Jingliang Su

Pigeonpox is a significant infectious disease caused by Pigeonpox virus (PPV), which severely impacts the pigeon industry. Current control methods primarily rely on heterologous vaccines, such as those derived from avian poxviruses, but their protection is limited, creating an urgent need for the development of a specific vaccine. In this study, 720 samples collected from several regions of China between 2022 and 2023 were tested for PPV, followed by virus isolation, identification, and genetic evolutionary analysis. Based on these findings, complete genome sequencing and attenuation of the representative BJ-02 isolate were conducted, and the potential of this strain as a candidate for an attenuated vaccine was preliminarily evaluated. The survey showed PCR positive rates of 9.05%, 16.11%, and 12.50% in samples from Beijing, Guangdong, and Hainan, respectively. Six viral strains were isolated, all of which produced typical lesions on chorioallantoic membranes (CAM) and chicken embryo fibroblasts (CEF). Phylogenetic analysis based on the P4b gene revealed that the six viruses clustered within the same evolutionary branch, closely related to PPV and penguinpox virus strains from South Africa, India, and Taiwan, China. Complete genome sequencing of the BJ-02 strain showed its genomic structure to be similar to that of other fowlpox viruses, with some differences. After serial passage in CAM, PEF and CEF, the BJ-02 SD55 high-passage strain adapted well to in vitro culture, exhibited significantly reduced pathogenicity in chicken embryos and pigeons, and showed no reversion to virulence after five consecutive back-passages. Animal immunization tests demonstrated that the BJ-02 SD55 suspected attenuated strain induced specific antibodies and provided 100% protection against challenge with the virulent strain. In conclusion, PPV is widely prevalent in China. The BJ-02 strain, successfully isolated and attenuated through serial passage, demonstrates excellent immunogenicity and high safety, making it a promising candidate for a specific pigeonpox vaccine. Additionally, the complete genome characterization of BJ-02 contributes to the avian poxvirus genome database and provides critical data to support research on viral pathogenesis and the development of viral vector vaccines for avian and potentially mammalian species.

Background: The flavivirus Duck Tembusu virus (DTMUV) exhibits high pathogenicity and transmissibility, posing a severe threat to the poultry industry in China and Southeast Asia. Although the molecular mechanisms of DTMUV pathogenesis remain unclear, its potential to disrupt intracellular calcium ion (Ca² ⁺ ) homeostasis, a known driver of viral replication, has not been investigated. Here, we investigated the role and underlying mechanism of Ca² ⁺ homeostasis in DTMUV infection. Methods: Fluo-4AM staining and flow cytometry of duck embryo fibroblasts (DEFs) were used to detect cytoplasmic Ca² ⁺ . To modulate Ca² ⁺ levels and AMP-activated protein kinase (AMPK) activity, we used voltage-gated calcium channel (VGCC) blockers (verapamil, diltiazem hydrochloride), a Ca² ⁺ chelator (BAPTA-AM), and an AMPK inhibitor (Compound C). Viral entry, genomic RNA replication (targeting the DTMUV NS5 gene), viral yield, and release were evaluated via qRT-PCR and plaque assays. AMPK activation was detected using western blotting with anti-phospho-AMPKα (Thr172) and anti-AMPKα antibodies. Statistical analyses were performed using Student’s t-test or two-way analysis of variance. Results: DTMUV infection significantly increased cytoplasmic Ca² ⁺ in DEFs at 6, 8, 10, and 12 hours post-infection. This elevation was suppressed by treatment with verapamil or diltiazem hydrochloride, indicating that DTMUV induces extracellular Ca² ⁺ influx via VGCCs. Functional assays showed that reducing cytoplasmic Ca² ⁺ via treatment with VGCC blockers or BAPTA-AM specifically inhibited DTMUV RNA replication, but not viral entry or release, decreasing progeny virus production. Further mechanistic analysis revealed that DTMUV infection activates the AMPK pathway in a Ca² ⁺ -dependent manner, with Compound C-mediated AMPK inhibition dose-dependently suppressing viral RNA replication and progeny yield. Conclusions: DTMUV disrupts host Ca² ⁺ homeostasis to activate AMPK, which promotes viral RNA replication. This study provides novel insights into DTMUV pathogenesis and identifies Ca² ⁺ –AMPK signaling as a potential anti-DTMUV target.

Canine distemper virus (CDV) is the etiological agent of canine distemper. The virus can infect canids irrespective of age, sex, or breed, leading to a highly contagious and lethal disease that seriously threatens the health of canids, fur animals, and wildlife. Although vaccination can currently prevent CDV infection, developing effective emergency treatment drugs remains crucial. Nanobodies derived from camelid or shark heavy chain-only antibodies can effectively inhibit viral infections, suggesting their potential as therapeutic agents for treating CDV infection. In this study, we utilized a phage display nanobody library constructed from immunized alpacas and isolated a nanobody (Nb-6C6) that specifically binds to the CDV hemagglutinin (H) protein. Nb-6C6 was successfully expressed in mammalian cells and exhibited high binding affinity to CDV H (EC₅₀ = 0.174 µg/mL). Neutralization assays further revealed that Nb-6C6 could effectively neutralize CDV (IC₅₀ = 1.773 µg/mL). Fusion of Nb-6C6 with canine IgG Fc resulted in homodimers, significantly increasing its neutralizing activity by up to 4.6-fold. AlphaFold3 analysis indicated that the neutralizing capacity of Nb-6C6 against CDV is attributed to an interaction between residue D106 in the CDR3 region and the conserved residue R408 of the H protein. These findings suggest that the nanobody Nb-6C6 and its bivalent form exhibit high-affinity binding and potent neutralizing activity against CDV, highlighting their potential as promising therapeutic candidates for the treatment of CDV infection.

Introduction: Duck circovirus (DuCV) infection is currently recognized as an important immunosuppressive disease in commercial duck flocks in China. Specific antibodies against DuCV viral proteins are required to improve diagnostic assays and understand the pathogenesis of DuCV infection.Methods and results: To generate DuCV-specific monoclonal antibodies (mAbs), a recombinant DuCV capsid protein without the first 36 N-terminal amino acids was produced in Escherichia coli. Using the recombinant protein as an immunogen, a mAb was developed that reacted specifically with the DuCV capsid protein, expressed in E. coli and baculovirus systems. Using homology modeling and recombinant truncated capsid proteins, the antibody-binding epitope was mapped within the region of ¹⁴⁴IDKDGQIV¹⁵¹, which is exposed to solvent in the virion capsid model structure. To assess the applicability of the mAb to probe the native virus antigen, the murine macrophage cell line RAW267.4 was tested for DuCV replicative permissiveness. Immunofluorescence and Western blot analysis revealed that the mAb recognized the virus in infected cells and the viral antigen in tissue samples collected from clinically infected ducks.Discussion: This mAb, combined with the in vitro culturing method, would have widespread applications in diagnosing and investigating DuCV pathogenesis.

Goose astrovirus (GoAstV) has frequently been isolated in China since it was first identified as the etiological agent of visceral gout in goslings in 2017. However, the actual prevalence of GoAstV infection and its economic impact on commercial goose production remain poorly characterized. Here, virus detection and serological testing were conducted to determine the extent of GoAstV infection in commercial goose flocks. We detected GoAstV RNA in 2 (6/300) of dead-in-shell embryos and day-old hatched goslings by RT-PCR, indicating vertical transmission under natural conditions. Using a virus neutralization test, GoAstV antibodies were detected in 41.761.1 of serum samples from four commercial goose flocks, indicating that infections were common. To determine the virus types circulating in the commercial flocks, we isolated 15 GoAstVs from goose tissue samples from farms located in five provinces during 20182022. Genomic sequence analysis showed that all sequences were corresponded to GoAstV group 2 (GoAstV-2) but were assigned into three capsid subgroups based on sequence variations in the capsid protein. Representative isolates of capsid subgroups were also antigenically evaluated using cross-neutralization tests in LMH cell cultures. The antigenic relatedness values (R) calculated using the Horsfall formula were between 62 and 86, indicating that no significant antigenic differences exist between the isolates. Our findings indicate that GoAstV-2 viruses are an important cause of fatal gout in goose flocks, as well as hatchery contamination in China.

Tembusu virus (TMUV) associated disease is a growing cause of egg production decrease and encephalitis in domestic waterfowl, with expanding distribution. In previous studies, TMUV isolates were phylogenetically classified into two genetic lineages and different clusters with varied pathogenicity. However, little is known about the phenotypic and virulence characteristics of cluster 3 isolates within the duck TMUV lineage. In this study, the etiological agent causing egg drop in a laying chicken farm in southern China was investigated and a TMUV was isolated from pooled tissue samples. Genome sequencing and phylogenetic analysis grouped the isolate into TMUV cluster 3 with closest relation to the mosquito‐origin TMUV YN12193. Cross‐neutralization testing using convalescent sera revealed significant antigenic variation between the isolate and a representative strain of cluster 2.2. The experimental infection of SPF hens confirmed the ability of the isolate to replicate in multiple tissues and led to ovary damage. Additionally, high seroconversion rates (95.83%–100%) were detected in the three flocks following retrospective investigation. Our study demonstrates the occurrence of cluster 3 TMUV infection in laying chickens and that the virus exhibits significant antigenic variation compared with cluster 2 TMUV.

Tembusu virus (TMUV), a highly infectious pathogenic flavivirus, causes severe egg-drop and encephalitis in domestic waterfowl, while the determinants responsible for viral pathogenicity are largely unknown. In our previous studies, virulent strain JXSP_2-4 had been completely attenuated by successive passages in BHK-21 cells and the avirulent strain was designated as JXSP-310. Based on the backbone of JXSP_2-4, a series of chimeric viruses were generated according to the amino acid substitutions in NS5 and their infectivities were also analyzed in cell cultures and ducklings. The results showed that the viral titers of RNA-dependent RNA polymerase (RdRp) domain-swapped cheimeric mutant (JXSP-310^RdRp) in cells and ducklings were both markedly decreased compared with JXSP_2-4, indicating that mutations in the RdRp domain affected viral replication. There are R543K and V711A two amino acid substitutions in the RdRp domain. Further site-directed mutagenesis showed that single-point R543K mutant (JXSP-R543K) exhibited similar replication efficacy compared with JXSP_2-4 in cells, but the viral loads in JXSP-R543K-infected ducklings were significantly lower than that of JXSP_2-4 and higher than JXSP-310^RdRp. Surprisingly, the single-point V711A mutation we introduced rapidly reverted. In addition, qRT-PCR and Western blot confirmed that the mutations in the RdRp domain significantly affected the replication of the virus. Taken together, these results show that R543K substitution in the RdRp domain impairs the in vivo growth of TMUV, but sustaining its attenuated infectivity requires the concurrent presence of the V711A mutation.

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 10³ TCID₅₀/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

Murine Norovirus (MNV) is one of the most known viruses among viruses in mice. Because of the high prevalence of MNV in frequently used laboratory animals in biomedical researches, there is a significant impact of MNV. There may be different prevalence degrees and molecular characteristics of MNV in different regions around the world. Here, we reported an MNV strain “designated HBTS-1806” isolation from commercial mice's feces that caused a detectable cytopathic effect (CPE) in RAW264.7 cells. According to electron microscopy, the virus was 50–70 nm in diameter. The complete genome of HBTS-1806 is 7383 nucleotides with a structure similar to that of MNV reference strains. According to phylogenetic analysis on the basis of the whole genome, HBTS-1806 shared nucleotide sequence identities of 90.2–95.4% with other Chinese isolates reported. Analysis of amino acid sequence on the basis of ORF1 and ORF2 suggested that the isolated strain may be derived from recombination. Although no gross lesions or histopathological changes were found from mice infected with 5 × 10⁵ TCLD₅₀ of MNV by oral gavage inoculation, the intestinal virus loads lasted 12 weeks, suggesting a persistent infection strain of MNV isolate in China.