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Porcine reproductive and respiratory syndrome virus (PRRSV) is globally prevalent and seriously harms the economic efficiency of pig farming. Because of its immunosuppression and high incidence of mutant recombination, PRRSV poses a great challenge for disease prevention and control. Nonstructural protein 2 (NSP2) is the most variable functional protein in the PRRSV genome and can generate NSP2N and NSP2TF variants due to programmed ribosomal frameshifts. These variants are broad and complex in function and play key roles in numerous aspects of viral protein maturation, viral particle assembly, regulation of immunity, autophagy, apoptosis, cell cycle and cell morphology. In this paper, we review the structural composition, programmed ribosomal frameshift and biological properties of NSP2 to facilitate basic research on PRRSV and to provide theoretical support for disease prevention and control and therapeutic drug development.

Viral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea. Specific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods. This novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region. The mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.

Urban stray cats are cats without owners that survive in the wild for extended periods of time. They are one of the most common stray animals in cities, and as such, monitoring the pathogens carried by urban stray cats is an important component of urban epidemiological surveillance. In order to understand the prevalence of respiratory diseases in urban stray cats in Shanghai and provide scientific evidence for the development of targeted prevention and control strategies for respiratory diseases in stray cats, we collected 374 ocular, nasal, and oropharyngeal swabs from urban stray cats in Shanghai from January 2022 to December 2022. After RNA extraction, we used real-time PCR to detect six respiratory pathogens, including influenza A virus, feline calicivirus, feline herpesvirus type 1, Mycoplasma, Chlamydia, and Bordetella bronchiseptica. The results showed that among the 374 samples, 146 tested positive, with a positivity rate of 39.04%. The highest positivity rate was observed for Mycoplasma felis at 18.72% (70/374), followed by Chlamydia felis at 11.76% (44/374), feline calicivirus at 3.74% (14/374), feline herpesvirus 1 at 3.48% (13/374), Bordetella bronchiseptica at 1.34% (5/374), and influenza A virus was not detected. The highest positivity rate for Mycoplasma felis was in Minhang District at 31.94% (23/72), while Chlamydia felis and Bordetella bronchiseptica had the highest positivity rates in Jiading District at 23.53% (8/34) and 5.88% (2/34), respectively. The highest positivity rates for feline calicivirus and feline herpesvirus 1 were both observed in Qingpu District, at 14.46% (12/83) and 9.64% (8/83), respectively. A total of 36 samples showed mixed infections with two or more pathogens, with Mycoplasma felis being involved in 32 of these mixed infections, with the highest number of mixed infections being with Chlamydia felis at 25 samples. Respiratory pathogen positivity was detected throughout the year, with peak detection rates in summer and winter. The positivity rates of cat respiratory pathogens in different seasons showed statistical differences (χ2 = 27.73, p < 0.01). There was no statistical difference in the positivity rates of respiratory pathogens between cats of different genders (χ2 = 0.92, p > 0.05). The positivity rates of respiratory pathogens in cats of different age groups showed statistical differences (χ2 = 44.41, p < 0.01). Mycoplasma felis and Chlamydia felis were the main pathogens causing respiratory infections in stray cats, with Mycoplasma felis showing a much higher positivity rate than other respiratory pathogens and often co-infecting with Chlamydia felis and feline calicivirus. The positivity rate of Mycoplasma felis was high in summer, autumn, and winter, with no statistical difference between seasons. These results indicate a serious overall prevalence of respiratory pathogens in urban stray cats in the Shanghai area, showing seasonal trends and mixed infections with other pathogens. These findings suggest the need for comprehensive prevention and control measures to address respiratory pathogen infections in urban stray cats in the Shanghai area.

H9N2 influenza viruses have been circulating in China since 1994, but a systematic investigation of H9N2 in Shanghai has not previously been undertaken. Here, using 14 viruses we isolated from poultry and pigs in Shanghai during 2002 and 2006-2014, together with the commercial vaccine A/chicken/Shanghai/F/1998 (Ck/SH/F/98), we analyzed the evolution of H9N2 influenza viruses in Shanghai and showed that all 14 isolates originated from Ck/SH/F/98 antigenically. We evaluated the immune protection efficiency of the vaccine. Our findings demonstrate that H9N2 viruses in Shanghai have undergone extensive reassortment. Various genotypes emerged in 2002, 2006 and 2007, while during 2009-2014 only one genotype was found. Four antigenic groups, A-D, could be identified among the 14 isolates and a variety of antigenically distinct H9N2-virus-derived avian influenza viruses (AIVs) circulated simultaneously in Shanghai during this period. Challenge experiments using vaccinated chickens indicated that the vaccine prevented shedding of antigenic group A and B viruses, but not those of the more recent groups C and D. Genetic analysis showed that compared to the vaccine strain, representative viruses of antigenic groups C and D possess greater numbers of amino acid substitutions in the hemagglutinin (HA) protein than viruses in antigenic groups A and B. Many of these substitutions are located in antigenic sites. Our results indicate that the persistence of H9N2 AIV in China might be due to incomplete vaccine protection and that the avian influenza vaccine should be regularly evaluated and updated to maintain optimal protection.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/μL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

Currently, multiple recombinant variants of porcine reproductive and respiratory syndrome virus (PRRSV) are circulating in China. These variants exhibit differences in pathogenicity. To comprehensively understand the current status of the virus and its recombination patterns, a total of 677 PRRSV strains were used for evolutionary analysis, of which 673 strains were isolated from mainland China. The results indicated that current PRRSV strains in circulation in China are highly complex, with NADC30‐like strains having replaced highly pathogenic strain of PRRSV (HP‐PRRSV) strains as the dominant strains. An analysis of the patterns and hotspots of PRRSV‐2 recombinant strains from 2019 to 2023 revealed two main types: the strains with NADC30 as the primary parent were observed to have recombination hotspots concentrated in the Nsp1, Nsp4 to Nsp9, and ORF2 to ORF6 regions, while recombination frequencies were found to be lower in the Nsp2, Nsp10, and Nsp11 regions. In contrast, the other type of recombinant strain, primarily derived from HP‐PRRSV, exhibited recombination hotspots concentrated in the Nsp2 and ORF2 to ORF6 regions, while the recombination frequency in the Nsp8 and Nsp9 regions was relatively low. Further, a comprehensive analysis of the phylogenetic and recombination patterns of four PRRSV strains (ZJ‐2021‐1, JS‐2020‐1, GZ‐2022‐1, and SH‐2020‐2) indicated variations in genotyping precision among Nsp2, ORF5, and the entire genome. As demonstrated by the results of the animal experiments, there were differences in pathogenicity between ZJ‐2021‐1 and JS‐2020‐1. The pathogenicity of the recombinant strain exhibited similar characteristics to its parental skeleton. Our results provided reference data for exploring epidemiological characteristics and pathogenicity of recombinant PRRSV strains.

Our study was designed to investigate the original spectrum of feline respiratory tract infection and to provide a scientific basis for the clinical diagnosis and treatment of feline respiratory infections and for precise prevention and control measures. A total of 400 cats with upper respiratory tract infections from animal hospitals in 12 provinces in China were examined from November 2022 to October 2023 to investigate the epidemiology of feline calicivirus (FCV), feline herpes virus type 1 (FHV-1), influenza A virus (IAV), Mycoplasma felis, Chlamydia felis, and Bordetella bronchiseptica through loop-mediated isothermal amplification (LAMP) with microfluidic chip detection. The results showed that 396 of the 400 samples tested were positive for at least one of these pathogens, with an overall detection rate of 99.00%. The detection rates were as follows: FCV, 36.00% (144/400); M. felis, 34.00% (136/400); FHV-1, 21.50% (86/400); C. felis, 15.75% (63/400); B. b, 13.00% (52/400); IAV, 4.50% (18/400). There were no statistically significant differences in the detection rates of respiratory pathogens between different sexes, ages, seasons, breeds, or regions (P > 0.05). There were 88 mixed infections, giving a total mixed infection rate of 22.00% (88/400). It is worth noting that the detection rate of FCV at different ages and of FHV-1 in different sexes showed significant differences (P < 0.05). The highest rate of FCV infection was found in animals that were 1 to 2 years old, and the rate of FHV-1 infection in male cats was higher than that in female cats. The results showed that the spectrum of feline respiratory pathogens is complex, with diverse epidemiological characteristics and mixed infections, and some differences among different respiratory pathogens were found with regard to the sex, age, and breed of the cat. Studies should be continued to provide a scientific basis for precise prevention and control of feline respiratory diseases.

Background Avian influenza viruses represent a growing threat of an influenza pandemic. The co‐circulation of multiple H9N2 genotypes over the past decade has been replaced by one predominant genotype—G57 genotype, which displays a changed antigenicity and improved adaptability in chickens. Effective H9N2 subtype avian influenza virus vaccines for poultry are urgently needed. Objective In this study, we constructed H9N2 subtype avian influenza virus‐like particle (VLP) and evaluated its protective efficacy in specific pathogen‐free (SPF) chickens to lay the foundation for developing an effective vaccine against influenza viruses. Methods Expression of influenza proteins in VLPs was confirmed by Western blot, hemagglutination inhibition (HI), and neuraminidase inhibition (NI). The morphology was observed by electron microscopy. A group of 15 three‐week‐old SPF chickens was divided into three subgroups of five chickens immunized with VLP, commercial vaccine, and PBS. Challenge study was performed to evaluate efficacy of VLP vaccine. Results and Conclusions The hemagglutinin (HA) and neuraminidase (NA) proteins were co‐expressed in the infected cells, self‐assembled, and were released into the culture medium in the form of VLPs of diameter ~80 nm. The VLPs exhibited some functional characteristics of a full influenza virus, including hemagglutination and neuraminidase activity. In SPF chickens, the VLPs elicited serum antibodies specific for H9N2 and induced a higher HI titer (as detected by a homologous antigen) than did a commercial H9N2 vaccine (A/chicken/Shanghai/F/1998). Viral shedding from VLP vaccine subgroup was reduced compared with commercial vaccine subgroup and control subgroup.

Five H3N2 avian influenza viruses (AIVs) were isolated from live poultry markets (LPMs) and poultry slaughterhouses in Shanghai, China in 2013. All viruses were characterized by whole-genome sequencing with subsequent genetic comparison and phylogenetic analysis. The hemagglutinin cleavage site of all viruses indicated that the five strains were low-pathogenic AIVs. Phylogenetic analysis of all eight viral genes showed that the five H3N2 viruses clustered in the Eurasian lineage of influenza viruses. The eight genes showed evidence of reassortment events between these H3 subtype viruses and other subtype viruses, especially H5 and H7 subtypes, probably in pigeons, domestic ducks, and wild birds. These findings emphasized the importance of AIV surveillance in LPMs and poultry slaughterhouses for understanding the genesis and emergence of novel reassortants with pandemic potential.

为了解冠状病毒在猫科动物的感染情况，对采自上海某动物园的猫科动物144份样品进行检测，并对获得的阳性样品进行扩增和测序。结果显示：SARSCoV2荧光RTPCR检测全部为阴性；FCoV荧光RTPCR检测阳性3份。测序结果显示，检测到FCoV的ORF3、ORF7和S基因均呈遗传多样性，序列存在很大的个体间差异。3a、3b和3c基因主要表现为氨基酸突变。7a基因在第9位（F）、12位（V）和47位（S）均未发生氨基酸替换。它们均具有完整的7b基因。S基因的S1/S2裂解位点处有连续的RRS/ARRS序列模体，符合FECV裂解位点的氨基酸序列特征。在第1045位（M）、1047位（S）和1108位（D）均没有发生氨基酸的完全替换。上述结果显示，这3株病毒均为致病性低的FECV。基于ORF3c和S2基因的遗传进化树显示，SH1951与SH2003和SH2004处于同一分支，与血清型Ⅰ型毒株的亲缘关系最近；而基于ORF7b基因的进化树显示，SH1951与SH2003和SH2004处于不同的遗传进化分支，表明它们之间的亲缘关系较远。因此，需要加强对野猫的管理与监测，防止野猫携带的FECV或其他病毒传染给豹猫。