Members of the Coronaviridae
family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26–32 kb [1
]. These viruses are classified into two subfamilies: Letovirinae
, which contains the only genus: Alphaletovirus
; and Orthocoronavirinae
), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2
]. Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma- and delta-CoVs mainly infect birds, but some can also infect mammals [4
]. Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have caused severe disease with a 10% or 35% mortality, respectively [6
]. CoVs infection in domestic animals can also cause great economic losses, such as transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and HKU2-related CoV in pigs [7
Prior to the global SARS outbreak in 2002 to 2003, only 10 CoVs were reported. Since the outbreak, numerous CoVs have been discovered in animals, particularly, in bats [6
]. According to a recent report by the International Committee of Viruses on Taxonomy (ICTV), at least 17 out of 29 assigned alpha and beta-CoV species were identified from 11 out of 18 bat families [2
]. Phylogenetic analysis suggested that bats are major hosts for alpha- and beta-CoVs. Recombination of different CoVs occurred in bats, as previously reported. Bats play an important role in CoV evolution [4
Rhinolophus bats are widespread in China. At least 4 CoV species with high genetic diversity have been found in members of this family [11
]. Among these viruses, bat SARS-related coronaviruses (SARSr-CoVs) have been proved to be able to infect animal and human cells by using the same receptor as SARS-CoV [12
]. Recently, a new porcine disease was confirmed to be caused by BatCoV HKU2-related virus in Guangdong Province, China [8
]. These findings indicate that these bat species play important roles in CoV evolution and transmission.
Here, we report a novel species of alpha-CoV discovered in Rhinolophus bats in China, their unique genomic structures and a preliminary functional assessment of accessory genes, as well as this virus’ infectivity in different cells.
In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus
species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42
]. These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis
, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus
, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving.
Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.
Accessory genes are usually involved in virus-host interactions during CoV infection [43
]. In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44
]. Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47
]. SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48
], inhibition of cellular protein synthesis [49
], cell-cycle blockage [50
], and apoptosis induction [51
]. In this study, we found that BtCoV/Rh/YN2012 ORF9 shares ~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus
from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF
7a or ORF9
from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure.
Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses’ pathogenicity with a different pathway.
Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments.
Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs.