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.

Reemergence of Classical Swine Fever, Japan, 2018.

In September 2018, classical swine fever reemerged in Japan after 26 years, affecting domestic pigs and wild boars. The causative virus belongs to the 2.1 subgenotype, which caused repeated outbreaks in eastern and Southeast Asia. Intensive surveillance of swine and vaccination of wild boars will help control and eradicate this disease in Japan.

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.

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.

Development of a Novel Indirect ELISA for the Serological Diagnosis of African Swine Fever Using p11.5 Protein as a Target Antigen.

African swine fever is a hemorrhagic viral disease with a mortality rate of nearly 100% in pigs. Hence, it is classified as a notifiable disease by the World Organization for Animal Health. Because no field-available vaccine exists, African swine fever virus (ASFV) control and eradication solely depend on good farm biosecurity management and rapid and accurate diagnosis. In this study, we developed a new indirect serological enzyme-linked immunosorbent assay (ELISA) using recombinant p11.5 protein from ASFV as a solid-phase target antigen. The cutoffs were determined by receiver operating curve analysis performed with serum samples obtained from naïve and infected pigs. Based on the results of a commercially available serological ELISA, the relative sensitivity and specificity of our assay were 93.4% and 94.4% (N = 166; area under the curve = 0.991; 95% confidence interval = 0.982-0.999), respectively. Furthermore, to compare the performance of the serological ELISAs, we conducted the assays on a panel of sera collected from pigs and boars experimentally infected with different ASFV isolates. The results indicated the greater sensitivity of the newly developed assay and its ability to detect anti-ASFV antibodies earlier after virus inoculation.

A Spontaneously Occurring African Swine Fever Virus with 11 Gene Deletions Partially Protects Pigs Challenged with the Parental Strain.

African swine fever (ASF) is an infectious Suidae disease caused by the ASF virus (ASFV). Adaptation to less susceptible, non-target host cells is one of the most common techniques used to attenuate virulent viruses. However, this may induce many mutations and large-scale rearrangements in the viral genome, resulting in immunostimulatory potential loss of the virus in vivo. This study continuously maintained the virulent ASFV strain, Armenia2007 (Arm07), to establish an attenuated ASFV strain with minimum genetic alteration in a susceptible host cell line, immortalized porcine kidney macrophage (IPKM). A mutant strain was successfully isolated via repeated plaque purification in combination with next-generation sequencing analysis. The isolated strain, Arm07ΔMGF, which was obtained from a viral fluid at a passage level of 20, lacked 11 genes in total in the MGF300 and MGF360 regions and showed marked reduction in virulence against pigs. Moreover, all the pigs survived the challenge with the parental strain when pigs were immunized twice with 105 TCID50 of Arm07ΔMGF, although viremia and fever were not completely prevented after the challenge infection. These findings suggest that this naturally attenuated, spontaneously occurring ASFV strain may provide a novel platform for ASF vaccine development.

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.

Stability of recombinant bovine interferon-γ antiviral activity in the absence of stabilizing additives.

The stability of recombinant bovine interferon-γ (rbIFN-γ) produced by a baculovirus expression system was investigated under different storage conditions: freezing-thawing and storage for 30 days at temperatures of -80, 4, 25, and 37°C. Antiviral activity was not significantly decreased by freeze-thawing at least five times. Furthermore, although not statistically different, antiviral activity gradually decreased as temperature increased. These findings suggest that rbIFN-γ possesses high thermal and freeze-thaw stability.

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.

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact.

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD50)/ml, 101 HAD50/ml and 106 HAD50/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD50/ml and 101 HAD50/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD50/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 103 HAD50/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.

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.

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.

Non-cytopathic bovine viral diarrhea virus infection inhibits differentiation of bovine neural stem/progenitor cells into astrocytes in vitro.

Bovine viral diarrhea virus (BVDV) causes fetal brain malformations in ruminants when the fetuses are infected transplacentally in mid-pregnancy. In both cytopathic and non-cytopatic virus infections, viral lytic infection in actively replicating cells and interruption of vascular integrity have been suggested as the pathogenesis, but functional disturbance of infected neural developing cells has been unclear. In this study, we examined the effect of infection with non-cytopathic BVDV2 on the differentiation of neural stem/precursor cells isolated from the bovine fetus. In the process of differentiation to three types of neural cells, neurons, astrocytes and oligodendrocytes, virus infection significantly and selectively inhibited the differentiation of neural stem/precursor cells into the astrocytic lineage. This inhibition is possibly important for the pathogenesis of congenital brain malformations associated with non-cytopathic BVDV infection.

A cloned classical swine fever virus derived from the vaccine strain GPE- causes cytopathic effect in CPK-NS cells via type-I interferon-dependent necroptosis.

Classical swine fever viruses (CSFVs) do typically not show cytopathic effect (CPE) in cell culture, while some strains such as vaccine strain the GPE- induce CPE in the swine kidney-derived CPK-NS cell line cultured in serum-free medium. These latter strains commonly lack Npro-mediated inhibition of type-I interferon (IFN) induction. In order to explore the molecular mechanisms of GPE--induced CPE, we analyzed the cellular pathways involved. In CPK-NS cells infected with the attenuated-vaccine-derived vGPE- strain, both, apoptosis and necroptosis were induced. Necroptosis was type-I IFN-dependent and critical for visible CPE. In contrast, the parental virulent vALD-A76 strain did not induce any of these pathways nor CPE. We used reverse genetics to investigate which viral factors regulate these cell-death pathways. Interestingly, a mutant vGPE- in which the Npro function was restored to inhibit type-I IFN induction did not induce necroptosis nor CPE but still induced apoptosis, while an Npro-mutant vALD-A76 incapable of inhibiting type-I IFN production induced necroptosis and CPE. Although Erns of CSFV is reportedly involved in controlling apoptosis, apoptosis induction by vGPE- or apoptosis inhibition by vALD-A76 were independent of the unique amino acid difference found in Erns of these two strains. Altogether, these results demonstrate that type-I IFN-dependent necroptosis related to non-functional Npro is the main mechanism for CPE induction by vGPE-, and that viral factor(s) other than Erns may induce or inhibit apoptosis in vGPE- or vALD-A76 infected CPK-NS cells, respectively.

Genome Sequence of a Classical Swine Fever Virus of Subgenotype 2.1, Isolated from a Pig in Japan in 2018.

In 2018, classical swine fever virus (CSFV) was detected in Japan. Here, we report the whole-genome sequence of CSFV/JPN/1/2018. This virus is closely related to isolates in East Asia and is classified under subgenotype 2.1. This is the first detection of a CSFV of this lineage in Japan.

Experimental infection of pigs with a classical swine fever virus isolated in Japan for the first time in 26 years.

Following an outbreak of classical swine fever (CSF) in Japan, 2018, CSFV JPN/1/2018 was isolated from an infected pig sample. In this study, we carried out a comparative experimental infection in pigs using this strain and the highly virulent ALD strain and compared outcomes, including clinical manifestation, virus shedding patterns and antibody responses. Although pigs inoculated orally or intramuscularly with JPN/1/2018 developed hyperthermia and had decreased leucocyte numbers, they survived for the whole experimental period and showed less severe clinical signs than those infected with the ALD strain. We confirmed the presence of characteristic multifocal infarction of the margin of the spleen that arises following infection with JPN/1/2018, albeit that this finding was not observed in all infected pigs. Both viruses efficiently spread to contact pigs in a similar manner, suggesting in transmissibility between the two strains. Viral RNAs were detected in all clinical samples, especially whole blood samples, before the pigs developed hyperthermia until at least approximately 2 weeks after inoculation. Our findings will be valuable for the investigations into epidemic events occurring in Japan and for establishing diagnostic strategies and control measures against CSF.

END-phenomenon negative bovine viral diarrhea virus that induces the host's innate immune response supports propagation of BVDVs with different immunological properties.

Our previous study reported that persistently infected (PI) cattle of bovine viral diarrhea virus (BVDV) have co-infected with BVDV/END- and /END+ that promote and inhibit host's type-I interferon (IFN) production, respectively. However, the relationship between co-infection of immunologically distinct BVDVs and persistent infection as well as the biological significance of END- viruses remains unknown. Experiments using cultured cells revealed that END+ virus, which is unable to propagate in situations where the host's immune response is induced by IFN-α addition, is able to propagate under those conditions when co-infecting with END- virus. These results indicate that BVDV/END- can coexist with BVDV/END+ and that co-infection with END- viruses supports the propagation of END+ viruses. Our in vitro experiments strongly suggest that co-infection with END- virus is involved in the maintenance of persistent infection of BVDV.

Genetic and pathobiological characterization of bovine viral diarrhea viruses recently isolated from cattle in Japan.

The 475 strains of bovine viral diarrhea virus (BVDV) isolated from cattle in 12 prefectures of Japan in the last 7 years were phylogenetically classified as BVDV-1 or BVDV-2 on the basis of the nucleotide sequence of the 5'-untranslated region. BVDV-1 strains were further subtyped as 1a (101 strains), 1b (163), 1c (128), 1j (3), and So CP/75-like (1), and all of the 79 BVDV-2 strains belonged to subtype 2a. These 2a BVDVs contain two isolates that had high nucleotide identities with those of highly pathogenic BVDV-2 strains reported in North America (Pellerin et al., 1994). However, acute infection with severe mortality like North American outbreak was not observed and most of the present BVDV-2 strains were isolated from persistently infected (PI) cattle showing mild or no clinical sign. Moreover, it was revealed that 61.5% of the 39 PI cattle with cytopathogenic BVDVs did not show typical mucosal disease and 54.6% of the 405 PI animals only with non-cytopathogenic BVDVs were apparently healthy. The present results indicate that the prevention of the infection with an appropriate vaccine and active surveillance covering healthy cattle are required for the control of BVD.