Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins.

Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. However, little is known about its pathogenicity in pangolins. Using CT scans we show that SARSr-CoV-2 positive Malayan pangolins are characterized by bilateral ground-glass opacities in lungs in a similar manner to COVID-19 patients. Histological examination and blood gas tests are indicative of dyspnea. SARSr-CoV-2 infected multiple organs in pangolins, with the lungs the major target, and histological expression data revealed that ACE2 and TMPRSS2 were co-expressed with viral RNA. Transcriptome analysis indicated that virus-positive pangolins were likely to have inadequate interferon responses, with relative greater cytokine and chemokine activity in the lung and spleen. Notably, both viral RNA and viral proteins were detected in three pangolin fetuses, providing initial evidence for vertical virus transmission. In sum, our study outlines the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans.

Characterization of the induction kinetics and antiviral functions of IRF1, ISG15 and ISG20 in cells infected with gammacoronavirus avian infectious bronchitis virus.

Coronavirus infection induces a variety of cellular antiviral responses either dependent on or independent of type I interferons (IFNs). Our previous studies using Affymetrix microarray and transcriptomic analysis revealed the differential induction of three IFN-stimulated genes (ISGs), IRF1, ISG15 and ISG20, by gammacoronavirus infectious bronchitis virus (IBV) infection of IFN-deficient Vero cells and IFN-competent, p53-defcient H1299 cells, respectively. In this report, the induction kinetics and anti-IBV functions of these ISGs as well as mechanisms underlying their differential induction are characterized. The results confirmed that these three ISGs were indeed differentially induced in H1299 and Vero cells infected with IBV, significantly more upregulation of IRF1, ISG15 and ISG20 was elicited in IBV-infected Vero cells than that in H1299 cells. Induction of these ISGs was also detected in cells infected with human coronavirus-OC43 (HCoV-OC43) and porcine epidemic diarrhea virus (PEDV), respectively. Manipulation of their expression by overexpression, knockdown and/or knockout demonstrated that IRF1 played an active role in suppressing IBV replication, mainly through the activation of the IFN pathway. However, a minor, if any, role in inhibiting IBV replication was played by ISG15 and ISG20. Furthermore, p53, but not IRF1, was implicated in regulating the IBV infection-induced upregulation of ISG15 and ISG20. This study provides new information on the mechanisms underlying the induction of these ISGs and their contributions to the host cell antiviral response during IBV infection.

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.

Coronavirus RNA-dependent RNA polymerase interacts with the p50 regulatory subunit of host DNA polymerase delta and plays a synergistic role with RNA helicase in the induction of DNA damage response and cell cycle arrest in the S phase.

Disruption of the cell cycle is a common strategy shared by many viruses to create a conducible cellular microenvironment for their efficient replication. We have previously shown that infection of cells with gammacoronavirus infectious bronchitis virus (IBV) activated the theataxia-telangiectasia mutated (ATM) Rad3-related (ATR)/checkpoint kinase 1 (Chk1) pathway and induced cell cycle arrest in S and G2/M phases, partially through the interaction of nonstructural protein 13 (nsp13) with the p125 catalytic subunit of DNA polymerase delta (pol δ). In this study, we show, by GST pulldown, co-immunoprecipitation and immunofluorescent staining, that IBV nsp12 directly interacts with the p50 regulatory subunit of pol δ in vitro and in cells overexpressing the two proteins as well as in cells infected with a recombinant IBV harbouring an HA-tagged nsp12. Furthermore, nsp12 from severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 was also able to interact with p50. These interactions play a synergistic role with nsp13 in the induction of S phase arrest. The fact that subunits of an essential cellular DNA replication machinery physically associate with two core replication enzymes from three different coronaviruses highlights the importance of these associations in coronavirus replication and virus-host interaction, and reveals the potential of targeting these subunits for antiviral intervention.

Development of a multiplex RT-PCR method for the detection of four porcine enteric coronaviruses.

Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV M gene, PDCoV N gene, TGEV S gene and SADS-CoV RdRp gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 105 copies/µL, 6.48 × 105 copies/µL, 8.54 × 105 copies/µL and 7.79 × 106 copies/µL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.

Transcriptomic analysis reveals crucial regulatory roles of immediate-early response genes and related signaling pathways in coronavirus infectious bronchitis virus infection.

Coronavirus infection of cells differentially regulates the expression of host genes and their related pathways. In this study, we present the transcriptomic profile of cells infected with gammacoronavirus infectious bronchitis virus (IBV). In IBV-infected human non-small cell lung carcinoma cells (H1299 cells), a total of 1162 differentially expressed genes (DEGs), including 984 upregulated and 178 downregulated genes, was identified. These DEGs were mainly enriched in MAPK and Wnt signaling pathways, and 5 out of the 10 top upregulated genes in all transcripts were immediate-early response genes (IEGs). In addition, the induction of 11 transcripts was validated in IBV-infected H1299 and Vero cells by RT-qPCR. The accuracy, reliability and genericity of the transcriptomic data were demonstrated by functional characterization of these IEGs in cells infected with different coronaviruses in our previous publications. This study provides a reliable transcriptomic profile of host genes and pathways regulated by coronavirus infection.

Modulation of viral replication, apoptosis and antiviral response by induction and mutual regulation of EGR and AP-1 family genes during coronavirus infection.

Coronaviruses have evolved a variety of strategies to exploit normal cellular processes and signalling pathways for their efficient reproduction in a generally hostile cellular environment. One immediate-early response gene (IEG) family, the AP-1 gene family, was previously shown to be activated by coronavirus infection. In this study, we report that another IEG family, the EGR family, is also activated in cells infected with four different coronaviruses in three genera, i.e. gammacoronavirus infectious bronchitis virus (IBV), alphacoronaviruses porcine epidemic diarrhoea virus (PEDV) and human coronavirus-229E (HCoV-229E), and betacoronavirus HCoV-OC43. Knockdown of EGR1 reduced the expression of cJUN and cFOS, and knockdown of cJUN and/or cFOS reduced the expression of EGR1, demonstrating that these two IEG families may be cross-activated and mutual regulated. Furthermore, ERK1/2 was identified as an upstream kinase, and JNK and p38 as inhibitors of EGR1 activation in coronavirus-infected cells. However, upregulation of EGR family genes, in particular EGR1, appears to play a differential role in regulating viral replication, apoptosis and antiviral response. EGR1 was shown to play a limited role in regulation of coronavirus replication, and an anti-apoptotic role in cells infected with IBV or PEDV, but not in cells infected with HCoV-229E. Upregulation of EGR1 may also play a differential role in the regulation of antiviral response against different coronaviruses. This study reveals a novel regulatory network shared by different coronaviruses in the immediate-early response of host cells to infection.

Activation of the MKK3-p38-MK2-ZFP36 Axis by Coronavirus Infection Restricts the Upregulation of AU-Rich Element-Containing Transcripts in Proinflammatory Responses.

Coronavirus infections induce the expression of multiple proinflammatory cytokines and chemokines. We have previously shown that in cells infected with gammacoronavirus infectious bronchitis virus (IBV), interleukin 6 (IL-6), and IL-8 were drastically upregulated, and the MAP kinase p38 and the integrated stress response pathways were implicated in this process. In this study, we report that coronavirus infection activates a negative regulatory loop that restricts the upregulation of a number of proinflammatory genes. As revealed by the initial transcriptomic and subsequent validation analyses, the anti-inflammatory adenine-uridine (AU)-rich element (ARE)-binding protein, zinc finger protein 36 (ZFP36), and its related family members were upregulated in cells infected with IBV and three other coronaviruses, alphacoronaviruses porcine epidemic diarrhea virus (PEDV), human coronavirus 229E (HCoV-229E), and betacoronavirus HCoV-OC43, respectively. Characterization of the functional roles of ZFP36 during IBV infection demonstrated that ZFP36 promoted the degradation of transcripts coding for IL-6, IL-8, dual-specificity phosphatase 1 (DUSP1), prostaglandin-endoperoxide synthase 2 (PTGS2) and TNF-α-induced protein 3 (TNFAIP3), through binding to AREs in these transcripts. Consistently, knockdown and inhibition of JNK and p38 kinase activities reduced the expression of ZFP36, as well as the expression of IL-6 and IL-8. On the contrary, overexpression of mitogen-activated protein kinase kinase 3 (MKK3) and MAPKAP kinase-2 (MK2), the upstream and downstream kinases of p38, respectively, increased the expression of ZFP36 and decreased the expression of IL-8. Taken together, this study reveals an important regulatory role of the MKK3-p38-MK2-ZFP36 axis in coronavirus infection-induced proinflammatory response. IMPORTANCE Excessive and uncontrolled induction and release of proinflammatory cytokines and chemokines, the so-called cytokine release syndrome (CRS), would cause life-threatening complications and multiple organ failure in severe coronavirus infections, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and COVID-19. This study reveals that coronavirus infection also induces the expression of ZFP36, an anti-inflammatory ARE-binding protein, promoting the degradation of ARE-containing transcripts coding for IL-6 and IL-8 as well as a number of other proteins related to inflammatory response. Furthermore, the p38 MAP kinase, its upstream kinase MKK3 and downstream kinase MK2 were shown to play a regulatory role in upregulation of ZFP36 during coronavirus infection cycles. This MKK3-p38-MK2-ZFP36 axis would constitute a potential therapeutic target for severe coronavirus infections.

Phylogeography and evolutionary dynamics analysis of porcine delta-coronavirus with host expansion to humans.

From 2003 onwards, three pandemics have been caused by coronaviruses: severe acute respiratory syndrome coronavirus (SARS-CoV); middle east respiratory syndrome coronavirus (MERS-CoV); and, most recently, SARS-CoV-2. Notably, all three were transmitted from animals to humans. This would suggest that animals are potential sources of epidemics for humans. The emerging porcine delta-coronavirus was reported to infect children. This is a red flag that marks the ability of PDCoV to break barriers of cross-species transmission to humans. Therefore, we conducted molecular genetic analysis of global clade PDCoV to characterize spatiotemporal patterns of viral diffusion and genetic diversity. PDCoV was classified into three major lineages, according to distribution and phylogenetic analysis of PDCoV. It can be inferred based on the analysis results of the currently known PDCoV strains that PDCoV might originate in Asia. We also selected six special spike amino acid sequences to align and analyze to find seven significant mutation sites. The accumulation of these mutations may enhance dynamic movements, accelerating spike protein membrane fusion events and transmission. Altogether, our study offers a novel insight into the diversification, evolution, and interspecies transmission and origin of PDCoV and emphasizes the need to study the zoonotic potential of the PDCoV and comprehensive surveillance and enhanced biosecurity precautions for PDCoV.

Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.