Effects of host proteins interacting with non-structural protein nsp9 of porcine epidemic diarrhea virus on viral replication / 生物工程学报

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic virus that can cause acute intestinal infectious diseases in both piglets and fattening pigs. The virus encodes at least 16 non-structural proteins, including nsp9, which has been shown to bind to single-stranded RNA. However, its function and mechanism remain unclear. In this study, we aimed to identify potential host proteins that interact with PEDV nsp9 using immunoprecipitation combined with mass spectrometry. The interactions were then confirmed by co-immunoprecipitation (Co-IP) and confocal laser scanning fluorescence techniques. The results showed that nsp9 interacts with HSPA8, Tollip, HSPA9 and TOMM70. Among them, overexpression of HSPA8 resulted in caused first upregulated and then down-regulated expression of nsp9, and promoted the proliferation of PEDV. Overexpression of Tollip significantly upregulated the expression of nsp9 and inhibited the proliferation of PEDV. Overexpression of TOMM70 significantly reduced the expression of nsp9, but did not show significant effect on the proliferation of PEDV. Overexpression of HSPA9 did not show significant effect on the expression of nsp9 and the proliferation of PEDV. These findings may facilitate further investigating the role of nsp9-interacting proteins in PEDV infection.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis / 生物工程学报

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Preparation of bovine viral diarrhea disease virus 1 virus-like particles and evaluation of its immunogenicity in a guinea pig model / 生物工程学报

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-Erns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 106 TCID50 virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.

Development of an improved virus plaque assay based on avicel / 生物工程学报

Avicel is made of a mixture of microcrystalline cellulose (MCC) and carboxymethyl cellulose (CMC), and used for virus plaque assay. The avicel in common use is produced by FMC Biopolymer. Due to the relatively fixed proportion of MCC and CMC, avicel in common use is not suitable for plaque determination experiment of all types of viruses. In this study, we evaluated the effect of avicel made of different proportions of MCC and CMC on virus plaque assay, and developed an improved avicel virus plaque assay featured with simple and convenient operation, good practicability and high stability. To generate avicel overlays with different proportions of MCC and CMC, twelve different 2×avicel solutions were prepared. Their overall viscosity and bottom viscosity were measured to evaluate the ease of operation. The results showed that most of the 2×avicel solutions (except the 4.8% MCC+1.4% CMC and 4.8% MCC+1.0% CMC group) were easy to absorb and prepare nutrient overlap than 2×CMC solution. In order to find the best scheme to detect the titer of porcine epidemic diarrhea virus (PEDV), these avicel overlay solutions with different proportion of MCC and CMC were used as a replacement in the standard plaque assay. By comparing the size, clarity, stability and titer accuracy of virus plaque, we identified that 0.6% MCC and 0.7% CMC was the most preferable composition of avicel overlay for PEDV plaque assay. In conclusion, we developed an improved virus plaque assay based on avicel, which may facilitate the research of virus etiology, antiviral drugs and vaccines.

Immunization against porcine epidemic diarrhea virus and vaccine development / 生物工程学报

Porcine epidemic diarrhea (PED) is a major disease of pigs that inflicts heavy losses on the global pig industry. The etiologic agent is the porcine epidemic diarrhea virus (PEDV), which is assigned to the genus Alphacoronavirus in the family Coronaviridae. This review consists of five parts, the first of which provides a brief introduction to PEDV and its epidemiology. Part two outlines the passive immunity in new born piglets and the important role of colostrum, while the third part summarizes the characteristics of the immune systems of pregnant sows, discusses the concept of the "gut-mammary gland-secretory IgA(sIgA) axis" and the possible underpinning mechanisms, and proposes issues to be addressed when designing a PEDV live vaccine. The final two parts summarizes the advances in the R&D of PEDV vaccines and prospects future perspectives on prevention and control of PEDV, respectively.

Immunogenicity of engineered Lactobacillus plantarum expressing porcine epidemic diarrhea virus S1 gene / 生物工程学报

To investigate whether the engineered Lactobacillus plantarum expressing the porcine epidemic diarrhea virus (PEDV) S1 gene can protect animals against PEDV, guinea pigs were fed with recombinant L. plantarum containing plasmid PVE5523-S1, with a dose of 2×10⁸ CFU/piece, three times a day, at 14 days intervals. Guinea pigs fed with wild type L. plantarum and the engineered L. plantarum containing empty plasmid pVE5523 were used as negative controls. For positive control, another group of guinea pigs were injected with live vaccine for porcine epidemic diarrhea and porcine infectious gastroenteritis (HB08+ZJ08) by intramuscular injection, with a dose of 0.2 mL/piece, three times a day, at 14 days intervals. Blood samples were collected from the hearts of the four groups of guinea pigs at 0 d, 7 d, 14 d, 24 d, 31 d, 41 d and 48 d, respectively, and serum samples were isolated for antibody detection and neutralization test analysis by enzyme-linked immunosorbent assay (ELISA). The spleens of guinea pigs were also aseptically collected to perform spleen cells proliferation assay. The results showed that the engineered bacteria could stimulate the production of secretory antibody sIgA and specific neutralizing antibody, and stimulate the increase of IL-4 and IFN-γ, as well as the proliferation of spleen cells. These results indicated that the engineered L. plantarum containing PEDV S1 induced specific immunity toward PEDV in guinea pigs, which laid a foundation for subsequent oral vaccine development.

Development of a blocking ELISA based on a single-domain antibody target the S1 protein of porcine epidemic diarrhea virus / 生物工程学报

The aim of this study was to develop a blocking enzyme-linked immunosorbent assay (bELISA) based on a biotinylated nanobody target the S1 protein of porcine epidemic diarrhea virus (PEDV) for detecting the anti-PEDV antibodies and evaluating the immune effect of the vaccine. The gene encoding the single-domain antibody sdAb3 target the PEDV S1 protein was amplified and the Avitag sequence was fused at its 3'-end. The PCR product was cloned into the expression vector pET-21b for expression and purification of the sdAb3-Avitag protein. The purified sdAb3-Avitag fusion protein was biotinylated and its activity was determined. Using the recombinant S1 protein as a coating antigen, a bELISA was established and optimized. Serum samples were tested in parallel by the bELISA and a commercial kit. The recombinant vector pET21b-sdAb3-Avitag was constructed to express the tagged sdAb3. After induction for expression, the biotin-labeled sdAb3 (sdAb3-Biotin) with high purity and good activity was obtained. For the optimized bELISA, the coating concentration of the S1 protein was 200 ng/well, the serum dilution was 1:2 and incubated for 2 h, the dilution ratio of the biotinylated sdAb3 was 1:8 000 and incubated for 30 min, the dilution of the enzyme-labeled antibody was 1:5 000 and incubated for 30 min. The bELISA had no cross reaction with the sera of major porcine viruses including transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus and showed good specificity and reproducibility. For a total of 54 porcine serum samples tested, the overall compliance rate of the bELISA with a commercial kit was 92.56%. This study developed a rapid and reliable bELISA method, which can be used for serosurveillance and vaccine evaluation for PEDV.

Prevalence of bovines persistently infected with bovine viral diarrhea virus (BVDV) in dairy cattle herds in Paraná State, Brazil

ABSTRACT: This study aimed to establish the prevalence of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) in dairy farms at Parana State, Brazil. Samples were collected from 6,465 female Holstein Friesian Dairy Cattle, including animals less than two years old, females over two years old who had not given birth at the farm, and mothers of calves diagnosed as persistently infected. The cattle came from 40 dairy herds distributed in 10 municipalities in the State of Paraná. The samples were obtained from May 2015 to August 2018. The diagnosis of PI animals was made with an antigen-capture ELISA test. We detected PI animals in fifteen herds sampled (37.5%), ranging from one to sixteen animals per herd. The prevalence in Parana States municipalities was 1.78%, ranging from 0.3 to 8.9% at positive herds. The analysis of the individual herds shows significant dissemination of the BVDV in Paranas municipalities, including endemic areas. With this, we highlight the need for measures to raise awareness among producers about the existence and importance of bovine viral diarrhea (BVD) in dairy herds, reinforcing the PI animals role in disease epidemiology and the economic impact caused by the maintenance of these farm animals.

RESUMO: Com o intuito de se estabelecer a prevalência de animais persistentemente infectados (PI) com o BVDV em propriedades leiteiras no estado do Paraná. Foram coletadas amostras de 6.465 bovinos, fêmeas, da raça Holandês Preto e Branco (HPB). Amostraram-se animais com idade inferior a dois anos, fêmeas com mais de dois anos que não haviam tido partos na propriedade, e mães de bezerros que foram diagnosticados como persistentemente infectados. Os bovinos foram provenientes de 40 rebanhos leiteiros, distribuídos em 10 municípios no Estado do Paraná. A coleta deu-se no período de maio de 2015 a agosto de 2018. O diagnóstico dos animais PI foi feito por meio do teste de ELISA de captura de antígeno. Animais PI foram detectados em quinze rebanhos amostrais (37,5%), oscilando entre um e dezesseis animais por rebanho. A prevalência nos municípios do estado Paraná foi de 1,78%, oscilando entre 0,3 a 8,9% nos rebanhos positivos. Com a alta prevalência de animais PI observada, quando analisados os rebanhos amostrais individualmente, é possível afirmar que há uma disseminação importante do BVDV em municípios paranaenses, destacando inclusive áreas endêmicas. Com isso, vê-se a necessidade de medidas de conscientização dos produtores sobre a existência e importância da BVD nos rebanhos, destacando o papel dos animais PI na epidemiologia da doença, bem como o impacto econômico causado pela manutenção desses animais nos rebanhos.

Prevalence of bovines persistently infected with bovine viral diarrhea virus (BVDV) in dairy cattle herds in Paraná State, Brazil / Prevalência de bovinos persistentemente infectados com o vírus da diarreia viral bovina (BVDV) em rebanhos leiteiros no estado do Paraná, Brasil

This study aimed to establish the prevalence of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) in dairy farms at Parana State, Brazil. Samples were collected from 6,465 female Holstein Friesian Dairy Cattle, including animals less than two years old, females over two years old who had not given birth at the farm, and mothers of calves diagnosed as persistently infected. The cattle came from 40 dairy herds distributed in 10 municipalities in the State of Paraná. The samples were obtained from May 2015 to August 2018. The diagnosis of PI animals was made with an antigen-capture ELISA test. We detected PI animals in fifteen herds sampled (37.5%), ranging from one to sixteen animals per herd. The prevalence in Parana State's municipalities was 1.78%, ranging from 0.3 to 8.9% at positive herds. The analysis of the individual herds shows significant dissemination of the BVDV in Parana's municipalities, including endemic areas. With this, we highlight the need for measures to raise awareness among producers about the existence and importance of bovine viral diarrhea (BVD) in dairy herds, reinforcing the PI animals' role in disease epidemiology and the economic impact caused by the maintenance of these farm animals.(AU)

Com o intuito de se estabelecer a prevalência de animais persistentemente infectados (PI) com o BVDV em propriedades leiteiras no estado do Paraná. Foram coletadas amostras de 6.465 bovinos, fêmeas, da raça Holandês Preto e Branco (HPB). Amostraram-se animais com idade inferior a dois anos, fêmeas com mais de dois anos que não haviam tido partos na propriedade, e mães de bezerros que foram diagnosticados como persistentemente infectados. Os bovinos foram provenientes de 40 rebanhos leiteiros, distribuídos em 10 municípios no Estado do Paraná. A coleta deu-se no período de maio de 2015 a agosto de 2018. O diagnóstico dos animais PI foi feito por meio do teste de ELISA de captura de antígeno. Animais PI foram detectados em quinze rebanhos amostrais (37,5%), oscilando entre um e dezesseis animais por rebanho. A prevalência nos municípios do estado Paraná foi de 1,78%, oscilando entre 0,3 a 8,9% nos rebanhos positivos. Com a alta prevalência de animais PI observada, quando analisados os rebanhos amostrais individualmente, é possível afirmar que há uma disseminação importante do BVDV em municípios paranaenses, destacando inclusive áreas endêmicas. Com isso, vê-se a necessidade de medidas de conscientização dos produtores sobre a existência e importância da BVD nos rebanhos, destacando o papel dos animais PI na epidemiologia da doença, bem como o impacto econômico causado pela manutenção desses animais nos rebanhos.(AU)

Co-infection by Neopora caninum and bovine viral diarrhea virus in cattle from Rio Grande do Sul, Brazil, destined to exportation / Coinfecção por Neospora caninum e vírus da diarreia viral bovina em bovinos do Rio Grande do Sul, Brasil, destinados à exportação

Reproductive tests in cattle are of great economic importance, given the impact it can have on the production system and may be caused by agents. Neospora caninum and Bovine Viral Diarrhea virus (BVDV) are considered of great importance as reproductive and should be considered responsible for keeping animals persistently infected. The present study included 479 calf serum samples for export in the state of Rio Grande do Sul (RS). All samples were screened for BVDV by an ELISA antigen. BVDV antigen-positive ELISA samples were isolated from BVDV in cell culture. An indirect immunofluorescence (IFT) technique was used to detect anti-N. caninum antibodies. Of the 479 export-treated serum samples, 361 were positive for BVDV antigens by ELISA and/or viral isolation test (361/479-75.36%), and 109 IFT-positive samples for N. caninum (109/479-22.75%). Despite detection of antibodies anti-N. caninum did not differ statistically between naturally infected BVDV and non-BVDV infected animals suggesting that there is no interference of BVDV infection on infection or detection rate of animals with N. caninum, positive animals in viral isolation and high DO in BVDV-Ag ELISA. may present active disease and consequent immunosuppression, contributing to a potential reactivation of N. caninum.(AU)

Testes reprodutivos em bovinos são de grande importância econômica, dado o impacto que podem ter no sistema de produção e podem ser causados por agentes. O Neospora caninum e o vírus da Diarreia Viral Bovina (BVDV) são considerados de grande importância como reprodutivos e devem ser considerados responsáveis por manter os animais persistentemente infectados. O presente estudo incluiu 479 amostras de soro de bezerro para exportação no estado do Rio Grande do Sul (RS). Todas as amostras foram rastreadas para BVDV por um antígeno ELISA. As amostras de ELISA positivas para o antigénio BVDV foram isoladas a partir de BVDV em cultura de células. Uma técnica de imunofluorescência indireta (IFT) foi utilizada para detectar anticorpos anti-N caninum. Das 479 amostras de soro tratadas para exportação, 361 foram positivas para antígenos de BVDV por ELISA e/ou teste de isolamento viral (361/479-75,36%) e 109 amostras positivas para IFT para N. caninum (109/479-22,75%). Apesar da detecção de anticorpos anti-N. caninum não diferiu estatisticamente entre animais infectados naturalmente BVDV e não BVDV sugerindo que não há interferência da infecção pelo BVDV na infecção ou taxa de detecção de animais com N. caninum, animais positivos em isolamento viral e alta DO em BVDV-Ag ELISA, pode apresentar doença ativa e consequente imunossupressão, contribuindo para uma potencial reativação de N. caninum.(AU)

Pestivirus spillover effect: molecular detection of bovine viral diarrhea virus in domestic and feral pigs / Efeito spillover de pestivírus: detecção molecular do vírus da diarreia viral bovina em suínos domésticos e javalis

Pestivirus infections are important in the livestock industries, with infection occurring in cattle, sheep and pigs. The Pestivirus genus of the family Flaviviridae, includes four recognized species: bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2), border disease virus (BDV), and classical swine fever virus (CSFV). All pestivirus species can infect pigs, therefore accurate and specific pestivirus detection and differentiation is of great importance to assure control measures in swine populations. The aim of the study was the molecular detection of different pestiviruses in domestic and feral pigs. A total of 527 samples (92 pigs and 435 wild boars) were tested for pestiviruses detection using molecular assays. Eleven positive samples (6 wild boars and 5 domestic pigs) were identified using panpestivirus primers targeting the 5'- UTR region of the pestivirus RNA genome. Further all the positive samples were sequentially tested for detection of CSFV, BVDV-1 and BVDV-2 using specific primers. All RNAs were identified as positives for BVDV-1 and no amplification signals were obtained from BVDV-2 and CSFV. The current detection of BVDV-1 in clinical swine specimens highlights the important risk factor of swine population as reservoir and consequently carrier for BVDV.(AU)

As infecções por pestivírus são importantes nas indústrias pecuárias, com infecções em bovinos, ovinos e suínos. O gênero Pestivirus da família Flaviviridae inclui quatro espécies reconhecidas: vírus da diarreia viral bovina 1 (BVDV-1), vírus da diarreia viral bovina 2 (BVDV-2), vírus da doença de fronteira (VDF) e vírus da peste suína clássica (VPSC). Todas as espécies de pestivírus podem infectar porcos, portanto a detecção e diferenciação precisas e específicas de pestivírus são de grande importância para garantir medidas de controle nas populações suínas. O objetivo do estudo foi a detecção molecular de diferentes pestivírus em suínos domésticos e javali. Um total de 527 amostras (92 porcos e 435 javalis) foram testados para detecção de pestivírus usando ensaios moleculares. Onze amostras positivas (6 javalis e 5 porcos domésticos) foram identificadas usando iniciadores de panpestivírus visando a região 5'-UTR do genoma do RNA do pestivírus. Além disso, todas as amostras positivas foram testadas sequencialmente para detecção de VPSC, BVDV-1 e BVDV-2 usando iniciadores específicos. Todos os RNAs foram identificados como positivos para BVDV-1 e nenhum sinal de amplificação foi obtido do BVDV-2 e CSFV. A detecção atual do BVDV-1 em amostras clínicas de suínos destaca o importante fator de risco da população suína como reservatório e consequentemente portador do BVDV.(AU)

The 40-91 aa sequence of porcine epidemic diarrhea virus ORF3 protein is the key structural domain controlling its location in cytoplasm / 生物工程学报

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.

Serological response against bovine herpesvirus and bovine viral diarrhea virus induced by commercial vaccines in Holstein heifers / Resposta sorológica contra herpesvírus bovino e vírus da diarreia viral bovina induzida por vacinas comerciais em novilhas Holandesas

Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%). For BVDV-1 Singer, it was also possible to detect Abs production in G1 (log2=5.8, 100% seroconversion rate) and G3 (log2=3.5, seroconversion rate = 60%), only after the booster dose (D42). Neutralizing Abs to BVDV-2 (SV253) were detected only in G3, observing 90% seroconversion associated with high titers of Abs (log2=6.7) after the 2nd dose of vaccine (D42). Heifers from G1 and G3 responded to BoHV-1 after the first dose (D21): G1 (log2=2.5, seroconversion rate = 67%) and G3 (log2=0.7, seroconversion rate = 80%). In D42, a higher magnitude response was observed in the heifers from G3 (log2=6.1, 100%) compared with G1 (log2=4.3, 100%) and G2 (log2=2.7, 60%). Based on the data obtained, it can be concluded that the commercial vaccine contained aluminum hydroxide (G1) was most effective in the induction of antibodies against BVDV-1. On the other hand, this vaccine did not induce the production of neutralizing Abs against BVDV-2. Only the heifers from G3 (Quil A, amphigen and cholesterol) generated neutralizing Abs against BVDV-2. The animals that received commercial vaccine containing oil emulsion as adjuvant (G2) had a weak/undetectable response against BVDV-1 and BVDV-2. The best protective response against BoHV-1 was observed in heifers vaccinated with the live modified thermosensitive virus.(AU)

A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%). Para o BVDV-1 Singer, somente após D42 foi observada a produção de Acs no G1 (log2=5,8; taxa de soroconversão de 100%) e G3 (log2=3,5; taxa de soroconversão = 60%). Os anticorpos contra BVDV-2 (SV253) foram detectados apenas nas novilhas do G3, observando-se taxa de soroconversão de 90% com altos títulos de anticorpos neutralizantes (log2=6,7) em D42. Novilhas G1 e G3 responderam ao BoHV-1 após a primeira dose (D21): G1 (log2=2,5; taxa de seroconversão = 67%) e G3 (log2=0,7; taxa de seroconversão = 80%). Em contrapartida, foi observada uma maior magnitude de resposta para as novilhas G3 (log2=6,1; 100%) em D42, em relação aos animais G1 (log2=4,3; 100%) e G2 (log2=2,7; 60%). Com base nos dados obtidos, foi possível concluir que a vacina composta por hidróxido de alumínio (G1) foi mais eficaz na produção de anticorpos contra o BVDV-1, em contrapartida esse produto não induziu anticorpos contra o BVDV-2. Apenas as novilhas do G3 (Quil A, amphigen e colesterol) geraram Acs neutralizantes contra o BVDV-2. Os animais que receberam a vacina em emulsão oleosa (G2) como adjuvante apresentaram uma resposta fraca/indetectável contra o BVDV-1 e BVDV-2. A melhor resposta protetiva contra o BoHV-1 foi observada nas novilhas vacinadas com a vacina viva modificada termosensível.(AU)

Generation and protective efficacy of a cold-adapted attenuated genotype 2b porcine epidemic diarrhea virus

The recent emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. In this study, we generated a cold-adapted live attenuated vaccine candidate (Aram-P29-CA) by short-term passage of a virulent PEDV isolate at successively lower temperatures in Vero cells. Whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. Animal inoculation experiments confirmed the attenuated phenotype of Aram-P29-CA virus in the natural host. Pregnant sows were orally administered P29-CA live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. The oral homologous prime-boost vaccination of P29-CA significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and PEDV fecal shedding after the challenge. Furthermore, strong antibody responses to PEDV were detected in the sera and colostrum of immunized sows and in the sera of their offspring. These results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against PEDV.

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus

ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).

Primera evidencia del Virus de Diarrea Viral Bovina (VDVB) genotipo 2 en Colombia / First evidence of Bovine Viral Diarrhea Virus (BVDV) genotype 2 in Colombia

RESUMEN El Virus de la Diarrea Viral Bovina (VDVB) es un patógeno que afecta los hatos bovinos. El virus ha sido clasificado en dos biotipos (citopático y no citopático) y en 3 genotipos (1, 2 y 3) según su secuencia nucleotídica. El propósito de este estudio fue determinar la presencia del VDVB genotipo 2 (VDVB-2) en Colombia, mediante el estudio de cuatro zonas representativas de producción ganadera por medio de RT-PCR en muestras de suero y cartílago de oreja. Para ello, se recolectaron los sueros preparto de 379 vacas, de 274 terneros precalostrales (antes de que se les diera calostro), y de 145 terneros de 25 días post-nacimiento. Adicionalmente, se tomaron 181 biopsias de cartílago de oreja de estos terneros. Se realizó RT-PCR a todas las muestras para determinar la presencia o ausencia del VDVB. Aquellas muestras que resultaron positivas se evaluaron adicionalmente mediante dos métodos para determinar su genotipo: a) una nueva RT-PCR con primers específicos para el VDVB-2, y b) una PCR diferente con la que se obtuvo un producto de 296 pb, el cual se sometió a digestión enzimática. Los resultados mostraron que 17 (4,48%) muestras de suero preparto fueron positivas para Pestivirus, de las cuales 6 correspondieron al VDVB-2 (1,58%). Ninguna de las muestras de suero obtenidas de los terneros resultó positiva para el VDVB-2. Finalmente, 18 (9,9%) biopsias de cartílago de oreja fueron positivas al VDVB, 14 (7,7%) de las cuales resultaron positivas para el VDVB-2. El presente estudio es la primera evidencia documentada de la presencia del VDVB-2 en bovinos de Colombia.

ABSTRACT The bovine viral diarrhea virus (BVDV) is a pathogen that affects cattle. The virus has been classified into two biotypes (cytopathic and non-cytopathic) and three genotypes (1, 2 or 3) according to their nucleotide sequence. The objective of this study was to determine the presence of the BVDV genotype 2 (BVDV-2) in Colombia, through the study of four representative areas of cattle production by means of RT-PCR conducted on serum and ear notches. For this purpose, sera were collected from 379 prepartum cows, 274 calves born to these cows (before they were given colostrum), and 145 25-day- old calves. Additionally, 181 ear notches were taken from these calves. RT-PCR was performed on all samples to determine the presence or absence of BVDV. Tha samples that tested positive were further assessed by two methods to determine their genotype: a) a new RT-PCR with specific primers for BVDV-2, and b) a different PCR obtaining a product of 296 bp, which was further subjected to enzymatic digestion. The results showed that 17 (4.48%) prepartum sera samples were positive for Pestiviruses, from which 6 corresponded to BVDV-2 (1.58%). None of the sera obtained from the calves were positive for BVDV-2. Finally, 18 (9.9%) ear notches were positive for BVDV, 14 (7.7%) of which were positive for BVDV-2. The present study is the first documented evidence of the presence of the BVDV-2 in cattle from Colombia.

Prevalence of coronavirus from diarrheic calves in the Republic of Korea

Objective: To investigate the prevalence of bovine coronavirus (BCoV), bovine rotavirus, and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and October of 2016 in the Republic of Korea. Methods: Samples were obtained from 50 normal and 93 diarrheic (56 semi-formed, 28 loose, and 9 watery feces) calves in six different regions of northern and southern Korea. These fecal samples were tested for BCoV, bovine rotavirus, and bovine viral diarrhea virus by RT-PCR. Results: Among the three pathogens examined, infection with BCoV was especially prominent in relation to diarrhea among calves aged 1-21 days [odds ratio (OR)=9.3, 95% confidence interval (CI): 1.1-78.9; P=0.02). Infection with BCoV alone (OR=2.9; 95% CI: 1.1-7.6; P=0.03) or co-infection of BCoV with bovine viral diarrhea virus (OR=3.6; 95% CI: 1.0-12.4; P=0.04) was significantly associated with the development of loose feces. Grazing and colostrum intake strongly reduced the occurrence of diarrhea as compared to housed calves (OR=0.2; 95% CI: 0.1-0.4; P=0.00) and calves that had not been fed colostrum (OR=0.2; 95% CI: 0.1-0.7; P=0.02), respectively. Conclusions: The present study suggests that BCoV is involved in calf diarrhea in the Republic of Korea. Therefore, grazing and colostrum intake is recommended for preventing and controlling calf diarrhea caused by BCoV.

Prevalence of coronavirus from diarrheic calves in the Republic of Korea

Objective:To investigate the prevalence of bovine coronavirus (BCoV),bovine rotavirus,and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and October of 2016 in the Republic of Korea.Methods:Samples were obtained from 50 normal and 93 diarrheic (56 semi-formed,28 loose,and 9 watery feces)calves in six different regions of northern and southern Korea.These fecal samples were tested for BCoV,bovine rotavirus,and bovine viral diarrhea virus by RT-PCR.Results:Among the three pathogens examined,infection with BCoV was especially prominent in relation to diarrhea among calves aged 1-21 days [odds ratio (OR)=9.3,95％ confidence interval (CI):1.1-78.9;P=0.02).Infection with BCoV alone (OR=2.9;95％ CI:1.1-7.6;P=0.03) or coinfection of BCoV with bovine viral diarrhea virus (OR=3.6;95％ CI:1.0-12.4;P=0.04) was significantly associated with the development of loose feces.Grazing and colostrum intake strongly reduced the occurrence of diarrhea as compared to housed calves (OR=0.2;95％ CI:0.1-0.4;P=0.00) and calves that had not been fed colostrum (OR=0.2;95％ CI:0.1-0.7;P=0.02),respectively.Conclusions:The present study suggests that BCoV is involved in calf diarrhea in the Republic of Korea.Therefore,grazing and colostrum intake is recommended for preventing and controlling calf diarrhea caused by BCoV.

N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Porcine epidemic diarrhea virus (PEDV) is a contagious coronavirus infecting pigs that leads to significant economic losses in the swine industry. Given that PEDV infection occurs in gut epithelial cells mainly via the fecal-oral route, induction of PEDV-specific immune responses in the mucosal compartment is required for protective immunity against viral infection. However, an effective mucosal vaccine against the currently prevalent PEDV strain is not available. In this study, we demonstrated that the N-terminal domain (NTD) of the spike (S) protein of PEDV represents a new vaccine candidate molecule to be applied via the mucosal route. We first established an Escherichia coli expression system producing the partial NTD (NTD231–501) of the PEDV S protein. Orally administered NTD231–501 protein specifically interacted with the apical area of M cells in the follicle-associated epithelium of Peyer's patch. Additionally, the NTD protein induced antigen-specific immune responses in both the systemic and mucosal immune compartments when administered orally. Collectively, we propose the NTD of the PEDV S protein to be a candidate mucosal vaccine molecule.

Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs

PURPOSE: The first aim of this study was to develop a novel inactivated porcine epidemic diarrhea virus (PEDV) vaccine using the recently isolated Korean PEDV QIAP1401 strain and to evaluate its protective efficacy in growing pigs. The second was to determine the optimum adjuvant formulation of the inactivated PEDV vaccine that induces protection against viral challenge. MATERIALS AND METHODS: To generate high titers of infectious PEDV, the QIAP1401 isolate was passaged in Vero cells. The experimental vaccines were prepared from a binary ethyleneimine-inactivated QIAP1401 strain passaged sequentially 70 times (QIAP1401-p70), formulated with four commercial adjuvants, and administered twice intramuscularly to growing pigs. Challenge studies using a virulent homologous strain of PEDV QIAP1401-p11, which was passaged 11 times after isolation, were performed to assess protection against disease progression and viral shedding during the 15-day observation period. The vaccine-induced antibody responses were measured in serum samples collected at predetermined time points by indirect enzyme-linked immunosorbent assay and virus neutralization test. RESULTS: The QIAP1401-p70 strain had 42 amino acid (aa) mutations, including a 25 aa deletion, and was selected as the inactivated PEDV vaccine candidate. Although none of the pigs that received the experimental vaccines were completely protected against subsequent viral challenge, they exhibited a significantly higher immune response than did non-vaccinated control pigs. Among the vaccine groups, the highest antibody responses were observed in the pigs that received an oil-based multiphasic water/oil/water (W/O/W) emulsion adjuvanted vaccine, which delayed the onset of clinical symptoms and viral shedding. CONCLUSION: A novel inactivated PEDV vaccine formulated with a W/O/W emulsion adjuvant was both immunogenic and protective against viral challenge.