Novel bovine viral diarrhea virus (BVDV) virus-like particle vaccine candidates presenting the E2 protein using the SpyTag/SpyCatcher system induce a robust neutralizing antibody response in mice.

Bovine viral diarrhea virus (BVDV) is a pathogen of commercial consequence in cattle. Although many modified live and killed vaccines are commercially available, their drawbacks precipitate the need for new effective vaccines. Virus-like particles (VLPs) are a safe and powerful technology used in several human and veterinary vaccines; however, it is difficult to produce large amounts of BVDV VLPs. In this study, we generated red-spotted grouper nervous necrosis virus (RGNNV) VLPs presenting the BVDV E2 protein (domain I to IIIb) of the Nose (BVDV-1) or KZ-91-CP (BVDV-2) strain by exploiting SpyTag/SpyCatcher technology. Mice immunized twice with 30 µg of RGNNV VLPs conjugated with 10 µg of E2 proteins of the Nose or KZ-91-CP strain with a 14-day interval elicited high (1:512,000 to 1:1,024,000) and moderate (1:25,600 to 1:102,400) IgG titers against E2 proteins of homologous and heterologous strains, respectively. In addition, this prime-boost regimen induced strong (1:800 to 1:3,200) and weak (~1:10) neutralization titers against homologous and heterologous BVDV strains, respectively. Our results indicate that conjugation of the E2 protein to RGNNV VLPs strongly enhances the antigenicity of the E2 protein and that RGNNV VLPs presenting the E2 protein are promising BVDV vaccine candidates.

Usability of Immortalized Porcine Kidney Macrophage Cultures for the Isolation of ASFV without Affecting Virulence.

Immortalized porcine kidney macrophage (IPKM) cells are highly susceptible to major African swine fever virus (ASFV) isolates. To clarify the compatibility of this cell line for ASFV isolation from biomaterials, animal experiments and in vitro isolation were performed. Pork products seized at international airports were subjected to virus inoculation in pigs (in vivo) and IPKM cell cultures (in vitro) to examine the viability and virulence of the contaminating viruses. Moreover, the viruses isolated using IPKM cells were inoculated into pigs to assess the virulence shift from the original materials. All pigs that were inoculated with either homogenate samples of seized pork product or IPKM-isolated ASFVs developed typical symptoms of ASF and died (or were euthanized) within the term of the animal experiments. The success rate of virus isolation in IPKM cells was comparable to that observed in porcine primary alveolar macrophage (PAM) cells. The IPKM cell line would be an ideal tool for the isolation and propagation of live ASFVs with high efficiency and enhanced usability, such as immortal, proliferative, and adhesive properties. The isolated viruses retained biologically similar characteristics to those of the original ones during isolation in vitro.

Mutations in the tumor suppressor gene p53 in cattle are associated with enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is a B-cell lymphoma caused by the bovine leukemia virus (BLV). Although an association between EBL and mutations in the bovine tumor suppressor gene TP53 (bTP53) has been suggested, the substantive incidence rate of bTP53 mutations in EBL cattle is still unclear. In this study, we investigated the complete sequence (exons 2-11) of bTP53 in tissue and peripheral blood leukocyte (PBL) samples obtained from 154 EBL cattle and 117 cattle without EBL (non-EBL cattle) to elucidate the correlation between bTP53 mutations and EBL. The detection frequencies of non-synonymous (NS) and deletion mutations in bTP53 in EBL cattle were significantly higher than those in non-EBL cattle in both tissue and PBL samples (p < 0.05). Among these mutations in EBL cattle, 73.7 % (42/54) were homologous to those of human TP53 (hTP53), which were previously detected in various tumors. It has been reported that 95.2 % (40/42) of these hTP53 mutations induced complete or partial loss of the transactivating function of its encoding protein, P53. Moreover, the BLV proviral load in tissue samples was significantly higher in cattle harboring bTP53 NS and deletion mutations than in cattle without these mutations in both EBL and BLV-infected non-EBL cattle (p < 0.05). Although the activity of the mutant variants of bP53 must be further investigated, our findings revealed that bTP53 mutations are involved in tumorigenesis in BLV-infected cells and EBL-associated carcinogenesis.

An immortalized porcine macrophage cell line competent for the isolation of African swine fever virus.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a fatal hemorrhagic disease of domestic pigs and wild boar. The virus primarily infects macrophage and monocyte host cells, these do not grow in vitro. Many attempts have been made to establish sustainable ASFV-sensitive cell lines, but which supported only low viral replication levels of limited, mostly artificially attenuated strains of ASFV. Here, we examined the competence of a novel cell line of immortalized porcine kidney macrophages (IPKM) for ASFV infection. We demonstrated that IPKM cells can facilitate high levels (> 107.0 TCID50/mL) of viral replication of ASFV, and hemadsorption reactions and cytopathic effects were observed as with porcine alveolar macrophages when inoculated with virulent field isolates: Armenia07, Kenya05/Tk-1, and Espana75. These results suggested that IPKM may be a valuable tool for the isolation, replication, and genetic manipulation of ASFV in both basic and applied ASF research.

Simultaneous evaluation of diagnostic marker utility for enzootic bovine leukosis.

BACKGROUND: Enzootic bovine leukosis (EBL) is a disease of cattle caused by bovine leukemia virus (BLV). More than 60% of BLV-infected cattle remain subclinical and are thus referred to as aleukemic (AL) cattle. Approximately 30% of infected cattle show a relatively stable increase in the number of B lymphocytes; these cattle are termed persistent lymphocytosis (PL) cattle. A small percentage of infected cattle develop BLV-induced B cell lymphoma (EBL) and are called EBL cattle. Due to the increase in the number of BLV-infected cattle, the number of EBL cattle has featured a corresponding increase over recent years in Japan. Several diagnostic criteria for EBL (e.g., enlarged superficial lymph nodes, protrusion of the eye, increased peripheral blood lymphocyte, etc.) are used for on-farm diagnosis and antemortem tests at slaughterhouses. Since the slaughter of EBL cattle for human consumption is not allowed, on-farm detection of EBL cattle is important for reducing the economic loss incurred by farms. Therefore, establishing new diagnostic markers to improve the efficiency and accuracy of the antemortem detection of EBL cattle is a critical, unmet need. To simultaneously evaluate the utility of candidate markers, this study measured the values of each marker using the blood samples of 687 cattle with various clinical statuses of BLV infection (EBL, PL, AL and non-infected cattle). RESULTS: Sensitivity (Se) and specificity (Sp) were highest for the serum thymidine kinase (TK) followed by the serum lactate dehydrogenase (LDH) isozyme 2. The number of peripheral blood lymphocytes and proviral load in peripheral blood had the lowest Se and Sp. The values of all markers other than TK were influenced by the sex of the tested cattle. CONCLUSIONS: Although tLDH and its isozymes (LDHs) may be influenced by the sex of the tested cattle, the high accuracy of TK and LDH2 as well as accessibility and simplicity of the protocol used to measure these enzymes recommend the utility of TK and LDHs for EBL cattle detection. Using these markers for screening followed by the application of existing diagnostic criteria may improve the efficiency and accuracy of EBL cattle detection on farms, thereby contributing to the reduction of economic losses in farms.

The effectiveness of colostral antibodies for preventing bovine leukemia virus (BLV) infection in vitro.

BACKGROUND: Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). The incidence of EBL in Japan is increasing annually; and the cases of EBL in cattle younger than 2 years old has been reported. Therefore, it is vital to find a method to control BLV infection, especially in young calves. In this study, to evaluate the protective ability of colostral antibodies against BLV infection, as well as the potential for BLV infection mediated by colostrum/milk, we investigated temporal fluctuations in the anti-BLV antibody titer and BLV proviral load (PVL) in colostrum/milk and peripheral blood of six infected dams during lactation. The association between PVL and antibody titer in colostrum and peripheral blood was then investigated using samples from a further twenty-seven cattle. Antibody concentrations were measured with a Syncytium-induction Inhibition Assay using colostral/milk whey and serum. PVL in peripheral blood and colostrum was measured by real-time PCR. RESULTS: Colostral antibodies showed high inhibitory activity until day 3 of lactation. The antibody titer and PVL in peripheral blood showed lesser changes than those in colostrum/milk throughout lactation. The colostral antibody titer was significantly higher than the serum antibody titer in all samples, whereas the colostrum PVL was significantly lower than the blood PVL. The blood PVL showed a significant correlation with serum antibody titer, colostrum PVL, and colostral antibody titer. However, there were no major correlations between the serum and colostral antibody titers. CONCLUSIONS: This is the first report investigating the temporal changes in colostral antibody titer in terms of inhibiting BLV infection in vitro. The results of antibody detection by Syncytium-induction Inhibition Assay suggested that the protective activity of the colostral antibodies against BLV infection would be conferred by anti-BLV gp51 antibody. The high antibody titer of colostral whey suggests that colostral whey could be a potential source of antibodies with a low risk of infection in neonatal calves.

Reciprocal complementation of bovine parainfluenza virus type 3 lacking either the membrane or fusion gene.

Two defective bovine parainfluenza virus type 3 (BPIV3) strains were generated, one lacking the membrane (M) protein gene and expressing EGFP (ΔM-EGFP) and the other lacking the fusion (F) protein gene and expressing mStrawberry (ΔF-mSB), by supplying deficient proteins in trans. When Madin-Darby bovine kidney (MDBK) cells were co-infected with ΔM-EGFP and ΔF-mSB at a multiplicity of infection (MOI) of 0.1, complemented viruses were easily obtained. Complemented viruses grew as efficiently as wild-type BPIV3 and could be passaged in MDBK cell cultures even at an MOI of 0.01, possibly due to multiploid virus particles containing genomes of both ΔM-EGFP and ΔF-mSB. This reciprocal complementation method using two defective viruses would be useful to express large or multiple proteins in cell cultures using paramyxovirus vectors.

A single L288I substitution in the fusion protein of bovine parainfluenza virus type 3 enhances virus growth in semi-suitable cell lines.

The bovine parainfluenza virus type 3 BN-CE vaccine strain was obtained by serial passage of the BN-1 strain in chicken embryonic fibroblasts (CEF). We previously identified a substitution (L288I) in the fusion (F) protein between the two strains. To examine the effect of the substitution on CEF adaptation and attenuation, we generated a recombinant BN-1 strain with the L288I substitution in the F protein (FL288I-EGFP). FL288I-EGFP replicated more efficiently than a recombinant BN-1 strain (wt-EGFP) in semi-suitable cell lines, suggesting that the L288I substitution was established in the BN-1 strain during the process of adaptation in CEF.

The role of neighboring infected cattle in bovine leukemia virus transmission risk.

A cohort study was conducted to evaluate the risk of bovine leukemia virus (BLV) transmission to uninfected cattle by adjacent infected cattle in 6 dairy farms. Animals were initially tested in 2010-2011 using a commercial ELISA kit. Uninfected cattle were repeatedly tested every 4 to 6 months until fall of 2012. The Cox proportional hazard model with frailty showed that uninfected cattle neighboring to infected cattle (n=53) had a significant higher risk of seroconversion than those without any infected neighbors (n=81) (hazard ratio: 12.4, P=0.001), implying that neighboring infected cattle were a significant risk factor for BLV transmission. This finding provides scientific support for animal health authorities and farmers to segregate infected cattle on farms to prevent spread of BLV.

Nationwide survey of bovine leukemia virus infection among dairy and beef breeding cattle in Japan from 2009-2011.

A nationwide survey of bovine leukemia virus (BLV) infection was conducted among dairy and beef breeding cattle in Japan from 2009-2011 using an enzyme-linked immunosorbent assay. Of a total of 20,835 cattle tested, 35.2% were seropositive for BLV and the animal type-level seroprevalences in dairy and beef breeding cattle were 40.9 and 28.7%, respectively. By the time animals were 1 year old, 21.0% of dairy and 13.7% of beef breeding cattle were considered infected. Our findings indicate that BLV is widespread among dairy and beef breeding cattle in Japan with the BLV seroprevalences approximately 10- and 4-fold higher, respectively, than previously reported for 1980-1982 in Japan.

The recent prevalence of bovine leukemia virus (BLV) infection among Japanese cattle.

A seroepidemiological survey of bovine leukemia virus (BLV) infection was conducted in Japan in 2007 using an enzyme-linked immunosorbent assay (ELISA) and an agar gel immunodiffusion (AGID) test. A total of 5420 cattle (dairy, 3966; breeding beef, 797; fattening beef, 657) from 209 farms in seven prefectures in Japan were tested. The overall prevalence of BLV infection was 28.6%. The prevalence of BLV infection in dairy cattle (34.7%) was higher than for both fattening beef cattle (7.9%) and breeding beef cattle (16.3%). Age-specific prevalence showed that BLV prevalence increased with age in all types of cattle and was notably different between dairy and beef cattle under 1 year of age. Among 207 farms, 141 herds (68.1%) had one or more positive animals. The proportion of these positive farms was significantly higher among dairy farms (79.1%) than among beef breeding farms (39.5%) and beef fattening farms (51.9%) (P<0.001). Dairy farms (40.5%) also showed a significantly higher within-herd prevalence than beef breeding (27.4%) and fattening (14.9%) farms (P=0.001). This study indicated that BLV is more widely spread in dairy cattle than in beef breeding cattle in Japan. Given the prevalence of BLV infection in dairy and beef cattle was 8- and 1.7-fold higher, respectively, than rates previously found in 1980-1982, BLV appears to be spreading particularly among the dairy cattle population during the last two decades. Further investigation is required to determine the risk factors necessary to control BLV infection that take into account the different farming practices that exist between dairy and beef sectors.

Development of enzyme-linked immunosorbent assay for detection of antibody against Caprine arthritis encephalitis virus using recombinant protein of the precursor of the major core protein, p55gag.

An indirect enzyme-linked immunosorbent assay (ELISA) by using recombinant Caprine arthritis encephalitis virus (CAEV) p55gag antigen (rELISA), an indirect ELISA by using whole CAEV (wELISA), and Western blot analysis by using the recombinant p55gag antigen (rWB) were developed for detection of CAEV-specific antibodies in goats. Seven hundred and forty-five sera from goats were tested by rELISA, wELISA, rWB, and agar gel immunodiffusion test (AGID), and the results were compared with those of WB analysis by using the whole CAEV antigen (wWB). The AGID test and rWB had similar sensitivities of 93.3% (95% confidence interval [CI]) and 93% (95% CI), respectively, and similar specificities of 96.0% (95% CI) and 96.3% (95% CI), respectively, compared with wWB. The wELISA had substantially lower sensitivity (80.4%) and specificity (78.0%) compared with wWB, and rELISA had the lowest sensitivity (78.2%) and specificity (61.1%) compared with wWB. The lack of adequate sensitivity and specificity for rELISA and wELISA suggests that these assays need considerable modification. However, the results for rWB show that this assay has excellent agreement with wWB and that it can be used as a confirmatory test for the presence of anti-CAEV antibodies.

Estimation of the within-herd transmission parameter of bovine leukemia virus.

In this study, we estimated the transmission parameter of bovine leukemia virus (BLV) infection within herds by following up the serological status of cattle in beef and dairy herds. Two consecutive serological tests using ELISA were conducted for cattle at four beef breeding farms and nine dairy farms with an interval of 5 months. Assuming that sero-converted animals were newly infected, transmission parameters were estimated using a hierarchical Bayesian Poisson model. All tested herds had at least one positive animal at the first testing, but sero-prevalence considerably varied between herds. It is estimated that 0.62 animals were infected by one infected animal introduced into a fully susceptible population for both beef breeding and dairy farms during the 5-month interval. The 95% credible interval of the transmission parameter for the combined herds was 0.37-0.89 over this period. Asymptomatically infected animals with BLV can become the source of lifelong infection in herds. These estimated transmission parameters indicated that the early removal of infected animals with periodical tests could reduce the dissemination of BLV infection within herds.

Risk factors associated with within-herd transmission of bovine leukemia virus on dairy farms in Japan.

BACKGROUND: Although several attempts have been made to control enzootic bovine leukosis (EBL) at the local level, a nationwide control program has not been implemented in Japan, except for passive surveillance. Effective control of EBL requires that the transmission routes of bovine leukemia virus (BLV) infection should be identified and intercepted based on scientific evidence. In this cross-sectional study, we examined the risk factors associated with within-herd transmission of BLV on infected dairy farms in Japan. Blood samples taken from 30 randomly selected adult cows at each of 139 dairy farms were tested by enzyme-linked immunosorbent assay (ELISA). Information on herd management was collected using a structured questionnaire. RESULTS: Infected farms were defined as those with more than one ELISA-positive animal and accounted for 110 (79.1%) of the 139 farms in the study. Completed questionnaires obtained from 90 of these 110 farms were used for statistical analysis. Seroprevalence, which was defined as the proportions of animals that tested positive out of all animals tested on the farm, was 17.1%, 48.1%, and 68.5% for the 25th, 50th, and 75th percentiles, respectively. A mixed logistic regression analysis implicated a loose housing system, dehorning, and a large number of horseflies in summer as risk factors (coefficient = 0.71, 1.11, and 0.82; p = 0.03, < 0.01, and 0.01, respectively) and feeding of colostrum to newborn calves from their dams as a protective factor (coefficient = -1.11, p = 0.03) against within-farm transmission of BLV on infected farms. CONCLUSION: Control of EBL in infected dairy farms in Japan will be improved by focusing particularly on these risk and protective factors.