The Applications of Nanopore Sequencing Technology in Animal and Human Virus Research.

In recent years, an increasing number of viruses have triggered outbreaks that pose a severe threat to both human and animal life, as well as caused substantial economic losses. It is crucial to understand the genomic structure and epidemiology of these viruses to guide effective clinical prevention and treatment strategies. Nanopore sequencing, a third-generation sequencing technology, has been widely used in genomic research since 2014. This technology offers several advantages over traditional methods and next-generation sequencing (NGS), such as the ability to generate ultra-long reads, high efficiency, real-time monitoring and analysis, portability, and the ability to directly sequence RNA or DNA molecules. As a result, it exhibits excellent applicability and flexibility in virus research, including viral detection and surveillance, genome assembly, the discovery of new variants and novel viruses, and the identification of chemical modifications. In this paper, we provide a comprehensive review of the development, principles, advantages, and applications of nanopore sequencing technology in animal and human virus research, aiming to offer fresh perspectives for future studies in this field.

Evaluation of canine adipose-derived mesenchymal stem cells for neurological functional recovery in a rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common condition in veterinary medicine that is difficult to manage.Veterinary regenerative therapy based on adipose mesenchymal stem cells seem to be an effective strategy for the treatment of traumatic brain injury. In this study, we evaluated therapeutic efficacy of canine Adipose-derived mesenchymal stem cells (AD-MSCs)in a rat TBI model, in terms of improved nerve function and anti-neuroinflammation. RESULTS: Canine AD-MSCs promoted neural functional recovery, reduced neuronal apoptosis, and inhibited the activation of microglia and astrocytes in TBI rats. According to the results in vivo, we further investigated the regulatory mechanism of AD-MSCs on activated microglia by co-culture in vitro. Finally, we found that canine AD-MSCs promoted their polarization to the M2 phenotype, and inhibited their polarization to the M1 phenotype. What's more, AD-MSCs could reduce the migration, proliferation and Inflammatory cytokines of activated microglia, which is able to inhibit inflammation in the central system. CONCLUSIONS: Collectively, the present study demonstrates that transplantation of canine AD-MSCs can promote functional recovery in TBI rats via inhibition of neuronal apoptosis, glial cell activation and central system inflammation, thus providing a theoretical basis for canine AD-MSCs therapy for TBI in veterinary clinic.

Hemicellulose-based nanoaggregate-incorporated biocompatible hydrogels with enhanced mechanical properties and sustained controlled curcumin release behaviors.

Local drug delivery has generated considerable interest due to its controlled and sustained drug release at the target site on demand. Nanoaggregate-incorporated composite hydrogels are desirable as local drug delivery systems; however, it is difficult to achieve sustained and controlled hydrophobic drug release and superior mechanical properties in one system. Herein, a "smart" composite hydrogel was synthesized by incorporating hemicellulose-based nanoaggregates into a double network consisting of alginate/Ca2+ and polyacrylic acid-co-dimethylaminoethyl methacrylate [P(AA-co-DMAEMA)]. Hemicellulose-based nanoaggregates were assembled from xylan-rich hemicellulose laurate methacrylate (XH-LA-MA) polymers and entrapped into the hydrogel framework via chemical fixation. Another composite hydrogel with physically embedded hemicellulose laurate (XH-LA) nanoaggregates was prepared as a comparison. Accordingly, covalently cross-linked XH-LA-MA nanoaggregates in hydrogels resulted in a denser pore structure and reinforced mechanical properties. Nanoaggregate diffusion analysis revealed that covalent bonding between the nanoaggregates and the hydrogel framework contributed to prolonged diffusion behavior. Curcumin (Cur)-loaded XH-LA-MA composite hydrogels enabled sustained Cur release in simulated body fluid and showed stimulus responsiveness toward ethylenediaminetetraacetic acid (EDTA) and/or glutathione (GSH). All the composite hydrogels were biocompatible, as verified by Cell Counting Kit-8 (CCK-8) assay against NIH/3T3 cells. These composite hydrogels hold great potential as a promising dosage form for biomedical applications.

Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells.

Introduction: Glucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms. Methods: The method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle. Results: Our study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication. Discussion: These findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.

Molecular Signatures in Swine Innate and Adaptive Immune Responses to African Swine Fever Virus Antigens p30/p54/CD2v Expressed Using a Highly Efficient Semliki Forest Virus Replicon System.

African swine fever virus (ASFV) causes a devastating viral hemorrhagic disease in domestic pigs and Eurasian wild boars, posing a foremost threat to the swine industry and pig farming. The development of an effective vaccine is urgently needed, but has been hampered by the lack of an in-depth, mechanistic understanding of the host immune response to ASFV infection and the induction of protective immunity. In this study, we report that immunization of pigs with Semliki Forest Virus (SFV) replicon-based vaccine candidates expressing ASFV p30, p54, and CD2v, as well as their ubiquitin-fused derivatives, elicits T cell differentiation and expansion, promoting specific T cell and humoral immunity. Due to significant variations in the individual non-inbred pigs in response to the vaccination, a personalized analysis was conducted. Using integrated analysis of differentially expressed genes (DEGs), Venn, KEGG and WGCNA, Toll-like receptor, C-type lectin receptor, IL17 receptor, NOD-like receptor and nucleic acid sensor-mediated signaling pathways were demonstrated to be positively correlated to the antigen-stimulated antibody production and inversely correlated to the IFN-γ secreting cell counts in peripheral blood mononuclear cells (PBMCs). An up-regulation of CIQA, CIQB, CIQC, C4BPA, SOSC3, S100A8 and S100A9, and down-regulation of CTLA4, CXCL2, CXCL8, FOS, RGS1, EGR1 and SNAI1 are general in the innate immune response post-the second boost. This study reveals that pattern recognition receptors TLR4, DHX58/DDX58 and ZBP1, and chemokines CXCL2, CXCL8 and CXCL10 may play important roles in regulating this vaccination-stimulated adaptive immune response.

F18:A-:B1 Plasmids Carrying blaCTX-M-55 Are Prevalent among Escherichia coli Isolated from Duck-Fish Polyculture Farms.

We determined the prevalence and molecular characteristics of blaCTX-M-55-positive Escherichia coli (E. coli) isolated from duck-fish polyculture farms in Guangzhou, China. A total of 914 E. coli strains were isolated from 2008 duck and environmental samples (water, soil and plants) collected from four duck fish polyculture farms between 2017 and 2019. Among them, 196 strains were CTX-M-1G-positive strains by PCR, and 177 (90%) blaCTX-M-1G-producing strains were blaCTX-M-55-positive. MIC results showed that the 177 blaCTX-M-55-positive strains were highly resistant to ciprofloxacin, ceftiofur and florfenicol, with antibiotic resistance rates above 95%. Among the 177 strains, 37 strains carrying the F18:A-:B1 plasmid and 10 strains carrying the F33:A-:B- plasmid were selected for further study. Pulse field gel electrophoresis (PFGE) combined with S1-PFGE, Southern hybridization and whole-genome sequencing (WGS) analysis showed that both horizontal transfer and clonal spread contributed to dissemination of the blaCTX-M-55 gene among the E. coli. blaCTX-M-55 was located on different F18:A-:B1 plasmids with sizes between ~76 and ~173 kb. In addition, the presence of blaCTX-M-55 with other resistance genes (e.g., tetA, floR, fosA3, blaTEM, aadA5 CmlA and InuF) on the same F18:A-:B1 plasmid may result in co-selection of resistance determinants and accelerate the dissemination of blaCTX-M-55 in E. coli. In summary, the F18:A-:B1 plasmid may play an important role in the transmission of blaCTX-M-55 in E. coli, and the continuous monitoring of the prevalence and transmission mechanism of blaCTX-M-55 in duck-fish polyculture farms remains important.

Construction and Evaluation of the Immunogenicity and Protective Efficacy of Recombinant Replication-Deficient Human Adenovirus-5 Expressing Genotype VII Newcastle Disease Virus F Protein and Infectious Bursal Disease Virus VP2 Protein.

Newcastle disease (ND) and infectious bursal disease (IBD) are two key infectious diseases that significantly threaten the health of the poultry industry. Although existing vaccinations can effectively prevent and treat these two diseases through multiple immunizations, frequent immunization stresses significantly impact chicken growth. In this study, three recombinant adenoviruses, rAd5-F expressing the NDV (genotype VII) F protein, rAd5-VP2 expressing the IBDV VP2 protein, and rAd5-VP2-F2A-F co-expressing F and VP2 proteins, were constructed using the AdEasy system. The F and VP2 genes of the recombinant adenoviruses could be transcribed and expressed normally in HEK293A cells as verified by RT-PCR and Western blot. The three recombinant viruses were shown to have similar growth kinetics as rAd5-EGFP. Compared with the PBS and rAd5-EGFP groups, SPF chickens immunized with recombinant adenoviruses produced higher antibody levels, more significant lymphocyte proliferation, and significantly higher CD4+/CD3+ and CD8+/CD3+ cells in peripheral blood. The survival rate of SPF chickens immunized with rAd5-F and rAd5-VP2-F2A-F after the challenge with DHN3 was 100%, and 86% of SPF chickens showed no viral shedding at 7 dpc. The survival rate of SPF chickens immunized with rAd5-VP2 and rAd5-VP2-F2A-F after the challenge with BC6/85 was 86%. rAd5-VP2 and rAd5-VP2-F2A-F significantly inhibited bursal atrophy and pathological changes compared to the rAd5-EGFP and PBS groups. This study provides evidence that these recombinant adenoviruses have the potential to be developed into safe and effective vaccine candidates for the prevention and control of ND and IBD.

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.

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.

Direct Interaction of Coronavirus Nonstructural Protein 3 with Melanoma Differentiation-Associated Gene 5 Modulates Type I Interferon Response during Coronavirus Infection.

Coronavirus nonstructural protein 3 (nsp3) is a multi-functional protein, playing a critical role in viral replication and in regulating host antiviral innate immunity. In this study, we demonstrate that nsp3 from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and avian coronavirus infectious bronchitis virus (IBV) directly interacts with melanoma differentiation-associated gene 5 (MDA5), rendering an inhibitory effect on the MDA5-mediated type I interferon (IFN) response. By the co-expression of MDA5 with wild-type and truncated nsp3 constructs, at least three interacting regions mapped to the papain-like protease (PLpro) domain and two other domains located at the N- and C-terminal regions were identified in SARS-CoV-2 nsp3. Furthermore, by introducing point mutations to the catalytic triad, the deubiquitylation activity of the PLpro domain from both SARS-CoV-2 and IBV nsp3 was shown to be responsible for the suppression of the MDA5-mediated type I IFN response. It was also demonstrated that both MDA5 and nsp3 were able to interact with ubiquitin and ubiquitinated proteins, contributing to the interaction between the two proteins. This study confirms the antagonistic role of nsp3 in the MDA5-mediated type I IFN signaling, highlighting the complex interaction between a multi-functional viral protein and the innate immune response.

Construction and Transcriptomic Study of Chicken IFNAR1-Knockout Cell Line Reveals the Essential Roles of Cell Growth- and Apoptosis-Related Pathways in Duck Tembusu Virus Infection.

For industrial vaccine production, overwhelming the existing antiviral innate immune response dominated by type I interferons (IFN-I) in cells would be a key factor improving the effectiveness and production cost of vaccines. In this study, we report the construction of an IFN-I receptor 1 (IFNAR1)-knockout DF-1 cell line (KO-IFNAR1), which supports much more efficient replication of the duck Tembusu virus (DTMUV), Newcastle disease virus (NDV) and gammacoronavirus infectious bronchitis virus (IBV). Transcriptomic analysis of DTMUV-infected KO-IFNAR1 cells demonstrated that DTMUV mainly activated genes and signaling pathways related to cell growth and apoptosis. Among them, JUN, MYC and NFKBIA were significantly up-regulated. Furthermore, knockdown of zinc-fingered helicase 2 (HELZ2) and interferon-α-inducible protein 6 (IFI6), the two genes up-regulated in both wild type and KO-IFNAR1 cells, significantly increased the replication of DTMUV RNA. This study paves the way for further studying the mechanism underlying the DTMUV-mediated IFN-I-independent regulation of virus replication, and meanwhile provides a potential cell resource for efficient production of cell-based avian virus vaccines.

Establishment and Cross-Protection Efficacy of a Recombinant Avian Gammacoronavirus Infectious Bronchitis Virus Harboring a Chimeric S1 Subunit.

Infectious bronchitis virus (IBV) is a gammacoronavirus that causes a highly contagious disease in chickens and seriously endangers the poultry industry. A diversity of serotypes and genotypes of IBV have been identified worldwide, and the currently available vaccines do not cross-protect. In the present study, an efficient reverse genetics technology based on Beaudette-p65 has been used to construct a recombinant IBV, rIBV-Beaudette-KC(S1), by replacing the nucleotides 21,704-22,411 with the corresponding sequence from an isolate of QX-like genotype KC strain. Continuous passage of this recombinant virus in chicken embryos resulted in the accumulation of two point mutations (G21556C and C22077T) in the S1 region. Further studies showed that the T248S (G21556C) substitution may be essential for the adaptation of the recombinant virus to cell culture. Immunization of chicks with the recombinant IBV elicited strong antibody responses and showed high cross-protection against challenges with virulent M41 and a QX-like genotype IBV. This study reveals the potential of developing rIBV-Beau-KC(S1) as a cell-based vaccine with a broad protective immunity against two different genotypes of IBV.

Evaluation of different combination of pam2CSK4, poly (I:C) and imiquimod enhance immune responses to H9N2 avian influenza antigen in dendritic cells and duck.

Current commercial H9 avian influenza viruses (AIVs) vaccines cannot provide satisfactory antibody titers and protective immunity against AIVs in duck. Toll like receptors (TLR) ligand as AIVs adjuvants can activate dendritic cells to improve immune responses in multiple animals, while the studies were absent in duck. Therefore, we investigated TLR ligands pam2CSK4, poly (I:C) and/or imiquimod enhance immune responses to inactivated H9N2 avian influenza antigen (H9N2 IAIV) in peripheral blood monocyte-derived dendritic cells (MoDCs) and duck. In vitro, we observed that transcription factor NF-κB, Th1/Th2 type cytokines (IFN-γ, IL-2 and IL-6) and the ability of catching H9N2 IAIV antigen were significantly up-regulated when H9N2 IAIV along with TLR ligands (pam2CSK4, poly (I:C) and imiquimod, alone or combination) in duck MoDCs. Also, the best enhancement effects were showed in combination of pam2CSK4, poly (I:C) and imiquimod group, whereas IFN-α showed no significant enhancement in all experimental groups. In vivo, the results demonstrated that the percentages of CD4+/ CD8+ T lymphocytes, the levels of Th1/Th2 type cytokines and H9N2 HI titers were significant enhanced in combination of pam2CSK4, poly (I:C) and imiquimod group. However, pam2CSK4 alone or combining with imiquimod showed no enhancement or additive effects on Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-6) and HI titers in Muscovy duck, respectively. Taken together, our results concluded that not all TLR ligands showed enhancement of immune responses to H9N2 IAIV in duck. The combination of poly (I:C), imiquimod and pam2CSK4 that can be an effectively adjuvant candidate for H9N2 AIVs inactivated vaccine in duck, which provide novel insights in explore waterfowl vaccine.

Avian IRF1 and IRF7 Play Overlapping and Distinct Roles in Regulating IFN-Dependent and -Independent Antiviral Responses to Duck Tembusu Virus Infection.

Avian interferon regulatory factors 1 and 7 (IRF1 and IRF7) play important roles in the host's innate immunity against viral infection. Our previous study revealed that duck tembusu virus (DTMUV) infection of chicken fibroblasts (DF1) and duck embryo fibroblasts (DEFs) induced the expression of a variety of IFN-stimulated genes (ISGs), including VIPERIN, IFIT5, CMPK2, IRF1, and IRF7. IRF1 was further shown to play a significant role in regulating the up-expression of VIPERIN, IFIT5, and CMPK2 and inhibiting DTMUV replication. In this study, we confirm, through overexpression and knockout approaches, that both IRF1 and IRF7 inhibit DTMUV replication, mainly via regulation of type I IFN expression, as well as the induction of IRF1, VIPERIN, IFIT5, CMPK2, and MX1. In addition, IRF1 directly promoted the expression of VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling were undermined. We also found that non-structural protein 2B (NS2B) of DTMUV was able to inhibit the induction of IFN-ß mRNA triggered by Newcastle disease virus (NDV) infection or poly(I:C) treatment, revealing a strategy employed by DTMUV to evade host's immunosurveillance. This study demonstrates that avian IRF7 and IRF1 play distinct roles in the regulation of type I IFN response during DTMUV infection.

Expression and Immunogenicity of Recombinant African Swine Fever Virus Proteins Using the Semliki Forest Virus.

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family that damages the immune system of pigs, resulting in the death or slaughter of millions of animals worldwide. Recent modern techniques in ASFV vaccination have highlighted the potential of viral replicon particles (RPs), which can efficiently express foreign proteins and induce robust cellular and humoral immune responses compared with the existing vaccines. In this study, we established a Semliki Forest virus (SFV) vector by producing replication-defective viral particles. This vector was used to deliver RPs expressing ASFV antigens. SFV-RPs expressing ASFV p32 (SFV-p32) and p54 (SFV-p54) were tested in baby hamster kidney (BHK-21) cells. Proteins expression was evaluated via western blotting and indirect immunofluorescence, while immunogenicity was evaluated in BALB/c mice. The resulting RPs exhibited high levels of protein expression and elicited robust humoral and cellular immune responses.

Revisiting the Mongolian Gerbil Model for Hepatitis E Virus by Reverse Genetics.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. A convenient small mammalian model for basic research and antiviral testing is still greatly needed. Although a small rodent, the Mongolian gerbil, was reported to be susceptible to swine genotype-4 HEV infection, whether the previous results were reliable and consistent needs to be validated by using biologically pure HEV stocks or infectious RNA. In this study, we revisited this gerbil infection model for human HEV of genotype 1, 3, or 4 (G1, G3, or G4) by HEV reverse genetics. Gerbils inoculated intrahepatically with capped G3 HEV RNA transcripts or intraperitoneally with infectious G3 cloned HEV produced robust infection, as evidenced by presence of HEV in livers, spleens, and feces for up to 7 weeks post inoculation, seroconversion, and pathological liver lesions. Furthermore, the value of the gerbil model in antiviral testing and type I IFN in host defense was assessed. We demonstrated the effectiveness of peg-IFNα-2a and ribavirin in inhibiting HEV replication in gerbils. By treatment with two molecule inhibitors of TBK1, we also revealed a role of RIG-I like receptor-interferon regulatory factor 3 in host anti-HEV innate immune sensing in this in vivo model. Finally, susceptibility of G4 HEV was demonstrated in intrahepatically inoculated gerbils with infectious HEV RNA transcripts, whereas no evidence for G1 HEV susceptibility was found. The availability of the convenient gerbil model will greatly facilitate HEV-specific antiviral development and assess the mechanism of host immune response during HEV infection. IMPORTANCE HEV infects >20 million people annually, causing acute viral hepatitis as well as chronic hepatitis, neurological diseases, and pregnancy-associated high mortality, which require therapeutic intervention. The HEV antiviral research is largely limited by the lack of a convenient small animal model. Here we revisit the Mongolian gerbil model for three genotypes of human HEV by infectious HEV clones and recognized standards of experimental procedures. Fecal virus shedding, seroconversion, and pathological liver lesions could be detected in HEV-inoculated gerbils. We demonstrate the effectiveness and usefulness of this model in testing antiviral drugs, and in assessing the mechanism of host innate immune response upon HEV infection. This conventional rodent model will aid in future antiviral development and delineating mechanism of host immune response.

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.

A highly pathogenic Marek's disease virus isolate from chickens immunized with a bivalent vaccine in China.

Marek's disease virus (MDV) is an important oncogenic poultry pathogen that can generally be controlled by vaccination. However, MDV infections still occur occasionally on vaccinated farms, possibly due to genetic variation among MDV strains or management-related issues. In this study, a novel MDV strain, designated LZ1309, was isolated from a poultry flock that had been vaccinated with the HVT and CVI988 vaccine strains. Animal experiments showed that LZ1309 infection led to high morbidity (100%) and mortality (90%). Moreover, existing vaccines provided only partial protection against LZ1309, with protection rates of 68.4%, 85%, and 90% for HVT, CVI988, and HVT plus CVI988, respectively. This study demonstrates the presence of a more virulent strain of MDV in vaccinated chickens in China that poses a new potential threat to poultry farms. In future studies, the development of new treatment strategies should be of high priority.

A novel spike subunit 1-based enzyme-linked immunosorbent assay reveals widespread porcine torovirus infection in eastern China.

Toroviruses (ToVs), closely related but genetically distinct from coronaviruses, are known to infect horses, cows, pigs, goats and humans, mainly causing enteritic disorders. However, due to the lack of an adaptive culture system, porcine ToV (PToV) has received less attention. In this study, we developed a novel serological detection method based on the PToV envelope spike subunit 1 (S1) protein for the first time, and compared it to an existing indirect enzyme-linked immunosorbent assay (ELISA) based on the nucleocapsid protein. By using the S1-based ELISA, we carried out the first seroepidemiological survey of PToV in China, assaying both specific IgG and IgA responses in 1,037 serum samples collected from diarrheic pigs in eastern China. There was a relatively high incidence of seropositivity in pigs of different ages, especially one-week-old piglets and sows (78% and 43%), the former probably reflecting maternal antibodies. Furthermore, 3/20 (15%) of faecal samples collected from one PToV-seropositive swine herd in Zhejiang province tested positive by RT-PCR. The complete PToV genome was sequenced from one of these samples, and its phylogenetic relationship with other full-length PToV sequences available in GenBank was determined. Our data provide the first serological evidence for PToV infection in pigs from China, which will help elucidate the potential pathogenicity of PToV in pigs.