Bungowannah Pestivirus Chimeras as Novel Double Marker Vaccine Strategy against Bovine Viral Diarrhea Virus

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are beneficial tools for the eradication of animal diseases in regions with a high prevalence of the designated disease. Bovine viral diarrhea virus (BVDV)-1 (syn. Pestivirus A) is a flavivirus that infects predominantly cattle resulting in major economic losses. An increasing number of countries have implemented BVDV eradication programs that focus on the detection and removal of persistently infected cattle. No efficient marker or DIVA vaccine is yet commercially available to drive the eradication success, to prevent fetal infection and to allow serological monitoring of the BVDV status in vaccinated farms. Bungowannah virus (BuPV, species Pestivirus F), a related member of the genus Pestivirus with a restricted prevalence to a single pig farm complex in Australia, was chosen as the genetic backbone for a marker vaccine candidate. The glycoproteins E1 and E2 of BuPV were substituted by the heterologous E1 and E2, which are major immunogens, of the BVDV-1 strain CP7. In addition, the candidate vaccine was further attenuated by the introduction of a deletion within the Npro protein coding sequence, a major type I interferon inhibitor. Immunization of cattle with the chimeric vaccine virus BuPV_ΔNpro_E1E2 CP7 (modified live or inactivated) followed by a subsequent experimental challenge infection confirmed the safety of the prototype strain and provided a high level of clinical protection against BVDV-1. The serological discrimination of vaccinated cattle could be enabled by the combined detection of BVDV-1 E2- in the absence of both BVDV NS3- and BVDV Erns-specific antibodies. The study demonstrates for the first time the generation and application of an efficient BVDV-1 modified double marker vaccine candidate that is based on the genetic background of BuPV accompanied by commercially available serological marker ELISA systems.

Kesesuaian Uji Antigen Capture Enzyme Linked Immunosorbant Assay dan Nested Multiplex Polymerase Chain Reaction untuk Diagnosis Rutin Bovine Viral Diarrhea

Bovine Viral Diarrhea (BVD) is one of the main causes of impaired productivity and reproduction of cows. Antigen capture Elisa (ACE) is one of the serological technique that is sensitive, reliable and used regularly for detecting persistent BVD infection individually which simpler than multiplex nested PCR. The aim of this study was to determine the agreement between ACE and multiplex nested PCR as a routine laboratory diagnostic technique to detect the presence of BVD infection. A total of 128 cow serum samples consisting of 63 positive and 65 negative samples based on ACE were used in this study. The samples were collected from active and passive surveillance in dairy and beef cattle conducted by Balai Besar Veteriner (BBVet) Wates. The serum samples were then tested molecularly using multiplex nested PCR against BVD. The result showed 48 out of 63 BVDV-1 positive samples were found positive BVD antigen whereas 57 of 65 BVDV-1 negative samples were negative using multiplex nested PCR, . The agreement value between the two different assays based on statistic analysis using Kappa method was 0.64 and classified a good one. The result concluded that the ACE BVD assay was equally suitable as routine diagnosis to determine BVD infected cattle in the farm. Keywords: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR. Abstrak Bovine Viral Diarrhea (BVD) merupakan salah satu penyebab gangguan produktivitas dan reproduksi sapi. Antigen capture ELISA (ACE) merupakan salah satu teknik serologis yang sensitif, dapat diandalkan dan digunakan secara teratur untuk mendeteksi infeksi BVD persisten secara individual yang lebih sederhana daripada multiplex nested PCR. Tujuan penelitian ini adalah untuk mengetahui kesesuaian antara uji ACE dan multiplex nested PCR sebagai teknik diagnostik laboratorium rutin untuk mendeteksi adanya infeksi BVD. Sebanyak 128 sampel serum sapi yang terdiri dari 63 sampel positif dan 65 negatif berdasarkan ACE BVDV Antigen Test Kit/Serum Plus (Idexx®) digunakan dalam kajian ini. Sampel serum sapi merupakan koleksi dari surveilans aktif dan pasif pada sapi perah dan potong yang dilakukan Balai Besar Veteriner (BBVet) Wates. Sampel serum kemudian diuji secara molekuler menggunakan multiplex nested PCR terhadap BVD. Hasil penelitian menunjukkan bahwa dengan teknik multiplex nested PCR, 48 dari 63 sampel positif BVDV-1 ditemukan positif untuk antigen BVD sedangkan 57 dari 65 sampel negatif BVDV-1 negatif untuk antigen BVD. Analisis statitik berdasarkan perhitungan metoda Kappa menunjukkan nilai kesesuaian antara dua uji sebesar 0,64 dan tergolong bagus. Hasil penelitian menunjukkan kesimpulan bahwa uji ACE BVD sesuai sebagai diagnosis rutin untuk menentukan ternak yang terinfeksi BVD di peternakan. Kata kunci: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR.

Co-Administration of a Plasmid Encoding CD40 or CD63 Enhances the Immune Responses to a DNA Vaccine Against Bovine Viral Diarrhea Virus in a Mouse Model

Bovine viral diarrhea virus (BVDV) causes substantial economic losses in the livestock industry worldwide. Plasmids encoding the BVDV E2 protein are potential DNA vaccines against BVDV, but their immunogenicity has been insufficient. Here, we investigated the adjuvant effect of CD40 and CD63 on the immune responses to a BVDV E2 DNA vaccine in a mouse model. We constructed pUMVC4a-based plasmids encoding the BVDV E2 protein (pE2), mouse CD40 (pCD40), or mouse CD63 (pCD63). Protein expression by each plasmid was confirmed through Western blot analysis and immunofluorescence staining of cultured cell lines. BALB/c mice were immunized intradermally twice with pE2 in combination with, or without, pCD40 or pCD63, with 3 weeks between the two doses. pE2 with pCD40 induced significantly higher neutralizing antibody titers against BVDV than pE2 alone. Furthermore, pE2 with pCD40 or pCD63 induced significantly increased lymphocyte proliferation and IFN-γ production in response to BVDV ex vivo, compared with E2 alone. These results suggest that a plasmid encoding CD40 or CD63 can be used as an adjuvant to enhance immune responses to DNA vaccines against BVDV.

Antiviral Effect of Cerium Dioxide Nanoparticles on the Model of the Causative Agent of Bovine Viral Diarrhea

Cerium dioxide nanoparticles (CeO2) have a wide range of biological properties, including antiviral activity. Preparations based on cerium oxide nanoparticles can be effective against animal RNA viruses, which have the greatest epizootic significance for Ukraine and potentially against human viruses, including SARS-CoV-2. In our studies, we determined the effect of cerium dioxide nanoparticles on bovine viral diarrhea virus (BVDV), a pestivirus that is the etiological agent of bovine viral diarrhea. To determine the antiviral efficacy of CeO2, a cytopathogenic strain of BVDV “BK-1” was used as an etiological agent of bovine viral diarrhea, which induces a cytopathic effect on cow embryo lungs (CEL) cell culture. When evaluating the antiviral efficacy of CeO2, it was determined that the MPC for CEL cell culture is 0.057 μg/cm3, and the CC50 is 0.077 μg/cm3. The antiviral activity of CeO2 was evaluated by the IC50 index, which was 0.036 μg/cm3 and the selectivity index, which after co-administration of CeO2 with BVDV was 2.14. There was a decrease in the titer of infectious activity of the virus during treatment with CeO2 by 2.09 lg TCD50/cm3.

Modeling the Effect of Bovine Viral Diarrhea Virus in Australian Beef Herds

Bovine viral diarrhea virus (BVDV) is an economically important disease in Australian beef farming. The disease typically results in low-level production losses that can be difficult to detect for several years. Simulation modeling can be used to support the decision to control BVDV; however, current BVDV simulation models do not adequately reflect the extensive farming environment of Australian beef production. Therefore, the objective of this study was to develop a disease simulation model to explore the impact of BVDV on beef cattle production in south-east Australia. A dynamic, individual-based, stochastic, discrete-time simulation model was created to simulate within-herd transmission of BVDV in a seasonal, self-replacing beef herd. We used the model to simulate the effect of herd size and BVDV introduction time on disease transmission and assessed the short- and long-term impact of BVDV on production outputs that influence the economic performance of beef farms. We found that BVDV can become established in a herd after a single PI introduction in 60% of cases, most frequently associated with the breeding period. The initial impact of BVDV will be more severe in smaller herds, although self-elimination is more likely in small herds than in larger herds, in which there is a 23% chance that the virus can persist for >15 years following a single incursion in a herd with 800 breeders. The number and weight of steers sold was reduced in the presence of BVDV and the results demonstrated that repeat incursions exacerbate long-term production losses, even when annual losses appear marginal. This model reflects the short- and long-term production losses attributed to BVDV in beef herds in southeast Australia and provides a foundation from which the influence and economic utility of BVDV prevention in Australian beef herds can be assessed.

Antiviral Effect of Ginsenoside Rb2 and Rb3 Against Bovine Viral Diarrhea Virus and Classical Swine Fever Virus in vitro

Bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) are members of the genus Pestivirus that cause disease in wild and domestic animals and are responsible for extensive economic losses of livestock and biological industry. BVDV is also a significant laboratory contaminant. Currently, no effective antiviral therapeutics are available to control their infection. Ginsenosides, as major pharmacological ingredients in the plants of ginseng, have various biological activities. In the present work, the antiviral activity of 9 ginsenosides and 3 other saponins from Araliaceae plants was investigated against Pestivirus. Ginsenoside Rb2 and Rb3 showed low cytotoxicity and obvious antiviral effect. They were able to inhibit the replication and proliferation of BVDV and CSFV. In addition, our results suggest that the possible antiviral mechanism of Rb2 might be related to its ability to affect the translation of these viruses. Obtained results suggest that ginsenoside Rb2 and Rb3 have a potential for effective treatment against Pestivirus infection.

An Importance of Long-Term Clinical Analysis to Accurately Diagnose Calves Persistently and Acutely Infected by Bovine Viral Diarrhea Virus 2

Bovine viral diarrhea virus (BVDV) infection results in a wide variety of clinical manifestations and is a pathogen that is able to cause huge economic losses in the cattle industry worldwide. It is important to identify cattle that are persistently infected (PI) by BVDV within the herd as early as possible because PI animals are the main reservoir of the virus. In contrast, cattle who are acutely infected (AI) with BVDV show various clinical signs, but most cattle show either mild symptoms or are asymptomatic. In general, AI and PI animals can be distinguished by repeat testing within an interval of at least 21 days. However, we found a rare case of a BVDV2-infected AI animal with long-term viral presence, making it indistinguishable from PI through two tests within an interval of 21 days. As a result, we diagnosed one infected animal as AI after 35 days from the initial sample collection via multiple analyses. Our findings recommend performing an additional test using samples that have been collected after 14–21 days from the second sample collection in cases where it is difficult to accurately differentiate an AI diagnosis from a PI diagnosis after only two tests. Additionally, our analysis exhibits that monitoring the number of copies of viruses with similar genomes in the sera by means of quantitative real-time RT-PCR through several sample collections periods might be useful to distinguish AI from PI. Furthermore, our data suggest that the AI animals with a long-term viral presence who show test results similar to those of PI animals might be the result of a coincidental combination of various factors that are present in cattle fields. These findings provide useful information that can be used to improve the diagnosis of BVDV in the field.

Th17 cell differentiation induced by cytopathogenic biotype BVDV-2 in bovine PBLCs

Background Bovine viral diarrhea virus (BVDV) is a major pathogen that causes bovine viral diarrhea/mucosal disease (BVD-MD), which has become a global infectious disease due to its wide spread and the lack of effective treatment. The process of BVDV infection is complex. Once infected, host immune cells are activated and modulated. As a major immune cell, peripheral blood lymphocyte cells (PBLCs) are the primary target of BVDV. In order to further understand the mechanism of BVDV- host interaction, the expression profiles of host lymphocytes mRNAs associated with BVDV infection were investigated by transcriptomic sequencing analysis. Results The transcriptomic sequencing analysis was performed on bovine PBLCs infected with CP BVDV-2 GS2018 after 12 h of infection. Gene expression profiling demonstrated that 1052 genes were differentially expressed in GS2018 infected PBLCs compared with the control group. Of these genes, 485 genes were up-regulated and 567 were down-regulated. The 19 differential expressed genes (DEGs) were selected for validation using quantitative real-time PCR and the results were consistent with the results of RNA-Seq. Gene ontology enrichment and KEGG pathway analysis showed that 1052 DEGs were significantly enriched in 16 pathways, including cytokine-cytokine receptor interaction, IL17, PI3K-Akt, MAPK and TNF signaling pathway. PPI network analysis showed that IL17A, IFN-γ and TNF-α interacted with various proteins and may play crucial roles in BVDV-2 infection. Of note, we confirmed that GS2018 induced Th17 cell differentiation in PBLCs and persistently increased the expression levels of IL17A. In turn, the replication of GS2018 was inhibited by IL17A. Conclusion In this study, the transcription changes of DEGs related to host immune responses in bovine PBLCs were caused by CP BVDV-2 infection. In particular, the effector molecules IL17A of Th17 cells were significantly up-regulated, which inhibited viral replication. These results will contribute to exploration and further understanding of the host immune response mechanism and interaction between host and BVDV-2.

Inactivation of Enveloped Bovine Viral Diarrhea Virus and Non-Enveloped Porcine Parvovirus Using Low-Pressure Non-Thermal Plasma

As the worldwide population has been experiencing since 2020, viruses represent a serious threat to global well-being. To avoid viral transmission through surgery or medical examination, sterilization of medical material is needed. From emerging sterilization processes, the use of non-thermal plasma (NTP) arises as a promising technique to efficiently reduce microbial burden on medical devices, including new complex polymers as thermosensitive ones. Thus, we evaluated the antiviral efficacy of a low-pressure NTP process taking place in a sealed bag. For this purpose, two different plasmas, O2 100% plasma and Ar 80%–O2 20% plasma, were tested against two viruses: the bovine viral diarrhea virus and the porcine parvovirus, surrogates of human hepatitis C virus and human parvovirus B19, respectively. The efficacy of both NTP treatments on viral load can be detected after only five minutes. Moreover, the longer the NTP treatments last, the more the load decreases. The most effective load reduction was obtained with a 120-min O2 plasma treatment inducing a minimum of four-log viral load reduction. So, this process demonstrated strong virucidal capacity inside a sealed bag and represents a very interesting opportunity in the field of fragile medical devices sterilization or disinfection.