In 1999, we isolated a strain of virus with high hemagglutinin titer from the lung of a dead common cotton-eared marmoset during a respiratory-disease outbreak in an animal laboratory. In the epidemic, almost all the marmosets became ill, and about 1/3 died. Virological and morphological analysis and sequence determination of part of the HN gene indicated that the virus responsible for the outbreak belonged to the family Paramyxoviridae, and so it was temporarily designated paramyxovirus Tianjin strain. In our previous work, a fragment of 375 bp was amplified from viral RNA by RT-PCR and sequenced, then sequence similarity searches were conducted using the BLAST service at the National Center for Biotechnology Information (NCBI). The results showed that the fragment had highest similarity with the part of the HN gene of Sendai virus (SeV) (10).
SeV is a member of the Respirovirus genus in the Paramyxovirinae subfamily. It usually causes out-breaks of lethal pneumonia in mouse colonies, the natural host, but is thought to be nonpathogenic in humans (7). During the epidemic, we found that the experimental mice bred in the same animal laboratory with marmosets had never suffered from the respi-ratory disease. Moreover, we failed to detect anti-bodies against the Tianjin strain in laboratory mice using the ELISA test. We postulate that this strain may be a variant of SeV whose host changed from rodents to marmoset.
In order to understand the genomic structure and taxonomic position of the strain, its complete genome was sequenced and analyzed. Characterization of genome and phylogenetic analysis with other members of the family Paramyxoviridae demonstrated that the Tianjin strain should be assigned to the genus Respirovirus within the subfamily Paramyxovirinae, and was most closely related to SeV (4, 5, 11). The genome of Tianjin strain consists of six structural genes in the order 3'-NP-P/C-M-F-HN-L-5', coding for the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large (L) protein, respectively. In the present paper, the amino acid sequences of six structural proteins of Tianjin strain were analyzed by using the bioinformatics methods. Our findings may provide a useful basis for the vaccine studies.
The deduced amino acid sequences of the full-length NP, P, M, F, HN, and L genes of the Tianjin strain were used to infer phylogenetic relationships with other Paramyxoviridae. Representative trees illus-trating the relationships observed are shown in Fig. 1. In the P and L protein trees, the genera Rubulavirus, Morbillivirus, Respirovirus, Henipavirus, Avulavirus, Pneumovirus and Metapneumovirus separated into distinct clusters. This result is consistent with the most recent taxonomy approved by the ICTV executive committee. Moreover, the Tianjin strain formed a single cluster with SeV, hPIV-1, hPIV-3 and bPIV-3 and was more closely related to SeV than to any other virus in the same genus. The NP, M, F and HN protein trees were similar in showing that the Tianjin strain was positioned in the Respirovirus genus and was most closely related to SeV (data not shown). Furthermore, phylogenetic analysis based on 6 structural proteins of the Tianjin strain and 14 strains of SeV clearly showed that the viruses were roughly divided into four phylogenetic clusters, the first containing Ohita M1, MVC11and Hamamatsu E0, E30cl2, E15cl2, E50cl9 and E30M15cl5, the second containing Pi, Nagoya, Z, F1-R, mutant ts-f1 and mutant T-5 revertant, and the third represented by BB1 strain. The Tianjin strain formed an independent branch (P, M and L protein trees shown in Fig. 2). This result suggested that Tianjin strain was most likely a new genotype of SeV. However, trees of the different proteins were not identical. The NP, P, F, HN and L protein trees were similar in showing Tianjin strain was most closely related to BB1 strain, whereas in the M protein trees, Tianjin strain was more closely related to the branch represented by Ohita and Hamamatsu strain than to BB1 strain. Similarity comparison of M proteins between the Tianjin strain and known SeVs also demonstrated that the Tianjin strain shared higher identity value with Ohita and Hamamatsu strain than with the BB1 strain (Table 1). This suggests a somewhat different evolutionary pathway of the Tianjin strain M protein compared to the NP, P, F, HN, and L proteins.
Figure 1. The Neighbor-Joining trees based on the complete P (A) and L protein (B) sequences of Tianjin strain (see arrow) and other Paramyxoviridae. The numbers on the nodes represent bootstrap values (for 1000 replications).
Figure 2. The Neighbor-Joining trees based on the complete P (A), M (B) and L protein (C) sequences of Tianjin strain (see arrow) and 14 known SeVs. The numbers on the nodes represent bootstrap values (for 1000 replications).
Table 1. Similarities comparisons of amino acid sequences of some proteins between Tianjin strain and 14 known SeVs (%)
Similarities comparisons of Tianjin strain protein sequences with those of 14 known SeVs clearly showed that L protein was relatively well conserved, sharing 96% to 98% amino acid identity with the known SeVs, followed by the M (93.1%~97.1%), NP (92.2%~96.0%), F (90.4%~95.4%), and HN proteins (89.4%~95.0%). The Tianjin strain P protein had relatively low levels of amino acid identity with the P proteins of SeVs, ranging from 78.7% to 91.9% (Table 1). This pattern was consistent with previous descriptions in which the P protein of SeV was poorly conserved.
When the alignments were carried out on the protein sequences of Tianjin strain and analogous proteins of 14 known SeVs, 110 unique amino acid substitutions were found, 15 in NP, 29 in P, 6 in M, 13 in F, 18 in HN, and 29 in L (see Fig. 3, which shows the alignment of M proteins). Among these unique amino acid substitutions, there were totally 54 conservative substitutions, 9 in NP, 8 in P, 2 in M, 10 in F, 10 in HN, and 15 in L. The presence of these unique amino acid substitutions suggested that the Tianjin strain maybe had a significant difference in biological, pathological, immunological, or epidemio-logical characteristics from the known SeVs. To establish the relationship between the substitutions and viral biological properties or pathogenesis, gene-ration of recombinant SeV carrying the mutation by reverse genetic technology would be necessary.