Molecular Characterization of Infectious Bronchitis Virus Strain HH06 Isolated in a Poultry Farm in Northeastern China.

Spike (S) glycoprotein is an important virulent factor for coronaviruses (CoVs), and variants of CoVs have been characterized based on S gene analysis. We present phylogenetic relationship of an isolated infectious bronchitis virus (IBV) strain with reference to the available genome and protein sequences based on network, multiple sequence, selection pressure, and evolutionary fingerprinting analysis in People's Republic of China. One hundred and elven strains of CoVs i.e., Alphacoronaviruses (Alpha-CoVs; n = 12), Betacoronaviruses (Beta-CoVs; n = 37), Gammacoronaviruses (Gamma-CoVs; n = 46), and Deltacoronaviruses (Delta-CoVs; n = 16) were selected for this purpose. Phylogenetically, SARS-CoV-2 and SARS-CoVs clustered together with Bat-CoVs and MERS-CoV of Beta-CoVs (C). The IBV HH06 of Avian-CoVs was closely related to Duck-CoV and partridge S14, LDT3 (teal and chicken host). Beluga whale-CoV (SW1) and Bottlenose dolphin-CoVs of mammalian origin branched distantly from other animal origin viruses, however, making group with Avian-CoVs altogether into Gamma-CoVs. The motif analysis indicated well-conserved domains on S protein, which were similar within the same phylogenetic class and but variable at different domains of different origins. Recombination network tree indicated SARS-CoV-2, SARS-CoV, and Bat-CoVs, although branched differently, shared common clades. The MERS-CoVs of camel and human origin spread branched into a different clade, however, was closely associated closely with SARS-CoV-2, SARS-CoV, and Bat-CoVs. Whereas, HCoV-OC43 has human origin and branched together with bovine CoVs with but significant distant from other CoVs like SARS CoV-2 and SARS-CoV of human origin. These findings explain that CoVs' constant genetic recombination and evolutionary process that might maintain them as a potential veterinary and human epidemic threat.

Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease.

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

Time-dependent viral interference between influenza virus and coronavirus in the infection of differentiated porcine airway epithelial cells.

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

The Cell Tropism of Porcine Respiratory Coronavirus for Airway Epithelial Cells Is Determined by the Expression of Porcine Aminopeptidase N.

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.

Prokaryotic expression of IBV N protein and development of indirect IBV N protein-mediated ELISA

Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study, according to IBV gene sequences published in Gen Bank, specific primers were designed to clone N gene by RT-PCR, and this gene was inserted into p ET-30a(+)vector resulting in a prokaryotic expression plasma p ET-30a-N. The results of SDS-PAGE and Western Blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum. Using purified recombinant N protein as a coating antigen, the indirect ELISA protocol was established and optimized, in which N protein was 2.5mug.m Lsup-1/sup of concentration, sample serum of 1:40 dilution. For clinical specimen, the IBV antibodies could be detected by this method efficiently and got nearly the same results as those of IBV-mediated ELISA. It would provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.