Respiratory tract and intestinal diseases are common threats to feline health. Coinfection with multiple pathogens is not rare among clinical infectious cases. Rapid diagnosis of these coinfections is of great significance for timely and effective clinical treatment. In this study, two novel multiplex polymerase chain reactions (mPCRs) were established for simultaneous detection of four pathogens associated with the feline intestinal tract (feline coronavirus (FCoV), feline astrovirus (FeAstV), feline panleukopenia virus (FPV) and feline kobuvirus (FeKoV)) and five pathogens associated with the respiratory tract (feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), feline leukemia virus (FeLV), Chlamydia felis (C. felis) and influenza A virus (IAV)). The results of sensitivity analysis revealed that the detection limits for FeKoV, FPV, FeAstV, FCoV, IAV, C. felis, FeLV, FHV-1 and FCV were 10³, 10⁴, 10³, 10³, 10³, 10⁴, 10⁴, 10⁵ and 10⁵ copies/µL, respectively. Moreover, the specificity of the two mPCRs was high. When the two mPCRs were applied to clinical samples, the assay worked well. In conclusion, we established two mPCR methods that provide an excellent tool for the diagnosis and monitoring of pathogens associated with the feline respiratory and intestinal tracts.

As companion animals, dogs are susceptible to various subtypes of influenza A virus (IAV), with the H3N2 and H3N8 subtypes of canine influenza virus (CIV) stably circulating among canines. Compared to the H3N8 CIV, the H3N2 CIV is more widely prevalent in canine populations and demonstrates increased adaptability to mammals, potentially facilitating cross-species transmission. Therefore, a comprehensive elucidation of the mechanisms underlying H3N2 CIV adaptation to mammals is imperative. In this study, we serially passaged the GD14-WT strain in murine lungs, successfully establishing a lethal H3N2 CIV infection model. From this model, we isolated the lethal strain GD14-MA and identified the key lethal mutations PA(S184N) and PB2(E627K). Moreover, the GD14-ma[PA(S184N)+PB2(E627K)] strain exhibited markedly enhanced pathogenicity in dogs. Viral titers in lung tissues from infected dogs and mice showed that GD14-ma[PA(S184N)+PB2(E627K)] does not increase its pathogenicity to mice and dogs by upregulating viral titers compared to the GD14-WT strain. Notably, sequence alignments across all H3N2 IAVs showed an increasing prevalence of the PA (S184N) and PB2 (E627K) mutations from avian to human hosts. Finally, single-cell RNA sequencing of infected mouse lung tissues showed that GD14-ma[PA(S184N)+PB2(E627K)] effectively evaded host antiviral responses, inducing a robust inflammatory reaction. Considering the recognized role of the PB2 (E627K) mutation in the mammalian adaptation of IAVs, our findings underscore the importance of ongoing surveillance for the PA (S184N) mutation in H3N2 IAVs. IMPORTANCE: Since the 21st century, zoonotic viruses have frequently crossed species barriers, posing significant global public health challenges. Dogs are susceptible to various influenza A viruses (IAVs), particularly the H3N2 canine influenza virus (CIV), which has stably circulated and evolved to enhance its adaptability to mammals, including an increased affinity for the human-like SAα2,6-Gal receptor, posing a potential public health threat. Here, we simulated H3N2 CIV adaptation in mice, revealed that the synergistic PA(S184N) and PB2(E627K) mutations augment H3N2 CIV pathogenicity in dogs and mice, and elucidated the underlying mechanisms at the single-cell level. Our study provides molecular evidence for adapting the H3N2 CIV to mammals and underscores the importance of vigilant monitoring of genetic variations in H3N2 CIV.