Identification and complete genome analysis of three novel paramyxoviruses, Tuhoko virus 1, 2 and 3, in fruit bats from China

Abstract

Among 489 bats of 11 species in China, three novel paramyxoviruses [Tuhokovirus 1, 2 and 3 (ThkPV-1, ThkPV-2 and ThkPV-3)] were discovered in 15 Leschenault's rousettes. Phylogenetically, the three viruses are most closely related to Menangle and Tioman virus. Genome analysis showed that their 3'-leader sequences are unique by possessing GA instead of AG at the 5th and 6th positions. Unlike Menangle and Tioman virus, key amino acids for neuraminidase activity characteristic of rubulavirus attachment proteins are present. The genome of ThkPV-1 represents the largest rubulavirus genome. Unique features between the three viruses include perfect complementary 5'-trailer and 3'-leader sequence and a unique cysteine pair in attachment protein of ThkPV-1, G at +1 position in all predicted mRNA sequences of ThkPV-2, and amino acid substitutions in the conserved N-terminal motif of nucleocapsid of ThkPV-3. Analysis of phosphoprotein gene mRNA products confirmed mRNA editing. Antibodies to the viruses were detected in 48-60% of Leschenault's rousettes.

Fig. 1

(A) Phylogenetic analysis of amino acid sequences of the 502-bp fragment of L gene of paramyxoviruses identified from bats in the present study. The tree was constructed by neighbor-joining method using Jukes-Cantor correction and bootstrap values calculated from 1000 trees. The scale bar indicates the estimated number of substitutions per 20 amino acids. The different strains of ThkPV-1, ThkPV-2 and ThkPV-3 from different bat samples are named as ThkPV/batA or ThkPV/batR representing alimentary or respiratory sample from the corresponding bat number, respectively. The three strains each from ThkPV-1, ThkPV-2 and ThkPV-3 with genome sequences determined are shown in bold. Paramyxoviruses from bats are shaded in gray. CdiPV, Canine distemper virus (NC_001921); PdiPV, Phocine distemper virus (Y09630); MeaPV, Measles virus (NC_001498); RinPV, Rinderpest virus (NC_006296); PprPV, Peste-des-petits ruminants virus (NC_006383); DmoPV, Dolphin morbillivirus (NC_005283); FdlPV, Fer-de-lance virus (NC_005084); SenPV, Sendai virus (NC_001552); HpiPV-1, Human parainfluenza virus 1 (NC_003461); HpiPV-3, Human parainfluenza virus 3 (NC_001796); SpiPV-3 (EU439429), Swine parainfluenza 3; BpiPV-3, Bovine parainfluenza virus 3 (NC_002161); AsaPV, Atlantic salmon paramyxovirus (EU156171); NarPV, Nariva virus (FJ362497); TupPV, Tupaia paramyxovirus (NC_002199); MosPV, Mossman virus (NC_005339); NipPV, Nipah virus (NC_002728); HenPV, Hendra virus (NC_001906); JPV, J-virus (NC_007454); BeiPV, Beilong virus (NC_007803); AviPV-4, Avian paramyxovirus 4 (FJ177514); AviPV-3, Avian paramyxovirus 3 (EU403085); AviPV-6, Avian paramyxovirus 6 (NC_003043); AviPV-7, Avian paramyxovirus 7 (FJ215864); GooPV, Goose paramyxovirus SF02 (NC_005036); PigPV-1, Pigeon paramyxovirus 1 (AJ880277); NdiPV, Newcastle disease virus (NC_002617); AviPV-9, Avian paramyxovirus 9 (EU910942); AviPV-8, Avian paramyxovirus 8 (FJ215864); AviPV-2, Avian parayxovirus 2 (EU338414); MumPV, Mumps virus (NC_002200); PruPV, Porcine rubulavirus (NC_009640); MapPV, Mapuera virus (NC_009489); HpiPV-4b, Human parainfluenza virus 4b (EU627591); SimPV-41, Simian virus 41 (NC_006428); HpiPV-2, Human parainfluenza virus 2 (NC_003443); SimPV-5, Simian virus 5 (NC_006430); TioPV, Tioman virus (NC_004074); MenPV, Menangle virus (NC_007620). (B) A Leschenault's rousette bat with grayish brown, soft, fine and silky fur, long and narrow muzzle, large and conspicuous eyes, and simple ears without antitragus or tragus.

Fig. 2

Alignment of genome terminal sequences of ThkPV-1, ThkPV-2 and ThkPV-3. Dots indicate identical residues. (A) Alignment of the 3' leader and 5' trailer sequences of ThkPV-1, ThkPV-2 and ThkPV-3. (B) Alignment of 3' leader sequences of ThkPV-1, ThkPV-2, ThkPV-3 and other rubulaviruses.

Fig. 3

Western blot analysis against purified (His)₆-tagged recombinant N protein from ThkPV-1 (A), ThkPV-2 (B), and ThkPV-3 (C). Western blot analysis was performed using serum samples from 23 Leschenault's rousette bats and two non-Leschenault's rousette bats (B25b and B26). Prominent immunoreactive protein bands of about 65 kDa, consistent with the expected size of 66.2, 66.7 and 65.5 kDa of the recombinant N proteins of ThkPV-1, ThkPV-2 and ThkPV-3, respectively, were detected with serum samples from Lechenault's rousette bats but not with the other two non-Leschenault's rousette bats. Lane numbers correspond to the bat numbers in Table 1.