofTianjin strain.