Comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus

Abstract

In this territory-wide molecular epidemiology study of coronaviruses (CoVs) in Hong Kong involving 1,541 dead wild birds, three novel CoVs were identified in three different bird families (bulbul CoV HKU11 [BuCoV HKU11], thrush CoV HKU12 [ThCoV HKU12], and munia CoV HKU13 [MuCoV HKU13]). Four complete genomes of the three novel CoVs were sequenced. Their genomes (26,396 to 26,552 bases) represent the smallest known CoV genomes. In phylogenetic trees constructed using chymotrypsin-like protease (3CL(pro)), RNA-dependent RNA polymerase (Pol), helicase, spike, and nucleocapsid proteins, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 formed a cluster distantly related to infectious bronchitis virus and turkey CoV (group 3a CoVs). For helicase, spike, and nucleocapsid, they were also clustered with a CoV recently discovered in Asian leopard cats, for which the complete genome sequence was not available. The 3CL(pro), Pol, helicase, and nucleocapsid of the three CoVs possessed higher amino acid identities to those of group 3a CoVs than to those of group 1 and group 2 CoVs. Unique genomic features distinguishing them from other group 3 CoVs include a distinct transcription regulatory sequence and coding potential for small open reading frames. Based on these results, we propose a novel CoV subgroup, group 3c, to describe this distinct subgroup of CoVs under the group 3 CoVs. Avian CoVs are genetically more diverse than previously thought and may be closely related to some newly identified mammalian CoVs. Further studies would be important to delineate whether the Asian leopard cat CoV was a result of interspecies jumping from birds, a situation analogous to that of bat and civet severe acute respiratory syndrome CoVs.

FIG. 1.

Phylogenetic analysis of amino acid sequences of the 393-bp fragment (excluding primer sequences) of RNA-dependent RNA Pol of CoVs identified from dead wild birds in the present study. The tree was constructed by the neighbor joining method using Kimura's two-parameter correction and bootstrap values calculated from 1,000 trees. The scale bar indicates the estimated number of substitutions per 50 amino acids. The three novel CoVs identified in the present study are shown in bold. The four genomes completely sequenced are highlighted in gray. HCoV-229E (NC_002645); PEDV, porcine epidemic diarrhea virus (NC_003436); TGEV, porcine transmissible gastroenteritis virus (NC_002306); FIPV, feline infectious peritonitis virus (AY994055); PRCV, porcine respiratory CoV (DQ811787); HCoV-NL63 (NC_005831); bat-CoV HKU2 (EF203064); bat-CoV HKU4 (NC_009019); bat-CoV HKU5 (NC_009020); bat-CoV HKU6 (DQ249224); bat-CoV HKU7 (DQ249226); bat-CoV HKU8 (NC_010438); bat-CoV HKU9 (NC_009021) BtCoV 1A, bat CoV 1A (NC_010437); BtCoV 1B, bat CoV 1B 1B (NC_010436); BtCoV/512/2005, bat CoV 512/2005 (NC_009657); HCoV-HKU1 (NC_006577); HCoV-OC43 (NC_005147); MHV (NC_006852); BCoV, bovine CoV (NC_003045); SDAV, rat sialodacryoadenitis CoV (AF124990); AntelopeCoV, sable antelope CoV (EF424621); GiCoV, giraffe CoV (EF424622); PHEV, porcine hemagglutinating encephalomyelitis virus (NC_007732); SARS-CoV (NC_004718); bat-SARS-CoV HKU3 (NC_009694); IBV (NC_001451); IBV-partridge, partridge CoV (AY646283); IBV-peafowl, peafowl CoV (AY641576); TCoV (NC_010800); ALC, Asian leopard cat CoV Guangxi/F230/2006 (EF584908); SW1 (NC_010646). BuCoV HKU11 (ChBu, chinese Bulbul, and RwBu, red-whiskered bulbul); ThCoV HKU12 (BlTh, blackbird, and GbTh, gray-backed thrush); MuCoV HKU13 (WrMu, white-rumped munia, and SbMu, scaly-breasted munia).

FIG. 2.

Genome organizations of BuCoV HKU11, ThCoV HKU12, MuCoV HKU13, and representative CoVs from each group. Papain-like proteases (PL1, PL2, and PL), 3CL^pro, RNA-dependent RNA Pol, hemagglutinin esterase (HE), spike (S), envelope (E), membrane (M), and nucleocapsid (N) are represented by gray boxes. Virus name abbreviations may be found in the Fig. 1 legend and the text.

FIG. 3.

Multiple alignment of conserved s2m's of BuCoV HKU11, ThCoV HKU12, MuCoV HKU13, Asian leopard cat CoV (ALC), IBV, SARS-CoV, and bat-SARS-CoV. The identical nucleotides are marked by asterisks.

FIG. 4.

Phylogenetic analyses of 3CL^pro, RNA-dependent RNA Pol, helicase (Hel), S, and N proteins of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13. The trees were constructed by using the neighbor joining method using Kimura's two-parameter correction and bootstrap values calculated from 1,000 trees. Three hundred eight, 958, 609, 1,735, and 592 amino acid positions in 3CL^pro, Pol, helicase, S, and N, respectively, were included in the analyses. The scale bar indicates the estimated number of substitutions per 10 amino acids. PEDV, porcine epidemic diarrhea virus; TGEV, porcine transmissible gastroenteritis virus; FIPV, feline infectious peritonitis virus; BtCoV 1A, bat CoV 1A; BtCoV 1B, bat CoV 1B; BtCoV/512/2005, bat CoV 512/2005; BCoV, bovine CoV; AntelopeCoV, sable antelope CoV; GiCoV, giraffe CoV; IBV-partridge, partridge CoV; IBV-peafowl, peafowl CoV; PHEV, porcine hemagglutinating encephalomyelitis virus; ALC, Asian leopard cat CoV Guangxi/F230/2006.