Classification And Phylogeography Of Hantaviruses Reservoir Hosts Rodentia In Longquan City, Zhejiang Province

Hemorrhagic fever with renal syndrome (HFRS) is an acute viral disease infected by hantaviruses. HFRS is mostly occurred in China and Longquan city is the mainly area with high incidence of HFRS in Zhejiang Province.Hantaviruses are enveloped RNA viruses belonging to the family Bunyaviridae. In2012, the International Committee on Taxonomy of Viruses has officially approved24species of hantavirus. And the reservoir hosts of hantaviruses are included some species of Rodentia, Insectivore and Chiroptera. So far, all the infectious hantaviruses are detected in rodents.Rodents are the most abundant and diverse order of mammals, which include30families and over2000species. They are broadly distributed and are highly diversified on morphology and ecology. According to previous studies, rodents originally appeared in the Palaeocene epoch about65million years ago. More importantly, at least60infectious pathogens have been found in rodents, most can infect human beings and livestocks and cause serious diseases. Currently, rodents live in or close to residential area and farming area which poses great threats to the public health.Earlier approaches for mammal taxonomy and identification are mainly based on morphology. However, morphological methods fail when the organisms are closely related or when the specimens are damaged. With the development of molecular techniques, the method combining both morphology and molecular methods has gradually become mainstream. In this study, we identified the rodents in Longquan of Zhejiang Province through morphological and molecular skills. Simultaneously, we detected the hantavirus in rodents. However, in here we only reported the classification and phylogenetic analysis.Purpose:Since1974, Longquan become one of the most severely affected regions by hemorrhagic fever. And human is infected with disease transmitted from rodents. So, it’s essential to study the animal taxonomy and phylogeny for disease control and prevention.Method:Trapped rodents were initially classified by morphology. Nucleic acids were extracted from samples. Cytb, CO1,16S and IRBP genes were amplified by PCR. Bioinformatics tools were used for computing the nucleotide composition, substitution pattern, as well as intraspecies and interspecies distance. Furthermore, maximum likelihood phylogenetic trees were reconstructed for genetic comparisons and phylogeographic analyses.Results:In2013, a total of640small mammals have been trapped and identified as rodents based on morphology. Namely, Apodemus agrarius, Micromys minutes, Rattus tanezumi, Rattus losea, Rattus norvigicus, Nivienter confuciacus and Microtus fortis. There are two mis-identifications after re-examination by PCR:Eothenomys melanogaster was mistaken for Microtus fortis and Chimarogale himalayica was mistaken for Apodemus agrarius.We obtained128sequences from PCR, which contained Cytb, CO1,16S and IRBP gene sequences. The analysis of nucleotide composition of sequences revealed that the ratio of A+T/G+C in mitochondrial gene Cytb, CO land16S was3/2which was in contrast with2/3in nuclear gene IRBP. Analysis of intraspecies and intersepcies distance revealed that the intraspecies was lower than2%for the four genes while the largest interspecies distance was25%in Cytb and CO1. Phylogenetic analyses suggested inconsistent topologies among the four genes. To solve this problem, we concatenated the mitochorial genes Cytb, CO1and16S and the resulting tree showed much better resolution and are consistent with previous taxonomy with the high bootstrap support. The phylogeographic analysis revealed that A. a, M. m, M.f and C. h had the obvious geographic clustering while the Rattus species did not. Conclusions:We suggested that A. a, R. n, R. t, R. l and M. f were the dominant rodent species in Longquan. The Cytb and CO1genes were regarded as good molecular marker for taxonomy. Combined three mitochorial gene to reconstructure phylogenetic tree are sufficient for a well-resolved taxonomy. Phylogeographic analysis indicted that most of the rodent species showed obvious geographic clustering with the exception of Rattus. A. a, R. n, R. t, R. l and M. f are the reservoir hosts of viruses which can cause human HFRS. Therefore, it is essential to monitor the rodents for disease control and prevention in Longquan.