Genetically Diverse Filoviruses in Rousettus and Eonycteris spp. Bats, China, 2009 and 2015

Genetically divergent filoviruses detected in Rousettus and Eonycteris spp. bats in China exhibited 61%–99% nt identity with reported filoviruses, based on partial replicase sequences, and they demonstrated lung tropism. Co-infection with 4 different filoviruses was found in 1 bat. These results demonstrate that fruit bats are key reservoirs of filoviruses

Isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of Severe Acute Respiratory Syndrome coronavirus

We report the isolation and characterization of a novel bat coronavirus which is much closer to the severe acute respiratory syndrome coronavirus (SARS-CoV) in genomic sequence than others previously reported, particularly in its S gene. Cell entry and susceptibility studies indicated that this virus can use ACE2 as a receptor and infect animal and human cell lines. Our results provide further evidence of the bat origin of the SARS-CoV and highlight the likelihood of future bat coronavirus emergence in humans

Bat Severe Acute Respiratory Syndrome-like coronavirus WIV1 encodes an extra accessory protein, ORFX, involved in modulation of the host immune response

Bats harbor severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) from which the causative agent of the 2002-2003 SARS pandemic is thought to have originated. However, despite the fact that a large number of genetically diverse SL-CoV sequences have been detected in bats, only two strains (named WIV1 and WIV16) have been successfully cultured in vitro. These two strains differ from SARS-CoV only in containing an extra open reading frame (ORF) (named ORFX), between ORF6 and ORF7, which has no homology to any known protein sequences. In this study, we constructed a full-length cDNA clone of SL-CoV WIV1 (rWIV1), an ORFX deletion mutant (rWIV1-ΔX), and a green fluorescent protein (GFP)-expressing mutant (rWIV1-GFP-ΔX). Northern blotting and fluorescence microscopy indicate that ORFX was expressed during WIV1 infection. A virus infection assay showed that rWIV1-ΔX replicated as efficiently as rWIV1 in Vero E6, Calu-3, and HeLa-hACE2 cells. Further study showed that ORFX could inhibit interferon production and activate NF-κB. Our results demonstrate for the first time that the unique ORFX in the WIV1 strain is a functional gene involving modulation of the host immune response but is not essential for in vitro viral replication. IMPORTANCE Bats harbor genetically diverse SARS-like coronaviruses (SL-CoVs), and some of them have the potential for interspecies transmission. A unique open reading frame (ORFX) was identified in the genomes of two recently isolated bat SL-CoV strains (WIV1 and -16). It will therefore be critical to clarify whether and how this protein contributes to virulence during viral infection. Here we revealed that the unique ORFX is a functional gene that is involved in the modulation of the host immune response but is not essential for in vitro viral replication. Our results provide important information for further exploration of the ORFX function in the future. Moreover, the reverse genetics system we constructed will be helpful for study of the pathogenesis of this group of viruses and to develop therapeutics for future control of emerging SARS-like infections

Serological Evidence of Bat SARS-Related Coronavirus Infection in Humans, China

[LETTER TO THE EDITOR

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus - Fig 8

<p><b>Analysis of receptor usage by immunofluorescence assay (A) and real-time PCR (B).</b> Virus infectivity of Rs4874, WIV1-Rs4231S and WIV1-Rs7327S was determined in HeLa cells with and without the expression of human ACE2. ACE2 expression was detected with goat anti-human ACE2 antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG. Virus replication was detected with rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein followed by cyanine 3 (Cy3)-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI (49,6-diamidino-2-phenylindole).The columns (from left to right) show staining of nuclei (blue), ACE2 expression (green), virus replication (red) and the merged triple-stained images, respectively.</p

Infection of Vero E6 cells by bat SARSr-CoV WIV1, Rs4874, WIV1-Rs4231S and WIV1-Rs7327S.

<p>(A) The successful infection was confirmed by immunofluorescent antibody staining using rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein. The columns (from left to right) show staining of nuclei (blue), virus replication (red), and both nuclei and virus replication (merged double-stain images). (B) The growth curves in Vero E6 cells with a MOI of 1.0 and 0.01.</p