Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Construction of infectious bronchitis virsus endoribonucleasensp15 defective recombinant virus by reverse genetic system

Infectious Bronchitis Virus (1BV) belongs to the y coronavirus, however, the function of IBV encoded endoribonuclease (non - structural protein 15, nsp15) has not been determined yet. To explore the function of nsp15 in the process of IBV replication, we mutated the IBV nsp15 endonuclease core residue His238 to Ala, constructed the nsp15-defective recombinant virus rIBV-nsp15-H238A, via in vitro ligation and recombination technology. Plaque assay and TCID50 were performed to measure virus titer, virus plaque size and growth curve. The IBV Beaudette-R genome was cloned as 5 fragments in vectors, BsaI or BsmBI restriction sites were added to the end of each fragment. After plasmid amplification, the cDNA fragments were obtained by enzymatic digestion, followed with in vitro ligation and transcription. Full - length genomic RNA was electroporated into Vero cells, together with N transcript, to rescue the recombinant viruses rIBV and rIBV - nsp15- H238A. Plaque assay was performed to detect and compare the viral titer and plaque size of these two recombinant viruses. Results showed that the virus titer of rIBV -nsp15 -H238A was 2.71x106PFU/mL, 3 times lower than that of rIBV (9.4x106PFU/mL). The plaque size of rIBV-nsp15-H238A was much smaller than that of rIBV, indicating that rIBV-nsp15-11238A replicates and spreads slower than rIBV. The growth curve of rIBV-nsp15-H238A was slower than that of rIBV. Our study demonstrates that nsp15 I-1238 is the key amino acid and plays an important role in the replication and spread of IBV. The construction of nsp15 defective recombinant virus provides a powerful tool for the study of the function of nsp15.

Non-Targeted Metabolomic Analysis of Chicken Kidneys in Response to Coronavirus IBV Infection Under Stress Induced by Dexamethasone.

Stress in poultry can lead to changes in body metabolism and immunity, which can increase susceptibility to infectious diseases. However, knowledge regarding chicken responses to viral infection under stress is limited. Dexamethasone (Dex) is a synthetic glucocorticoid similar to that secreted by animals under stress conditions, and has been widely used to induce stress in chickens. Herein, we established a stress model in 7-day-old chickens injected with Dex to elucidate the effects of stress on IBV replication in the kidneys. The metabolic changes, immune status and growth of the chickens under stress conditions were comprehensively evaluated. Furthermore, the metabolic profile, weight gain, viral load, serum cholesterol levels, cytokines and peripheral blood lymphocyte ratio were compared in chickens treated with Dex and infected with IBV. An LC-MS/MS-based metabolomics method was used to examine differentially enriched metabolites in the kidneys. A total of 113 metabolites whose abundance was altered after Dex treatment were identified, most of which were lipids and lipid-like molecules. The principal metabolic alterations in chicken kidneys caused by IBV infection included fatty acid, valine, leucine and isoleucine metabolism. Dex treatment before and after IBV infection mainly affected the host's tryptophan, phenylalanine, amino sugar and nucleotide sugar metabolism. In addition, Dex led to up-regulation of serum cholesterol levels and renal viral load in chickens, and to the inhibition of weight gain, peripheral blood lymphocytes and IL-6 production. We also confirmed that the exogenous cholesterol in DF-1 cells promoted the replication of IBV. However, whether the increase in viral load in kidney tissue is associated with the up-regulation of cholesterol levels induced by Dex must be demonstrated in future experiments. In conclusion, chick growth and immune function were significantly inhibited by Dex. Host cholesterol metabolism and the response to IBV infection are regulated by Dex. This study provides valuable insights into the molecular regulatory mechanisms in poultry stress, and should support further research on the intrinsic link between cholesterol metabolism and IBV replication under stress conditions.

Characterization of the Protective Efficacy Against QX Strain of a Recombinant Infectious Bronchitis Virus With H120 Backbone and QX Spike Gene.

Infectious bronchitis virus (IBV) has been prevalent in chicken farms for many years, and its control relies on extensive vaccine administration. The continuous emergence of new variants and the low cross-protection efficiency prompt the development of new vaccines. In this study, we develop a reverse genetics technique based on the classical vaccine strain H120 genome, via in vitro ligation method. Using the H120 genome as the backbone, we constructed the recombinant virus rH120-QX(S) by replacing the H120 S gene with the QX S gene, a prevalent strain in China. Biological characteristics of the rH120-QX(S) virus, such as 50% egg lethal dose (ELD50), 50% egg infectious dose (EID50), dwarf embryo, growth curve, and genetic stability, are measured, which are comparable to the parental virus H120. There are no clinical symptoms and tissue lesions in the trachea and kidney in the rH120-QX(S)-infected specific-pathogen-free (SPF) chickens, demonstrating that this recombinant virus does not confer pathogenicity. Furthermore, protection studies show that there is 100% homologous protection of rH120-QX(S) to the virulent QX strain, as shown by the absence of clinical signs and no lethality. Taken together, our results demonstrate that swapping the S gene onto the H120 genetic backbone is a precise and effective way to produce genetically defined IBV vaccine candidates.

Bile acids promote the caveolae-associated entry of swine acute diarrhea syndrome coronavirus in porcine intestinal enteroids.

Intestinal microbial metabolites have been increasingly recognized as important regulators of enteric viral infection. However, very little information is available about which specific microbiota-derived metabolites are crucial for swine enteric coronavirus (SECoV) infection in vivo. Using swine acute diarrhea syndrome (SADS)-CoV as a model, we were able to identify a greatly altered bile acid (BA) profile in the small intestine of infected piglets by untargeted metabolomic analysis. Using a newly established ex vivo model-the stem cell-derived porcine intestinal enteroid (PIE) culture-we demonstrated that certain BAs, cholic acid (CA) in particular, enhance SADS-CoV replication by acting on PIEs at the early phase of infection. We ruled out the possibility that CA exerts an augmenting effect on viral replication through classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling, innate immune suppression or viral attachment. BA induced multiple cellular responses including rapid changes in caveolae-mediated endocytosis, endosomal acidification and dynamics of the endosomal/lysosomal system that are critical for SADS-CoV replication. Thus, our findings shed light on how SECoVs exploit microbiome-derived metabolite BAs to swiftly establish viral infection and accelerate replication within the intestinal microenvironment.

Coronavirus Infection and Cholesterol Metabolism.

Host cholesterol metabolism remodeling is significantly associated with the spread of human pathogenic coronaviruses, suggesting virus-host relationships could be affected by cholesterol-modifying drugs. Cholesterol has an important role in coronavirus entry, membrane fusion, and pathological syncytia formation, therefore cholesterol metabolic mechanisms may be promising drug targets for coronavirus infections. Moreover, cholesterol and its metabolizing enzymes or corresponding natural products exert antiviral effects which are closely associated with individual viral steps during coronavirus replication. Furthermore, the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 infections are associated with clinically significant low cholesterol levels, suggesting cholesterol could function as a potential marker for monitoring viral infection status. Therefore, weaponizing cholesterol dysregulation against viral infection could be an effective antiviral strategy. In this review, we comprehensively review the literature to clarify how coronaviruses exploit host cholesterol metabolism to accommodate viral replication requirements and interfere with host immune responses. We also focus on targeting cholesterol homeostasis to interfere with critical steps during coronavirus infection.

Infectious Bronchitis Virus Infection Increases Pathogenicity of H9N2 Avian Influenza Virus by Inducing Severe Inflammatory Response.

Infectious bronchitis virus (IBV) and H9N2 avian influenza virus (AIV) are frequently identified in chickens with respiratory disease. However, the role and mechanism of IBV and H9N2 AIV co-infection remain largely unknown. Specific-pathogen-free (SPF) chickens were inoculated with IBV 2 days before H9N2 virus inoculation (IBV/H9N2); with IBV and H9N2 virus simultaneously (IBV+H9N2); with H9N2 virus 2 days before IBV inoculation (H9N2/IBV); or with either IBV or H9N2 virus alone. Severe respiratory signs, pathological damage, and higher morbidity and mortality were observed in the co-infection groups compared with the IBV and H9N2 groups. In general, a higher virus load and a more intense inflammatory response were observed in the three co-infection groups, especially in the IBV/H9N2 group. The same results were observed in the transcriptome analysis of the trachea of the SPF chickens. Therefore, IBV might play a major role in the development of respiratory disease in chickens, and secondary infection with H9N2 virus further enhances the pathogenicity by inducing a severe inflammatory response. These findings may provide a reference for the prevention and control of IBV and H9N2 AIV in the poultry industry and provide insight into the molecular mechanisms of IBV and H9N2 AIV co-infection in chickens.

The Interplay Between Coronavirus and Type I IFN Response

In the past few decades, newly evolved coronaviruses have posed a global threat to public health and animal breeding. To control and prevent the coronavirus-related diseases, understanding the interaction of the coronavirus and the host immune system is the top priority. Coronaviruses have evolved multiple mechanisms to evade or antagonize the host immune response to ensure their replication. As the first line and main component of innate immune response, type I IFN response is able to restrict virus in the initial infection stage;it is thus not surprising that the primary aim of the virus is to evade or antagonize the IFN response. Gaining a profound understanding of the interaction between coronaviruses and type I IFN response will shed light on vaccine development and therapeutics. In this review, we provide an update on the current knowledge on strategies employed by coronaviruses to evade type I IFN response.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.