Bovine Parainfluenza Virus Type Research Articles

Critical role of G3BP1 in bovine parainfluenza virus type 3 (BPIV3)-inhibition of stress granules formation and viral replication.

It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.

Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro.

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties.

Bovine parainfluenza virus type 3 infections induce ER stress-mediated autophagy to facilitate virus replication

Bovine Parainfluenza Virus Type 3 (BPIV3) serves as a crucial pathogen in cattle, adept at triggering severe respiratory symptoms. This investigation explores the intricate interplay of endoplasmic reticulum stress (ER stress), unfolded protein response (UPR), and autophagy upon BPIV3 infection. In this study, we initially confirm a substantial increase in glucose regulatory protein 78 (GRP78) expression, accompanied by noticeable morphological changes and significant expansion of the ER lumen observed through transmission electron microscopy upon BPIV3 infection. Our findings indicate that ER Stress is induced during BPIV3 infection in vitro. Subsequently, we illustrate that BPIV3 triggers ER Stress to facilitate viral replication through heightened autophagy through treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) and utilizing small interfering RNA (siRNA) technology to knock down GRP78. Additionally, we observe that the activation of ER stress initiates the UPR via PERK and ATF6 pathways, with the IRE1 pathway not contributing to the regulation of ER stress-mediated autophagy. Moreover, intervention with the PERK inhibitor GSK2606414, ATF6 inhibitor Ceapin-A7, and siRNA technology successfully reverses BPIV3-induced autophagy. In summary, these findings propose that BPIV3 induces ER stress to enhance viral replication through increased autophagy, with the PERK and ATF6 pathways playing a significant role in ER stress-mediated autophagy.

Prevalence, Molecular Characteristics and Virulence Identification of Bovine Parainfluenza Virus Type 3 in China.

Bovine parainfluenza virus type 3 (BPIV-3) is one of the major pathogens of the bovine respiratory disease complex (BRDC). BPIV-3 surveillance in China has been quite limited. In this study, we used PCR to test 302 cattle in China, and found that the positive rate was 4.64% and the herd-level positive rate was 13.16%. Six BPIV-3C strains were isolated and confirmed by electron microscopy, and their titers were determined. Three were sequenced by next-generation sequencing (NGS). Phylogenetic analyses showed that all isolates were most closely related to strain NX49 from Ningxia; the genetic diversity of genotype C strains was lower than strains of genotypes A and B; the HN, P, and N genes were more suitable for genotyping and evolutionary analyses of BPIV-3. Protein variation analyses showed that all isolates had mutations at amino acid sites in the proteins HN, M, F, and L. Genetic recombination analyses provided evidence for homologous recombination of BPIV-3 of bovine origin. The virulence experiment indicated that strain Hubei-03 had the highest pathogenicity and could be used as a vaccine candidate. These findings apply an important basis for the precise control of BPIV-3 in China.

Isolation and Genomic Characterization of a Chinese Genotype C Bovine Parainfluenza Virus Type 3 from Cattle and Its Pathogenicity in C57BL/6 Mice

Bovine parainfluenza virus type 3 (BPIV-3), also known as bovine respirovirus 3, is a common respiratory pathogen associated with bovine respiratory disease (BRD). BPIV-3 has currently circulated worldwide; however, data on the prevalence and genetic characteristics of BPIV-3 are still scarce and limited. In this study, the BPIV-3 strain SC was identified and isolated from cattle presenting with clinical signs of BRD in China. Animal experiments indicated that BPIV-3 SC can successfully infect C57BL/6 mice and induce weight loss, lung inflammatory cell infiltration, and inflammatory cytokine expression in mice. In addition, the complete genome of BPIV-3 SC was obtained using next-generation sequencing and was 15,473 bp in length. Phylogenetic analysis indicated that BPIV-3 SC belonged to genotype C, which clustered in the same large clade consisting of a population of Chinese genotype C strains but was found to be different from the other strains upon further differentiation. Compared to other Chinese genotype C strains, the BPIV-3 SC showed 70 unique nucleotide mutations and 13 unique amino acid mutations in the HN, P, and L proteins, suggesting a unique genetic evolution of BPIV-3 SC. In conclusion, we isolated and characterized a differential Chinese genotype C BPIV-3, which contributed to an understanding of the prevalence and evolution of BPIV-3 in China.

Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

A Comparative Study of the Effects of Al(OH)3 and AlPO4 Adjuvants on the Production of Neutralizing Antibodies (NAbs) against Bovine parainfluenza Virus Type 3 (BPIV3) in Guinea Pigs

Aluminum-containing adjuvants are extensively used for inactive human and animal vaccines owing to their favorable immunostimulative and safe properties. Nonetheless, there is controversy over the effects of different aluminum salts as an adjuvant for the bovine parainfluenza virus type 3 (BPIV3) vaccine. In order to find a suitable adjuvant, we studied the effects of two adjuvants (i.e., aluminum hydroxide [Al(OH)3] and aluminum potassium sulfate [AlPO4]) on the production of neutralizing antibodies (NAbs) for an experimental BPIV3 vaccine. The animals under study (Guinea pigs) were randomly assigned to five groups of experimental vaccines containing Al(OH)3 (AH), AlPO4 (AP), Al(OH)3-AlPO4 mixture (MIX), commercial vaccine (COM), and control (NS). The treatment groups were immunized with two doses of vaccine 21 days apart (on days 0 and 21), and the control group received normal saline under the same conditions. The animals were monitored for 42 days, and blood samples were then taken. The results indicated that all vaccines were able to induce the production of NAbs at levels higher than the minimum protective titer (0.6). An increase in titer was observed throughout the monitoring period. Moreover, an increase in both the level and mean titer of NAbs obtained from the vaccine containing Al(OH)3 adjuvant was significantly higher than in the other studied groups (P≤0.005). The comparison of NAbs titer in other groups did not display a significant difference. Considering the speed of rising and the optimal titer of NAbs production in the experimental vaccine, the Al(OH)3 adjuvant is a suitable candidate for preparing a vaccine against BPIV3 for immunization.

Isolation and Identification of a Genotype C Bovine Parainfluenza Virus Type 3 and Its Pathogenicity in Albino Guinea Pigs

Bovine parainfluenza virus type 3 (BPIV3) is a significant pathogen in cattle, causing a range of respiratory symptoms from mild to severe. In this study, we isolated a BPIV3 (BPIV3/SX/2021) strain using Madin-Darby bovine kidney cells from a lung tissue sample obtained in Shaanxi province. The full length of the BPIV3/SX/2021 strain is 15,474 nt, encoding six structural and three accessory proteins. A phylogenetic analysis revealed that SX2021strain belongs to BPIV3 genotype C, and the isolate shares the highest sequence homology with the other genotype C strains isolated in China. A genetic comparison of the BPIV3c strain isolated in China showed that some amino acid residues have mutations in the F and HN genes that affect the protein structure. Animal challenge experiments showed that experimental infection of guinea pigs with the BPIV3c strain SX2021 displayed depression and dyspnea clinical signs. Histopathological analysis of the lung and trachea showed that morphological changes consisted of lesions of bronchial epithelial cell shedding, alveoli septa thickening, focal fiber hyperplasia, and macrophage infiltration. The virus load analysis indicated that the SX2021 strain displayed a higher replication level in the lungs and trachea. These results provide strong evidence that the evolution of BPIV3, and the isolated BPIV3 strain SX2021 provide useful data to determine the pathogenesis of the virus.

Development and evaluation of a monoclonal antibody-based blocking ELISA to detect antibodies against the E2 protein of bovine viral diarrhea virus-1

With the rapid development of cattle industry, bovine viral diarrhea virus (BVDV) is becoming widespread in China, which causes serious economic losses to the industry. Effective vaccination and viral surveillance are critical for the prevent and control of BVDV infection. In the present study, the immunogenic domain of E2 protein of BVDV-1 was expressed by prokaryotic pET-28a vector. Monoclonal antibodies (mAbs) against E2 protein were prepared and systemically examined by western blot, immunofluorescence assay, blocking ELISA (bELISA) and virus neutralization test (VNT). The mAb 1E2B3, which showed good reactivity and neutralizing activity to BVDV-1 strains, was selected for ELISA establishment. After a series of screening and optimization, a novel bELISA for highly sensitive and specific detection of BVDV-1 antibodies was established, using HRP-labeled 1E2B3 and recombinant E2 protein. ROC analysis of 91 positive and 84 negative reference bovine serum samples yielded the area under the curve (AUC) of 0.9903. A diagnostic specificity of 96.43 % and a sensitivity of 95.6 % were achieved when the cutoff value was set at 24.31 %. There was no cross reaction to the positive sera of classical swine fever virus (CSFV), BVDV-2, border disease virus (BDV), bovine parainfluenza virus type 3 (BPIV3), infectious bovine rhinotracheitis virus (IBRV), foot-and-mouth disease virus (FMDV), Mycoplasma bovis (M.bovis) and Brucella. The total agreement rate of bELISA with VNT was 93.96 % (249/265). In addition, the result of bELISA was positively correlated with neutralizing antibody titer, and the bELISA could well distinguish the serum samples before and after BVDV vaccination. These results indicate that the established bELISA in this study is specific, sensitive, simple and convenient, which provides technical support for the vaccine efficacy evaluation, prevention and control of BVD in the future.

Immunogenicity and protective efficacy of a recombinant adenovirus containing the fusion protein of bovine parainfluenza virus type 3 genotype C in mice

Bovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen of cattle that causes substantial economic losses. A replicating-defective recombinant human adenovirus type 5 (HAd5), carrying a fusion protein of BPIV3 genotype C (HAd5-F), was constructed and evaluated for its immunogenicity and protective efficacy in mice. After intramuscular injection with the HAd5-F, the IgG titers against F proteins increased to 1:102,400, and virus-neutralizing titers increased to 1:256, significantly higher than those in the group injected with inactivated BPIV3C in mice (p<0.05). The splenic CD4+/CD8+T lymphocytes and IFN-γ+/IL-4+ cytokine percentages were more significant in the HAd5-F group than those in the control group. A BPIV3C challenge in a mouse model was used to assess protective efficacy of the HAd5-F. The viral loads in the lungs and tracheas of mice immunized with the HAd5-F were significantly lower than those in the control group (p<0.0001). There were no significant histopathological alterations in the lungs of mice vaccinated with the HAd5-F. These findings suggested that the HAd5-F elicited excellent immunity against BPIV3C infection.

Isolation and Molecular Characterisation of Respirovirus 3 in Wild Boar.

Paramyxoviruses are important pathogens affecting various animals, including humans. In this study, we identified a paramyxovirus in 2004 (180608_2004), isolated from a sample of the femoral marrow bone of a wild boar carcass imported from Australia. Antigenic and morphological characteristics indicated that this virus was similar to members of the family Paramyxoviridae. The complete genome phylogenetic analysis grouped this virus into genotype A of bovine parainfluenza virus type 3 (BPIV-3), recently renamed bovine respirovirus type 3 (BRV3), which also includes two swine paramyxoviruses (SPMV)-Texas-81 and ISU-92-isolated from encephalitic pigs in the United States in 1982 and 1992, respectively. The wild boar 180608_2004 strain was more closely related to both the BRV3 shipping fever (SF) strain and the SPMV Texas-81 strain at the nucleotide and amino acid levels than the SPMV ISU-92 strain. The high sequence identity to BRV3 suggested that this virus can be transferred from cattle to wild boars. The potential for cross-species transmission in the Respirovirus genus makes it essential for intensified genomic surveillance.

Prevalence and Molecular Characterization of Bovine Parainfluenza Virus Type 3 in Cattle Herds in China.

Bovine parainfluenza virus type 3 (BPIV3) is a common respiratory pathogen that causes respiratory illness in cattle and makes a major contribution to the bovine respiratory disease complex (BRDC); however, data on the prevalence and molecular features of BPIV3 are still scarce in China. To investigate the epidemiological characteristics of BPIV3 in China, between September 2020 and June 2022, 776 respiratory samples were received from 58 BRDC-affected farms located in 16 provinces and one municipality. Those were screened for BPIV3 using a reverse transcription insulated isothermal PCR (RT-iiPCR) assay. Meanwhile, the HN gene and complete genome sequence of strains from different provinces were amplified, sequenced, and analyzed. The tests showed that 18.17% (141/776) of samples tested were positive for BPIV3, which originated from 21 farms in 6 provinces. Moreover, 22 complete HN gene sequences and 9 nearly complete genome sequences were obtained from the positive samples. Phylogenetic analysis based on the HN gene and complete genome sequences revealed that the sequences were clustered in one large clade for all Chinese BPIV3 genotype C strains, while overseas strain sequences of BPIV3 genotype C clustered into other clades. Moving beyond the known complete genome sequences of BPIV3 in GenBank, a total of five unique amino acid mutations were found in N protein, F protein, and HN protein in Chinese BPIV3 genotype C strains. Taken together, this study reveals that BPIV3 genotype C strains, the dominant strains in China, have a broad geographical distribution and some unique genetic characteristics. These findings contribute to our understanding of the epidemiological characteristics and genetic evolution of BPIV3 in China.

Generation of a photocontrollable recombinant bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (BPIV3) is a promising vaccine vector against various respiratory virus infections, including the human PIV3, respiratory syncytial virus, and severe acute respiratory syndrome-coronavirus 2 infections. In this study, we combined the Magnet system and reverse genetic approach to generate photocontrollable BPIV3. An optically controllable Magnet gene was inserted into the H2 region of the BPIV3 large protein gene, which encodes an RNA-dependent RNA polymerase. The generated photocontrollable BPIV3 grew in specific regions of the cell sheet only when illuminated with blue light, suggesting that spatiotemporal control can aid in safe clinical applications of BPIV3.

Pathogenic infection characteristics and risk factors for bovine respiratory disease complex based on the detection of lung pathogens in dead cattle in Northeast China.

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 and beef 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.

Parainfluenza and corona viruses in a fallow deer (Dama dama) with fatal respiratory disease.

Parainfluenza virus type 3 (PIV-3) and coronaviruses (CoV) are commonly found in respiratory tracts of ruminants and capable of causing clinical disease. Here, we investigated the cause of ill-thrift and sudden death in a five-month-old male fallow deer which occurred in December 2019. The calf was one of the five calves in a herd of 170 deer that, along with three adult hinds, died during a 2-week period. The deer calves were in a shed, sharing airspace with young cattle that had been reported to be coughing. Significant gross pathology was observed in the respiratory and alimentary tracts of the deer calf and histopathology of the lung and trachea was suggestive of likely involvement of PIV-3. Strong and specific cytoplasmic labeling of bronchiolar epithelium and terminal airway, alike those seen with PIV-3 pneumonia in cattle, was observed using a polyclonal bovine PIV-3 antibody. Metagenomic analysis detected a PIV-3 and a CoV in the lung tissue. The PIV-3 L protein gene had the highest sequence identity with those of bovine PIV-3 (83.1 to 98.4%) and phylogenetically clustered with bovine PIV-3 in the genotype C. The CoV spike protein gene shared 96.7% to 97.9% sequence identity with those of bovine CoVs, but only 53.1% identity with SARS-CoV-2 reference virus. We believe this is the first report of PIV-3 and CoV co-infection in fallow deer and their association with fatal pneumonia; major pathology caused by PIV-3.

First Isolation and Characteristics of Bovine Parainfluenza Virus Type 3 from Yaks.

The yaks belong to the genus Bos within the family Bovidae that live in the Tibet Plateau and is an indispensable economic resource for the local herders. Respiratory tract infections are common diseases in yaks caused by various pathogens; however, there have been no reports of bovine parainfluenza virus type 3 (BPIV3) infection. This study was conducted to investigate the pathogens and analyze their characteristics from the four yak lung samples with severe respiratory tract infection symptoms in the yak farm. Results showed that out of four lung samples, three were identified as BPIV3-positive by RT-PCR. A BPIV3 strain (106.5 TCID50/mL) was successfully isolated from the BPIV3-positive lung samples using Madin–Darby bovine kidney cells. The isolate caused systemic infection in the BALB/c mice and induced pathological changes in the lungs. Moreover, three complete BPIV3 genomes were amplified from the clinical samples. Phylogenetic trees based on the complete genomes, hemagglutinin-neuraminidase protein (HN), phosphoprotein (P), and large polymerase subunit protein (L) amino acid sequences showed that the complete BPIV3 genomes belonged to BPIV3 genotype C, and clustered into a large branch with the Chinese strains, although the three yak BPIV3 strains were clustered into a small branch. Compared to known BPIV3 genotype C strains in GenBank, the three genomes of yak BPIV3 showed four identical amino acid mutations in the HN, P and L proteins, suggesting a unique genetic evolution of BPIV3 in yaks. This study first isolated and characterized the BPIV3 from yaks, which contributed to the understanding of the infection and evolution of BPIV3 in yaks in the Tibet Plateau.

The Prevalence, Coexistence, and Correlations between Seven Pathogens Detected by a PCR Method from South-Western Poland Dairy Cattle Suffering from Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is a very important disease that contributes to economic losses in dairy and beef cattle breeding worldwide. The molecular testing of material from 296 calves showing BRD symptoms from 74 dairy herds located in south-western Poland was performed in 2019–2021. Molecular tests were performed using a commercial kit “VetMAXTM Ruminant Respiratory Screening Kit” (Thermo Fisher Scientific) for the simultaneous detection of genetic material of seven pathogens responsible for BRD. At least one pathogen was detected in 95.95% of herds. The overall prevalence was: Pasteurella multocida 87.84%, Mannheimia haemolytica 44.59%, bovine coronavirus (BcoV) 32.43%, Mycoplasma bovis 29.73%, Histophilus somni 28.38%, bovine parainfluenza virus type 3 (BPIV-3) 13.51%, and bovine respiratory syncytial virus (BRSV) 10.81%. Twenty-nine configurations of pathogen occurrences were found. Bacterial infections were the most frequently recorded as 56.7% of all results. Coinfections mainly consisted of two pathogens. Not a single purely viral coinfection was detected. The most frequent result was a single P. multocida infection accounting for 18.31% of all results. The statistically significant correlation (p = 0.001) with the highest strength of effect (ϕ 0.38) was between M. bovis and H. somni.

Serodiagnosis and Risk Factors Associated with Infectious Agents of Reproductive Diseases in Bovines of Chiquinquirá, District of Boyacá (Colombia)

The productivity of cattle farms is affected by infectious and noninfectious factors that generate economic losses and cause reproductive failure represented by low conception rates, embryonic mortality, abortions, and fetal mummification. The infectious agents that most impact the reproductive health of the bovine species from conception to birth are bovine herpesvirus type 1 (BoHV-1) causing infectious bovine rhinotracheitis (IBR), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (PI3), Neospora caninum and Leptospira spp. The objective of this study was to diagnose the presence of BoHV-1, bovine viral diarrhea (BVD), PI3, Neospora caninum, and Leptospira spp. by serology and identify the risk factors associated with infectious agents of reproductive interest in bovines of Boyacá (Colombia). A descriptive cross-sectional study was developed, with simple random sampling, where a sample size of 601 female cattle of Holstein, Jersey, and Normande breeds of different age groups was determined. Blood samples were taken and processed using the indirect ELISA technique (SYNBIOTICS®, SERELISA® BVD p80 Ab Mono Blocking, Ingezim R.12.NC.K, PRIMACHECK VPI-3®) and the MAT test for the diagnosis of bovine leptospirosis. The data were processed with the statistical program Epi Info™. The highest apparent seroprevalence was established for infectious bovine rhinotracheitis (61.1%), followed by BVD (37.6%), PI3 (40.9%), neosporosis (51.1%), and leptospirosis (14.8%). Variables such as age >4 years and Holstein breed for IBR and >4 years for BVD were established risk factors. Considering our results, we suggest implementing prevention and control plans that include vaccination as a prophylactic measure and biosecurity tools that reduce the probability of contagion and transmission of pathogens.

Quantitative proteomic analysis shows involvement of the p38 MAPK pathway in bovine parainfluenza virus type 3 replication

BackgroundBovine parainfluenza virus type 3 (BPIV3) infection often causes respiratory tissue damage and immunosuppression and further results in bovine respiratory disease complex (BRDC), one of the major diseases in dairy cattle, caused huge economical losses every year. However, the pathogenetic and immunoregulatory mechanisms involved in the process of BPIV3 infection remain unknown. However, the pathogenetic and immunoregulatory mechanisms involved in the process of BPIV3 infection remain unknown. Proteomics is a powerful tool for high-throughput identification of proteins, which has been widely used to understand how viruses interact with host cells.MethodsIn the present study, we report a proteomic analysis to investigate the whole cellular protein alterations of MDBK cells infected with BPIV3. To investigate the infection process of BPIV3 and the immune response mechanism of MDBK cells, isobaric tags for relative and absolute quantitation analysis (iTRAQ) and Q-Exactive mass spectrometry-based proteomics were performed. The differentially expressed proteins (DEPs) involved in the BPIV3 invasion process in MDBK cells were identified, annotated, and quantitated.ResultsA total of 116 proteins, which included 74 upregulated proteins and 42 downregulated proteins, were identified as DEPs between the BPIV3-infected and the mock-infected groups. These DEPs included corresponding proteins related to inflammatory response, immune response, and lipid metabolism. These results might provide some insights for understanding the pathogenesis of BPIV3. Fluorescent quantitative PCR and western blotting analysis showed results consistent with those of iTRAQ identification. Interestingly, the upregulated protein MKK3 was associated with the p38 MAPK signaling pathway.ConclusionsThe results of proteomics analysis indicated BPIV3 infection could activate the p38 MAPK pathway to promote virus replication.

Epidermal growth factor receptor (EGFR) promotes uptake of bovine parainfluenza virus type 3 into MDBK cells

Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. The pathways of cell entry are highly related to viral transmission and pathogenicity. In previous studies, we demonstrated that macropinocytosis and clathrin-dependent endocytosis play critical roles in the entry of BPIV3 into MDBK cells. Macropinocytosis is special endocytic process which need to activate signaling pathways that remodle the actin cytoskeleton. Parainfluenza viruses (PIVs) initiate infection by binding to sialic acid receptors on cell surfaces. Nevertheless, sialic acids are not able to transmit signals across the plasma membrane, indicating the necessity for additional signaling receptors. Here, we have demonstrated that specific inhibitors and siRNAs targeting EGFR inhibit the entry of BPIV3 into MDBK cells. BPIV3 productive infection in MDBK cells led to activation of EGFR. Inactivation of EGFR suppressed BPIV3-induced rearrangement of the F-actin cytoskeleton. In addition, PI3K-Akt and ERK1/2 were activated in an EGFR-dependent manner during BPIV3 infection. Specific inhibitors targeting these canonical downstream effectors of EGFR could significantly reduce viral entry efficacy. Moreover, we also demonstrated that the important regulators of macropinocytosis, Rac1 and Pak1, are downstream mediators of EGFR during BPIV3 internalization. These results indicated that EGFR is a host-entry cofactor used by BPIV3 to enter MDBK cells.