Advances in Veterinary Virology

The newly identified porcine Kobuvirus (PKV) has raised concerns owing to its association with diarrheal symptom in pigs worldwide. The process involving the emergence and global spread of PKV remains largely unknown. Here, the origin, genetic diversity, and geographic distribution of PKV were determined based on the available PKV sequence information. PKV might be derived from the rabbit Kobuvirus and sheep were an important intermediate host. The most recent ancestor of PKV could be traced back to 1975. Two major clades are identified, PKVa and PKVb, and recombination events increase PKV genetic diversity. Cross-species transmission of PKV might be linked to interspecies conserved amino acids at 13–17 and 25–40 residue motifs of Kobuvirus VP1 proteins. Phylogeographic analysis showed that Spain was the most likely location of PKV origin, which then spread to pig-rearing countries in Asia, Africa, and Europe. Within China, the Hubei province was identified as a primary hub of PKV, transmitting to the east, southwest, and northeast regions of the country. Taken together, our findings have important implications for understanding the evolutionary origin, genetic recombination, and geographic distribution of PKV thereby facilitating the design of preventive and containment measures to combat PKV infection. Full article

Bovine leukemia virus (BLV) infection causes endemic bovine leukemia and lymphoma, resulting in lower carcass weight and reduced milk production by the infected cattle, leading to economic losses. Without effective measures for treatment and prevention, high rates of BLV infection can cause problems worldwide. BLV research is limited by the lack of a model system to assay infection. To overcome this, we previously developed the luminescence syncytium induction assay (LuSIA), a highly sensitive and objectively quantifiable method for visualizing BLV infectivity. In this study, we applied LuSIA for the high-throughput screening of drugs that could inhibit BLV infection. We screened 625 compounds from a chemical library using LuSIA and identified two that markedly inhibited BLV replication. We then tested the chemical derivatives of those two compounds and identified BSI-625 and -679 as potent inhibitors of BLV replication with low cytotoxicity. Interestingly, BSI-625 and -679 appeared to inhibit different steps of the BLV lifecycle. Thus, LuSIA was applied to successfully identify inhibitors of BLV replication and may be useful for the development of anti-BLV drugs. Full article

Goose astrovirus (GAstV) is an important pathogen causing visceral gout and high mortality in goslings, which has broken out and spread across China. In 2021, a disease characterized by urate deposition on the visceral surface and 30% mortality occurred in commercial adult Landaise geese in Zhejiang Province, China. A systematic study identified an infecting astrovirus, designated ZJCX, that was efficiently isolated from a diseased goose with a chicken hepatocellular carcinoma cell line (LMH). In contrast to other GAstVs originating from goslings, ZJCX caused cytopathogenic effects in LMH cells, and the crystalline arrangement of viral particles was observed through transmission electron microscopy. Indeed, phylogenetic analysis and nucleotide homology comparison revealed that ZJCX isolate belongs to the genotype II cluster of GAstVs and displays 97.8–98.4% identity with other GAstV II strains. However, several specific mutations occurred in the polyprotein and capsid protein regions. Moreover, a pathogenicity assessment of ZJCX with a gosling model was conducted, and typical visceral gout was reproduced and led to 18% mortality. The viral loads of ZJCX in the blood, kidney, and liver were detected with specific primers after inoculation, which demonstrated that the kidney and liver presented viral loads peaking at seven days post-inoculation (dpi). Biochemical parameter examination showed that AST, ALT, γ-GT, UA, and BUN levels were significantly increased by GAstV, whereas body weight was reduced. Overall, this study indicated that the GAstV isolate could infect adult geese, and the results regarding the viral loads and biochemical parameters induced by ZJCX provide insight into GAstV pathogenicity. Full article

Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut’s immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4⁺ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4⁺ T cells among piglets’ peripheral blood mononuclear cells (PBMCs) and caused CD4⁺ T cells to undergo a transformation into Foxp3⁺ regulatory T cells (Treg cells) via activating CD4⁺ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-β induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-β, which enhanced the differentiation of CD4⁺ T cells into Treg cells through the activation of ERK in CD4⁺ T cells. This information sheds light on PCV2′s function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection. Full article

Throughout the last decade, H5N6 avian influenza viruses (AIVs) circulating in poultry and infecting humans have caused increasing global concerns that they might become a pandemic threat to global health. Since AIVs could occasionally cause asymptomatic infections in geese, virus monitoring in such a host should be critical to the control of cross-species infection. In addition, previous studies showed that clade 2.3.4.4h H5N6 AIVs could infect mammals without adaptation. However, the pathogenicity and transmissibility of goose-origin clade 2.3.4.4h H5N6 AIVs in mammals remain unknown. In this study, two H5N6 AIVs were isolated from a domestic chicken (A/chicken/Hebei CK05/2019 (H5N6)) and a goose (A/goose/Hebei/GD07/2019(H5N6)). This study is the first to evaluate the pathogenicity and transmissibility of goose-origin clade 2.3.4.4h H5N6 AIVs in mammals by comparison with chicken-origin 2.3.4.4h H5N6 AIVs. The CK05 virus had an affinity for α-2,3-receptors, while the GD07 virus had an affinity for both α-2,3-and α-2,6-receptors. The GD07 virus had a higher replication capacity in vitro and more severe pathogenicity in mice than the CK05 virus. The CK05 virus could not be transmitted effectively among guinea pigs, whereas the GD07 virus could be transmitted through direct contact among guinea pigs. The results of this study indicated the potential health threat of clade 2.3.4.4h H5N6 AIVs to mammals and emphasized the importance of continuous monitoring of H5N6 AIVs, especially in waterfowl. Full article

Swine influenza virus (SIV) circulates worldwide, posing substantial economic loss and disease burden to humans and animals. Vaccination remains the most effective way to prevent SIV infection and transmission. In this study, we evaluated the protective efficacy of a recombinant, baculovirus-insect cell system-expressed bivalent nanoparticle SIV vaccine in mice challenged with drifted swine influenza H1N1 and H3N2 viruses. After a prime-boost immunization, the bivalent nanoparticle vaccine (BNV) induced high levels of hemagglutination inhibition (HAI) antibodies, virus-neutralization (VN) antibodies, and antigen-specific IgG antibodies in mice, as well as more efficient cytokine levels. The MF59 and CPG1 adjuvant could significantly promote both humoral and cellular immunity of BNV. The MF59 adjuvant showed a balanced Th1/Th2 immune response, and the CPG1 adjuvant tended to show a Th1-favored response. The BALB/c challenge test showed that BNV could significantly reduce lung viral loads and feces viral shedding, and showed fewer lung pathological lesions than those in PBS and inactivated vaccine groups. These results suggest that this novel bivalent nanoparticle swine influenza vaccine can be used as an efficacious vaccine candidate to induce robust immunity and provide broad protection against drifted subtypes in mice. Immune efficacy in pigs needs to be further evaluated. Full article

This study characterized the susceptibility and dynamic of porcine deltacoronavirus infection in grower pigs under experimental conditions using a combination of syndromic and laboratory assessments. Seven-week-old conventional pigs (n = 24) were randomly distributed into PDCoV- (n = 12) and mock-inoculated (n = 12) groups. Serum was collected at −7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 days post-inoculation (DPI) to evaluate viremia (RT-qPCR) and antibody response (S1-based ELISA). Viral shedding and potential infectivity were determined using pen-based oral fluids and feces collected every other day between DPI 0 and 42. Pigs showed no clinical signs or viremia throughout the study. Active virus shedding was detected in feces (6-22 DPI) and oral fluids (2-30 DPI), peaking at DPI 10. IgG was first detected at DPI 10, being statistically significant after DPI 14 and increasing thereafter, coinciding with the progressive resolution of the infection. Likewise, a significant increase in proinflammatory IL-12 was detected between DPI 10 and 21 in PDCoV-inoculated pigs, which could enhance innate resistance to PDCoV infection. This study demonstrated that active surveillance based on systematic sampling and laboratory testing combining molecular and serological tools is critical for the accurate detection of subclinical circulation of PDCoV in pigs after weaning. Full article

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019–2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019–2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020–2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt. Full article

Porcine circovirus type 4 (PCV4) is a novel virus associated with porcine dermatitis and nephropathy syndrome (PDNS)-like signs identified firstly in China in 2019. However, the details of the molecular epidemiology of PCV4 are unclear at this time. A total of forty-two related sequences were selected from the GenBank database to explore the spread of PCV4 and its rule in genetic evolution. Of the selected strains, 41 were from south China in 2019 to 2021 and the other was a foreign representative strain. Phylogenetic tree construction, nucleotide and amino acid (aa) sequence alignment, gene recombination and antigen structure prediction were performed on the collected sequences using bioinformatics softwares. The 42 PCV4 strains were divided into two subgenotypes: PCV4a (35/42) and PCV4b (7/42), according to the constructed genetic evolution tree. PCV4a is the main epidemic strain, and it can be further divided into two different gene clusters: PCV4a-1 (22/35) and PCV4a-2 (13/35). The pairwise comparison analysis showed that the complete genome sequence similarity of the 42 PCV4 strains ranged between 97.9% and 100%, and the aa sequences of the Cap proteins of 42 PCV4 strains had three major heterogenic or hypervariable regions—27–28, 96 and 212—all located near the antigenic epitope of the Cap protein. The results of this study can provide some basis for further studying the spread and epidemic growth of PCV4, and the prevention and control of PCV4 infection in China. Full article

Avian influenza viruses of the H9 subtype cause significant losses to poultry production in endemic regions of Asia, Africa and the Middle East and pose a risk to human health. The availability of reliable and updated diagnostic tools for H9 surveillance is thus paramount to ensure the prompt identification of this subtype. The genetic variability of H9 represents a challenge for molecular-based diagnostic methods and was the cause for suboptimal detection and false negatives during routine diagnostic monitoring. Starting from a dataset of sequences related to viruses of different origins and clades (Y439, Y280, G1), a bioinformatics workflow was optimized to extract relevant sequence data preparatory for oligonucleotides design. Analytical and diagnostic performances were assessed according to the OIE standards. To facilitate assay deployment, amplification conditions were optimized with different nucleic extraction systems and amplification kits. Performance of the new real-time RT-PCR was also evaluated in comparison to existing H9-detection methods, highlighting a significant improvement of sensitivity and inclusivity, in particular for G1 viruses. Data obtained suggest that the new assay has the potential to be employed under different settings and geographic areas for a sensitive detection of H9 viruses. Full article

The highly virulent Newcastle disease virus (NDV) isolates typically result in severe systemic pathological changes and high mortality in Newcastle disease (ND) illness, whereas avirulent or low-virulence NDV strains can cause subclinical disease with no morbidity and even asymptomatic infections in birds. However, understanding the host’s innate immune responses to infection with either a highly virulent strain or an avirulent strain, and how this response may contribute to severe pathological damages and even mortality upon infection with the highly virulent strain, remain limited. Therefore, the differences in epigenetic and pathogenesis mechanisms between the highly virulent and avirulent strains were explored, by transcriptional profiling of chicken embryonic visceral tissues (CEVT), infected with either the highly virulent NA-1 strain or the avirulent vaccine LaSota strain using RNA-seq. In our current paper, severe systemic pathological changes and high mortality were only observed in chicken embryos infected with the highly virulent NA-1 strains, although the propagation of viruses exhibited no differences between NA-1 and LaSota. Furthermore, virulent NA-1 infection caused intense innate immune responses and severe metabolic disorders in chicken EVT at 36 h post-infection (hpi), instead of 24 hpi, based on the bioinformatics analysis results for the differentially expressed genes (DEGs) between NA-1 and LaSota groups. Notably, an acute hyperinflammatory response, characterized by upregulated inflammatory cytokines, an uncontrolled host immune defense with dysregulated innate immune response-related signaling pathways, as well as severe metabolic disorders with the reorganization of host–cell metabolism were involved in the host defense response to the CEVT infected with the highly virulent NA-1 strain compared to the avirulent vaccine LaSota strain. Taken together, these results indicate that not only the host’s uncontrolled immune response itself, but also the metabolic disorders with viruses hijacking host cell metabolism, may contribute to the pathogenesis of the highly virulent strain in ovo. Full article