The Discoveries Of Himalayan Subtype Of Tick-borne Encephalitis Virus And A Novel Torovirus Detected In Tibetan Antelope

Himalayan marmot(Marmota himalayana)and Tibetan antelope(Pantholops hodgsonii)are important wild animals which inhabited in Qinghai-Tibet Plateau in China,where non-native animals or humans may experience life-threatening acute mountain sickness.They occupy open-high elevation alpine and desert steppe habitats in the mountainous terrain with frequent occurrence at elevations between 3250 and 5500 m,and with a low partial pressure of oxygen and a high level of ultraviolet radiation.In this study,we used RNA-seq sequencing to initially conduct analysis of RNA viruses which carried by the two wild animals.Aa new subtype of TBEV in wild rodent Marmota himalayana in Qinghai-Tibet Plateau in China was detected,proposed as Himalayan subtype(Him-TBEV).Two complete genomes of Him-TBEV were obtained from respiratory samples of 200 marmots.The phylogenetic analysis using the E protein and polyprotein demonstrated that the two strains of Him-TBEV formed an independent branch,separated from Eu-TBEV,Sib-TBEV and FE-TBEV.The nomenclature of Him-TBEV as a new subtype was also supported by comparative analysis using nucleotide and amino acid sequences of E protein and polyprotein.For E protein,The Him-TBEV showed 892.7%-95.0%amino acid identities with other three subtypes,for polyprotein,the Him-TBEV showed 92.6%-94.2%amino acids identities with other three subtypes.Furthermore,of 69 amino acid substitutions profiles detected in complete polyprotein of 112 strains of TBEV,Him-TBEV subtype displayed unique amino acids in the 36 positions.Notably,for the subtype-specific amino acid position 206 of E protein,Him-TBEV shared the Val with Eu-TBEV,but differed from FE-TBEV and Sib-TBEV.The evolutionary analysis with BEAST suggested that Him-TBEV diverged from other subtypes of eastern TBEV group about 2469 years ago.We conducted genome codon bias analysis of Him-TBEV.The ENC value of the whole open reading frame was 55.32,which means codon usage bias is lower in Him-TBEV;ENC-plot and Neutrality-plot analyses together demonstrated that natural selection mainly influence the condon usage bias of Him-TBEV.According to RSCU values of codons,there are 19 codons of defined as the maj or preference codons in Him-TBEV.The codon usage pattern of Him-TBEV was most similar with the codon usage pattern of Human.The public health significance of discovery of Him-TBEV in Marmota himalayana must be carefully evaluated.In addition,we discovered a novel torovirus from fecal samples of Tibetan Antelope(Pantholops hodgsonii)in Qinghai-Tibet Plateau of China,which was proposed as Antelope Torovirus(AToV).Of 505 fecal samples,three were tested positive for AToV by RT-PCR.Real time RT-PCR showed viral load of the three positive samples were 2.10×109/g,9.30×109/g and 1.76×1010/g.Combined results of RNA-seq sequencing,RACE and RT-PCR,we obtained three complete genome sequences of AToV.According to the criterion for recognization of the new virus species of genera Torovirus,issued by the International Committee on Taxonomy of Viruses(ICTV),the results of phylogenetic analysis and alignment of amino acid sequence indicated that the AToV was a new species of virus in genus of Torovirus.The genome of AToV virus was 28,438 nt in length,encoding 6 open reading frames(ORFs),i.e.ORF1a,ORF1b,S ORF,M ORF,HE ORF and N ORF.The ORF1a and ORF1b encoded non-structural proteins(ppla and pp1b)which included domains of replicase.The S ORF,M ORF,HE ORFand N ORF encoded structural protein,which embedded in the envelope and formed the capsid of the virus.At the overlapping region of ORF la and ORF1b,we found elements of ribosomal frameshifting,including a slippery sequence and a RNA pseudoknot,which were also found in coronaviruses.ToV-like paticle of about 100 nm in diameter with typical surface projections were observed by transmission electron microscope.The feature of the particle was consisted with toroviruses.Phylogenetic analysis of AToV and other To Vs suggested that their most recent common ancestor deviried 333 years ago.Notably,the HE genes of BToV B150/B155 were derived from HE genes of three species of toroviruses,referring BToV,PToV and an unknown torovirus.The unknown torovirus was proved as AToV which identified in this study.