Bovine respiratory disease complex (BRDC) involves multiple pathogens, shows diverse lung lesions, and is a major concern in calves. Pathogens from 160 lung samples of dead cattle from 81 cattle farms in Northeast China from 2016 to 2021 were collected to characterize the molecular epidemiology and risk factors of BRDC and to assess the major pathogens involved in bovine suppurative or caseous necrotizing pneumonia. The BRDC was diagnosed by autopsy, pathogen isolation, PCR, or reverse transcription-PCR detection, and gene sequencing. More than 18 species of pathogens, including 491 strains of respiratory pathogens, were detected. The positivity rate of bacteria in the 160 lung samples was 31.77%, including Trueperella pyogenes (9.37%), Pasteurella multocida (8.35%), Histophilus somni (4.48%), Mannheimia haemolytica (2.44%), and other bacteria (7.13%). The positivity rate of Mycoplasma spp. was 38.9%, including M. bovis (7.74%), M. dispar (11.61%), M. bovirhinis (7.94%), M. alkalescens (6.11%), M. arginini (0.81%), and undetermined species (4.68%). Six species of viruses were detected with a positivity rate of 29.33%, including bovine herpesvirus-1 (BoHV-1; 13.25%), bovine respiratory syncytial virus (BRSV; 5.50%), bovine viral diarrhea virus (BVDV; 4.89%), bovine parainfluenza virus type-3 (BPIV-3; 4.28%), bovine parainfluenza virus type-5 (1.22%), and bovine coronavirus (2.24%). Mixed infections among bacteria (73.75%), viruses (50%), and M. bovis (23.75%) were the major features of BRDC in these cattle herds. The risk analysis for multi-pathogen co-infection indicated that BoHV-1 and H. somni; BVDV and M. bovis, P. multocida, T. pyogenes, or Mann. haemolytica; BPIV-3 and M. bovis; BRSV and M. bovis, P. multocida, or T. pyogenes; P. multocida and T. pyogenes; and M. bovis and T. pyogenes or H. somni showed co-infection trends. A survey on molecular epidemiology indicated that the occurrence rate of currently prevalent pathogens in BRDC was 46.15% (6/13) for BoHV-1.2b and 53.85% (7/13) for BoHV-1.2c, 53.3% (8/15) for BVDV-1b and 46.7% (7/15) for BVDV-1d, 29.41% (5/17) for BPIV-3a and 70.59% (12/17) for BPIV-3c, 100% (2/2) for BRSV gene subgroup IX, 91.67% (33/36) for P. multocida serotype A, and 8.33% (3/36) for P. multocida serotype D. Our research discovered new subgenotypes for BoHV-1.2c, BRSV gene subgroup IX, and P. multocida serotype D in China's cattle herds. In the BRDC cases, bovine suppurative or caseous necrotizing pneumonia was highly related to BVDV [odds ratio (OR) = 4.18; 95% confidence interval (95% CI): 1.6-10.7], M. bovis (OR = 2.35; 95% CI: 1.1-4.9), H. somni (OR = 8.2; 95% CI: 2.6-25.5), and T. pyogenes (OR = 13.92; 95% CI: 5.8-33.3). The risk factor analysis found that dairy calves 3 mo (OR = 5.39; 95% CI: 2.7-10.7) were more susceptible to BRDC. Beef cattle were more susceptible to bovine suppurative or caseous necrotizing pneumonia than dairy cattle (OR = 2.32; 95% CI: 1.2-4.4). These epidemiological data and the new pathogen subgenotypes will be helpful in formulating strategies of control and prevention, developing new vaccines, improving clinical differential diagnosis by necropsy, predicting the most likely pathogen, and justifying antimicrobial use.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocyte (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the immunomodulatory effects of PD-1 pathway on major PBL subsets are unclear and their underlying molecular mechanisms need to be further studied. Therefore, in this study, we examined PD-1 expression in bovine PBL subsets after BVDV infection in vitro and analyzed the effects of PD-1 blockade on the apoptosis and proliferation of CD4⁺ and CD8⁺ T cells and expression of PD-1 downstream signaling molecules. The results showed that PD-1 expression was enhanced on CD4⁺ and CD8⁺ T cells, but not on CD21⁺ B cells after cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection in vitro and PD-1 blockade significantly reduced the apoptosis of CD4⁺ and CD8⁺ T cells after these two strains infection. Remarkably, PD-1 blockade significantly increased the proliferation of CD4⁺ and CD8⁺ T cells after CP BVDV infection, but only significantly increased the proliferation of CD4⁺ T cells after NCP BVDV infection. In addition, we confirmed that PD-1-mediated PI3K/Akt/mTOR, caspase 9/caspase 3 and ERK pathways are involved in regulating the apoptosis and proliferation of CD4⁺ and CD8⁺ T cells during BVDV infection in vitro. Notably, ERK is involved in the regulation mechanism PD-1 mediated only when the cells are infected with CP BVDV. Our findings provide a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

Bovine viral diarrhea virus (BVDV), a positive-strand RNA virus of the genus Pestivirus in the Flaviviridae family, is the causative agent of viral diarrheal disease in bovine. BVDV has been used as a surrogate model for the hepatitis C virus (HCV) to evaluate the efficacy of antiviral drugs. The plant flavonol quercetin causes multiple health-promoting effects in humans and animals. It can be made into a variety of additives, and it exerts a variety of immunomodulatory effects with the potential to be used as an antiviral agent. However, quercetin’s antiviral effect and mechanism of action on BVDV are still unclear. Therefore, this study was designed to evaluate quercetin’s effect on BVDV virus replication in vitro and in vivo and elucidate its mechanism of action. A CCK-8 kit was used to analyze the toxicity of the quercetin to the MDBK cells. Western blot, qRT-PCR, TCID₅₀, and histological analysis were used to determine the mechanism of quercetin’s anti-BVDV activity. An oxidative stress kit was used to evaluate the effects of quercetin on ROS, antioxidant enzymes, and MDA indexes. The effect of quercetin on IL-2 and IFN-γ in the serum of mice was determined by using an ELISA kit. The results showed that quercetin inhibits Hsp70, blocks BVDV infection in the early stage of virus infection and inhibits BVDV replication by inhibiting oxidative stress or ERK phosphorylation. In addition, quercetin alleviated the decrease in IFN-γ and IL-2 in the serum of BVDV-infected mice. Quercetin ameliorated BVDV-induced histopathological changes. In summary, this study demonstrated for the first time the role of Hsp70 in BVDV infection and the potential application of quercetin in treating BVDV infection.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocytes (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the situation in vivo remains to be further studied and confirmed. Therefore, in this study, we established a BALB/c mouse model of acute BVDV infection with cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain NY-1). Then, we examined the mRNA and protein levels of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC) from BVDV-infected mice and analyzed the effects of PD-1 blockade on the proportions of CD3⁺, CD4⁺, and CD8⁺ T cell subsets, the apoptosis and proliferation of PBL, and the production of IL-2 and IFN-γ. We found that leukopenia, lymphocytopenia, and thrombocytopenia were developed in both CP and NCP BVDV-infected mice at day 7 of post-infection. The mRNA and protein expression of PD-1 and PD-L1 were significantly upregulated in CP and NCP BVDV-infected mice. Moreover, PD-1/PD-L1 upregulation was accompanied by leukopenia and lymphopenia. Additionally, PD-1 blockade inhibited PBL apoptosis and virus replication, restored the proportions of CD3⁺, CD4⁺, and CD8⁺ T cell subsets, and increased IFN-γ production and p-ERK expression in BVDV-infected mice. However, blocking PD-1 did not significantly affect PBL proliferation and IL-2 production in NCP BVDV-infected mice. Our findings further confirmed the immunomodulatory role of PD-1 in peripheral blood lymphocytopenia in vivo and provided a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.