Feline calicivirus (FCV; Caliciviridae) is a highly contagious RNA virus that causes upper respiratory tract infections and intestinal symptoms in cats. In 2023 and 2024, in Shanghai (SH), China, we collected oral swab samples from 189 domestic cats exhibiting symptoms of upper respiratory tract disease (URTD), to test for five designated respiratory pathogens. Among the 111 cats testing positive for these pathogens, the FCV-positivity rate was 52% (58/111). Six FCV strains (three from SH and three from Guangdong [GD]) were successfully isolated from respiratory specimens from domestic cats. Whole genome sequencing and phylogenetic analyses revealed that these strains exhibited the GII FCV genotype, suggesting that this is the dominant genotype in Asia. Within the first 36 h postinfection, the GD strains exhibited faster growth and higher replication titers than the SH strains. The GD23-02 genotype (GD) and SH23-13 genotype (SH) exhibited intriguing amino acid variation in the VP1 E-region. We therefore selected these strains for challenge experiments. Although both strains caused oral-ulcer symptoms, they presented distinct disease progression and clinical manifestations; SH23-13 exhibited traits typical of virulent systemic disease (VSD), whereas GD23-02 predominantly exhibited symptoms of oral respiratory disease (ORD). Notably, both strains consistently induced severe diarrhea and intestinal damage, demonstrating the harmful effects of FCV on intestinal health. GD23-02 exhibited broad-spectrum neutralization capabilities, with antibody titers of 1:168 against the F9 vaccine GI strain and 1:2521 against the SH23-13 GII strain, making this a promising candidate for future vaccine formulation. These findings reveal the genetic diversity and complex pathogenicity of FCV isolates and elucidate the associations between FCV strains and intestinal disease. We recommend incorporating FCV testing into future diagnostic evaluation of feline diarrhea. This testing will be essential in elucidating the tissue specificity of FCV and potential escalation in its virulence.

Avian leukosis (AL) is a common neoplastic disease in poultry caused by the Avian leukosis virus (ALV). As there are no specific drugs or vaccines for preventing and treating this disease, population purification is currently primarily achieved by culling affected individuals and selecting and breeding leukemia-free chicken flocks. To address the lack of a rapid and accurate ALV detection method, this study developed a lateral flow strip detection system based on Fe-Co nanozyme-antibody probes. Owing to the efficient signal amplification property of nanozymes, the system significantly improved detection sensitivity, with a minimum detection limit of 12.5 TCID50/mL for ALV and 0.20 ng/mL for its related proteins. Furthermore, this test strip was eight times more sensitive than the commercial enzyme-linked immunosorbent assay (ELISA) group-specific antigen detection kit. Specificity verification results showed that the detection test strip only produced a positive reaction to ALV and no cross-reactions with six common avian viruses, namely Newcastle disease virus (NDV), Marek's disease virus (MDV), infectious bursal disease virus (IBDV), fowlpox virus (FPV), infectious laryngotracheitis virus (ILTV), and avian influenza virus (AIV). Additionally, the strip exhibited excellent intra- and inter-batch repeatability, with the corresponding coefficient of variation (CV) being less than 10 %. Clinical sample detection data indicated that the results of the nanozyme-based test strip were consistent with those of the commercial double-antibody sandwich ELISA detection kit, achieving a coincidence rate of up to 100 %. Collectively, the results of this study provide a novel technical method characterized by high sensitivity, high specificity, and simple operation for the on-site rapid screening of ALV.

Marek's disease virus (MDV), an alphaherpesvirus, causes severe immunosuppression and T cell lymphomas in chickens, known as Marek's disease (MD), an economically important poultry disease primarily controlled by vaccination. Importantly, it also serves as a comparative model for studying herpesvirus-induced tumor formation in humans. MDV encodes more than 100 genes, most of which have unknown functions. MDV LORF1 is unique to serotype I MDV (MDV-1), lacking homologs in other herpesviruses, and has not been explored yet. To this end, an infectious bacterial artificial chromosome (BAC) harboring the complete genome of the MDV-1 very virulent strain Md5 was generated, and the rescued rMd5 maintained biological properties similar to the parental virus both in vitro and in vivo. Subsequently, rMd5ΔLORF1, a recombinant Md5 virus deficient in pLORF1 expression, was generated by a frameshift mutation in the LORF1 gene. Chickens infected with rMd5ΔLORF1 exhibited a lower mortality rate and delayed bursal atrophy than those infected with the parental rMd5 and the revertant virus (rMd5-reLORF1). Consistently, viral loads of rMd5ΔLORF1 were obviously lower than those of rMd5 or rMd5-reLORF1 in the bursa, but not in the spleen. Importantly, we found that pLORF1 deficiency impairs viral replication in bursal B cells. Furthermore, we showed that pLORF1 associated with the cellular membrane, interacted with MDV structural proteins, and exhibited punctate colocalization with tegument or capsid proteins in the cytoplasm. Taken together, this study demonstrates for the first time that the MDV-1 unique gene LORF1 is involved in MDV-induced bursal atrophy but not in tumor formation.

Bovine respiratory disease syndrome, primarily caused by bovine parainfluenza virus type-3 (BPIV-3), is characterized by a high incidence in calves. However, despite its significant clinical importance, the codon usage patterns and evolutionary dynamics of BPIV-3 hosts remain elusive. Hence, this study aimed to systematically analyze the codon usage bias of the BPIV-3 structural protein gene HN, and the roles of mutational pressure and natural selection in its evolution were evaluated. Herein, analysis of indicators such as effective codon number (ENC), relative synonymous codon usage, and codon adaptation index revealed a low codon bias for BPIV-3, with codon preferences showing significant differences from the host. Notably, ENC-GC content at the third codon spot analysis indicated that natural selection dominated codon usage. Additionally, phylogenetic analysis divided BPIV-3 into three main genotypes (namely a, b, and c), with genotype C exhibiting a higher codon adaptability to the host. Altogether, these findings reveal the host-BPIV-3 evolutionary interaction mechanisms, providing a theoretical basis for vaccine design and epidemiological surveillance.

Cyclic peptides exhibit advantages in binding protein targets with high affinity and competency in inhibiting protein-protein interactions (PPIs). Cyclic peptide phage display with over a billion variants is an invaluable tool in drug discovery. However, achieving efficient peptide cyclization on phages remains a challenge due to the limited availability of reaction sites, which also restricts scaffold diversity. Here, we report an isothiocyanate-derived crosslinker featuring dual reactive groups: a bro-mide that covalently attaches to cysteine thiols, and a thiocyanogen that selectively forms a thiourea bridge with either the N-terminal amino group or ε-amines of lysine, depending on pH. This strategy enables pH-modulated cyclization, with head-to-side chain cyclization at pH 6.5 and side chain-to-side chain ligation at pH 9.5, simultaneously generating thiourea scaffolds. To demonstrate the versatility and biocompatibility of this approach, we constructed cyclic peptide libraries using both cy-clization methods and successfully selected binders for several targets, including Cyp D, TNF, MDM2, and Keap1, with disso-ciation constants (KD) ranging from micromolar to nanomolar. Given the broad pharmacological potential of the thiourea moiety, this phage display library opens new chemical space with high scaffold diversity and the integration of a proven pharmacophore for the development of cyclic peptide therapeutics.

Duck enteritis virus (DEV) and duck hepatitis A virus (DHAV) are prevalent duck pathogens, causing significant economic losses in the duck industry annually. Using a fosmid-based rescue system, we generated two DEV recombinants, rDEV-UL26/27-P13C and rDEV-US7/8-P13C, in which the P1 and 3C genes from DHAV type 3 (DHAV-3) were inserted into the DEV genome between genes UL26 and UL27 or genes US7 and US8. We inserted a self-cleaving 2A-element between P1 and 3C, allowing the production of both proteins from a single open reading frame. P1 and 3C were simultaneously expressed in infected chicken embryo fibroblasts, with no difference in growth kinetics between cells infected with the recombinant viruses and those infected with the parent DEV. Both recombinant viruses induced neutralizing antibodies against DHAV-3 and DEV in ducks. A single dose of the recombinant viruses induced solid protection against lethal DEV challenge and completely prevented DHAV-3 infection as early as 7 days post-vaccination. These recombinant P1- and 3C-expressing DEVs provide potential bivalent vaccines against DEV and DHAV-3 infection in ducks.

Inclusion body hepatitis (IBH), hydropericardium syndrome, and gizzard erosion associated with fowl adenovirus (FAdV) infections are reported globally and resulted in significant poultry industry economic losses. In 2018, severe IBH appeared in Pakistan in a 17-week-old layer flock. Subsequently, a FAdV-11 strain (designated as PKFAd18) was isolated from liver samples and identified based on phylogenetic analyses of the serotype-specific L1 region of the capsid hexon gene. There is no complete genome sequence of the Pakistani FAdV-11. This study successfully sequenced the complete genome of PKFAd18. The full genome of PKFAd18 contains 43 840 base pairs (bp) with a G + C content of 53.9 %, which is comparable to other FAdV serotypes. Similar to other FAdV-11 strains, PKFAd18 has only one fiber, while FAdV-1 and FAdV-4 have two fibers. Notably, PKFAd18 showed unique characteristics compared to other FAdV-11 strains. A natural large genomic deletion (1215 bp) appeared in tandem repeat region two, relative to the ON-NP2 strain. Phylogenetic analyses of the PKFAd18 penton gene showed higher homology with FAdV-9, highlighting potential natural recombination between FAdV-11 and FAdV-9. Moreover, the pathogenicity of PKFAd18 studied in specific-pathogen-free chickens showed that PKFAd18 is capable of inducing severe IBH and could be responsible for IBH in Pakistan. Thus, the first complete genome of FAdV-11 in Pakistan was sequenced in this study, which enriches the diversity of knowledge about FAdV-11 and is useful for developing diagnostics and vaccines for IBH induced by FAdV-11 in Pakistan.

The prevalence of Marek's disease (MD) caused by Gallid herpesvirus-2 (GaHV-2) has been increasing in chickens in China despite universal vaccination. Among the possible reasons for this trend, of Reticuloendotheliosis virus (REV) contamination in vaccines could lead to co-infection and reduce the vaccine efficacy. Here, we report the epidemiological findings of our continuous surveillance of MD, and an examination of the effects of REV and/or Gal-N-2 co-infection. A total of 1230 samples were collected between 2011 and 2015 from 305 flocks covering many of the chicken-raising regions of China. Among these, 606 samples were determined to be GaHV-2-positive, 13.0% of which were found to be co-infected with REV from 18.8% of the flocks. One GaHV-2 strain (HS/ 1412), a REV strain (HS/1412R), and a GaHV-2 and REV-co-infected strain (HS/1412 GR) were isolated from different chickens of a GaHV-2 and REV co-infected flock. Pathogenicity tests showed that HS/1412 and HS/ 1412 GR caused disease in all chickens and that HS/1412R induced morbidity in 84.6% of the infected chickens. HS/1412 GR induced 100% mortality and 76.9% tumor formation, which were significantly higher frequencies than those observed with strain HS/1412 (38.5% and 15.4%, respectively) and HS/1412R (0% and 0%). These results indicate that co-infection with GaHV-2 and REV might explain the persistent, sporadic outbreaks of neoplastic disease in some commercial flocks, resulting in a significant economic burden to the poultry industry of China.

Attenuated strains of Marek's disease virus serotype 1 (MDV1), and the closely related herpesvirus of turkeys, are among the most potent vectors for development of recombinant vaccines for poultry. To investigate the effects of MDV1 strain characteristics on the protective efficacy of the recombinant vaccines, we developed two recombinant MDV1 vaccines for expressing the VP2 gene of infectious bursal disease virus (IBDV) based on two different MDV1 strains, the attenuated strain 814 and the Meq gene-deleted recombinant MDV1 strain rLMS△Meq, as the viral vectors. The r814-VP2 virus based on the 814 strain exhibited higher replication efficiency in cell culture while lower viral titers in chickens, compared to rLMS△Meq-VP2 derived from the rLMS△Meq strain. Further studies indicated that r814-VP2 produced higher levels of VP2 protein in cells and elicited stronger immune responses against IBDV in chickens than rLMS△Meq-VP2. After IBDV challenge, rLMS△Meq-VP2 provided 50% protection against mortality, and the birds that survived developed bursal atrophy and gross lesions. In contrast, r814-VP2 conferred complete protection not only against development of clinical signs and mortality, but also against the formation of bursal lesions. The results indicate that different MDV1 vector influences the protective efficacy of recombinant MDV1 vaccines. The r814-VP2 has the potential to serve as a bivalent vaccine against two important lethal pathogens of chickens.

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

A TaqMan real-time PCR assay for rapid determination of the foreign gene copy number in genome of recombinant P.pastoris strains was developed using gp90 gene of reticuloendotheliosis virus as target gene and Glyceraldehydes-3-phosphate dehydrogenase(GAP) gene of P.pastoris as reference gene.The double standard curves of reference gene and gp90 gene were mapped and the genomic DNA of P.pastoris transformants containing gp90 gene was analyzed by real-time PCR,then the gp90/reference ratio in the samples was calibrated as the gp90 gene copy numbers integrated into the genome of P.pastoris.Using this method,one copy of the foreign gene integrated into the yeast genome can be detected sensitively and had good reproducibility.Sixty recombinant P.pastoris transformants were detected by this real-time PCR method.It was found that 38 recombinant strains contained one copy of gp90 gene and 22 contained multiple copies.This study suggests that the real-time quantitative PCR is a good tool for detecting of foreign gene copy number in P.pastoris transformants and it will be useful for screening of P.pastoris transformants with high level productivity.

The recombinant clone of vvIBDV(very virulent Infectious bursal disease virus) HLJ-0504 lacking of VP5 gene was constructed by silencing the start codon(ATG-ATC) with site-directed mutagenesis.The full length cDNA of segment A was flanked by hammerhead ribozyme and hepatitis delta virus ribozyme,which was introduced into a eukaryotic expression vector PCAGGs under the strong chicken β actin promoter.The recombinant plasmid,designated as pCAGGHLJ0504A△VP5HRT,was co-transfected into DF-1 cells with pCAGGHLJ0504BHRT.The recombinant virus rHLJ0504△VP5 was successfully rescued,which was verified by RT-PCR,indirect immnuofluorescence assay and electron microscope.The rescued virus could be a very helpful platform for further construction of candidate vaccine strain.

To study the molecular basis of cell tropism of very virulent infectious bursal disease virus(vvIBDV),a infectious clone based on the backbone of vvIBDV HLJ-0504 were constructed by overlap extension PCR(SOE PCR) and site-direct mutation.Recombinant viruse was rescued by reverse-genetics approach using the T7 RNA polymerase II transcription system.Cell tropism and the replication kinetics of the rescued IBDV in CEF cells were evaluated by IFA,electron microscope and RT-PCR.The results showed that the recombination virus rHLJ0504HT with double mutations at Q253H and A284T was successfully rescued and replicated in the CEF cells which was otherwise non-permissive to the non-mutated viruses vvIBDV.Consequently,the synergetic mutation of Q253H and A284T of VP2 changed the cell tropism of vvIBDV.

To express the envelope(env) protein of the reticuloendotheliosis virus(REV),the env gene of REV HLJR0901 strain was cloned into prokaryotic expression vector pET-32a,and expressed in BL21(DE3) E.coli by inducing with IPTG.The expressed protein was identified by SDS-PAGE and immunogenicity confirmed by western blot.The recobminant protein was purified and injected into BALB/c mice to prepare anti-env polyclonal serum.IFA and ELISA assays showed that specific mouse anti-env serum with a titer of 1∶105 was produced.This study laid the foundation for the development of the diagnostic methods and subunit vaccine for REV.

To investigate the co-infection status of avian leukaemia viruses (ALV) in layer flocks in China, 184 samples were collected from suspected ALV-J infection cases in Ningxia, Hubei, Guangdong, Shandong, Liaoning, Jilin and Heilongjiang provinces. These samples were subjected to PCR, virus isolation and indirect immunofluorescence assay (IFA) to detect infection by subgroup J ALV (ALV-J) in layer chicken flocks and co-infection with Reticuloendotheliosis (REV), Marek's disease virus (MDV) or Chicken anemia virus (CAV). The results showed that infection of ALV-J exists in all seven provinces with 60.9 % samples detected positive in 82.1 % flocks. The co-infection with REV, MDV or CAV with ALV-J accounted for 13.6 %, 24.5 %, 22.8 %, respectively. Multiple infection by up to 4 different viruses was detected in some cases. These results indicated there was serious ALV-J infecton in layer flocks in China and co-infection with other immunosuppressive virus is prevalent, therefore it is strongly recommended more attention be paid on co-infection in clinical diagnosis of ALV-J infection in layer flocks in the future.