Improvement of bovine pestiviral diagnosis by the development of a cost-effective method for detecting viral RNA in fresh specimens and samples spotted in filter papers / Optimización del diagnóstico de Pestivirus bovinos mediante el desarrollo de una metodología económica para detectar ARN viral en muestras frescas y fijadas en papeles de filtro

Abstract Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease thatcauses severe economic losses in cattle. The aim of this study was to improve their diagnosisby developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocolfor collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developedRT-qPCR showed high sensitivity in detecting these viruses in different matrices: viral stocks,semen and serum samples. With regard to the possibility of using the technique to test serumpools, it was possible to identify a positive serum sample within a pool containing 30 sera.In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovinesamples was analyzed. The sampling method on two different filter papers using bovine blooddrops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNAfor up to 12 months under refrigeration.

Resumen Los Pestivirus bovinos son los agentes causales de la diarrea viral bovina, una enfermedad que genera importantes pérdidas económicas en el ganado vacuno. El objetivo de este trabajo fue mejorar su diagnóstico mediante el desarrollo de una RT-qPCR para detectar los Pestivirus bovinos A, B y H y disenar un protocolo de recolección, envío y conservación de ARN viral en papeles de filtro. La RT-qPCR desarrollada demostró alta sensibilidad en la detección de estos virus en diferentes matrices: stock viral, suero y semen. Respecto de la posibilidad de usar la técnica para testear pools de suero, fue posible identificar un suero positivo dentro de un pool compuesto por 30 sueros. Además de evaluar la qPCR en muestras frescas, se analizó el uso de papeles de filtro para sembrar muestras de bovinos. La metodología de toma de muestras en dos tipos de papeles de filtro usando gotas de sangre fue una alternativa útil para el diagnóstico y permitió conservar ARN viral por hasta 12 meses a temperaturas de refrigeración.

Improvement of bovine pestiviral diagnosis by the development of a cost-effective method for detecting viral RNA in fresh specimens and samples spotted in filter papers.

Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease that causes severe economic losses in cattle. The aim of this study was to improve their diagnosis by developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocol for collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developed RT-qPCR showed high sensitivity in detecting these viruses in different matrices: viral stocks, semen and serum samples. With regard to the possibility of using the technique to test serum pools, it was possible to identify a positive serum sample within a pool containing 30 sera. In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovine samples was analyzed. The sampling method on two different filter papers using bovine blood drops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNA for up to 12 months under refrigeration.

HoBi-like pestivirus in 2 cases of fatal respiratory disease of feedlot cattle in Argentina.

HoBi-like pestivirus (HoBiPeV) is an emerging virus that has been detected in cattle and other ruminants. We diagnosed 2 cases of fatal bovine respiratory disease complex (BRDC) associated with infection with HoBiPeV in a feedlot in Argentina. The main findings in 2 steers autopsied were interstitial bronchopneumonia (case 1) and fibrinous bronchopneumonia (case 2). HoBiPeV was detected by RT-PCR in lungs of both animals and by immunohistochemistry in case 2. Phylogenetic analysis showed that both strains clustered within the "Brazilian-Italian" clade. In case 2, Mannheimia haemolytica was isolated from the lung. There is scant information about the contribution of HoBiPeV to the pathogenesis of BRDC. To our knowledge, HoBiPeV has not been reported previously in association with M. haemolytica pneumonia. Our findings further support the involvement of HoBiPeV in cases of BRDC and contribute to understanding the synergy of this etiologic agent in the pathogenesis of BRD, which is critical for the development of appropriate preventive strategies.

Anthropogenic Infection of Domestic Cats With SARS-CoV-2 Alpha Variant B.1.1.7 Lineage in Buenos Aires.

SARS-CoV-2 reverse zoonosis, particularly to domestic animals, and the potential role of infected animals in perpetuating the spread of the virus is an issue of increasing concern. In this case report, we identified the natural infection of two cats by SARS-CoV-2, in Argentina, whose owner had been previously infected by SARS-CoV-2. Viral genetic material was detected in feline oropharyngeal (OP) and rectal (R) swab by RT-qPCR, and sequence analysis revealed that the virus infecting the owner and one cat were genetically similar. The alpha variant (B.1.1.7 lineage) was identified with a unique additional mutation, strongly suggesting human-to-cat route of transmission. This study reinforces the One Health concept and the importance of integrating human, animal, and environmental perspectives to promptly address relevant health issues.

A novel MHC-II targeted BVDV subunit vaccine induces a neutralizing immunological response in guinea pigs and cattle.

Bovine viral diarrhoea virus (BVDV) is a major cause of economic loss in the cattle industry, worldwide. Infection results in reduced productive performance, growth retardation, reduced milk production and increased susceptibility to other diseases leading to early culling of animals. There are two primary methods used to control the spread of BVDV: the elimination of persistently infected (PI) animals and vaccination. Currently, modified live or inactivated vaccines are used in BVDV vaccination programmes, but there are safety risks or insufficient protection, respectively, with these vaccines. Here, we report the development and efficacy of the first targeted subunit vaccine against BVDV. The core of the vaccine is the fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present on antigen-presenting cells. Industrial production of the vaccine is carried out using the baculovirus expression vector system (BEVS) using single-use manufacturing technologies. This new subunit vaccine induces strong BVDV-specific neutralizing antibodies in guinea pigs and cattle. Importantly, in cattle with low levels of natural BVDV-specific neutralizing antibodies, the vaccine induced strong neutralizing antibody levels to above the protective threshold, as determined by a competition ELISA. The APCH-E2 vaccine induced a rapid and sustained neutralizing antibody response compared with a conventional vaccine in cattle.

Analysis of irradiated Argentinean fetal bovine serum for adventitious agents.

Fetal bovine serum (FBS) used in cell culture may be contaminated with adventitious agents, which can affect the production of biologicals and the results of clinical laboratory tests. We carried out a retrospective study to determine the incidence of adventitious agent contamination of Argentinean irradiated FBS dating from 2015 to 2019. We analyzed FBS batches for mycoplasma and adventitious viruses (bovine pestiviruses, bovine adenovirus, bluetongue virus, bovine parainfluenza virus 3, rabies virus, bovine parvovirus, bovine herpesvirus 1, bovine respiratory syncytial virus, and reovirus). Cell passages followed by direct immunofluorescence were carried out to check viability of the mentioned adventitious agents. Also, molecular detection of mycoplasma and pestiviruses was performed on the FBS samples. The presence of neutralizing antibodies against pestiviruses was determined. Molecular analyses indicated that frequencies of mycoplasma and pestiviruses in FBS were 14% and 84%, respectively. All of the batches were seronegative for pestiviral antibodies. After cell passages, all FBS samples were negative for hemadsorbent agents and by immunofluorescence for all of the viral species analyzed; PCR assays were negative for mycoplasma and pestiviruses. Our results demonstrate that, of all adventitious agents tested, local FBS batches only had traces of mycoplasma and pestiviruses; gamma irradiation was effective in inactivating them.

Molecular Characterization of Pestiviruses in Fetal Bovine Sera Originating From Argentina: Evidence of Circulation of HoBi-Like Viruses.

Serological evidence suggests that HoBi-like viruses, an emerging species within the Pestivirus genus of the Flaviviridae family, are in circulation in Argentina. While HoBi-like viruses were first isolated from Brazilian fetal bovine serum (FBS), no survey of Argentine FBS has been conducted. Therefore, 124 local samples of non-irradiated FBS originating from Argentina were surveyed for the presence of pestiviruses using RT-PCR. Amplicons from pestivirus positive samples were genotyped. Four samples were positive for HoBi virus-specific RT-PCR, while the BVDV-positive samples (n = 45) were classified as BVDV-1b (82.2%), BVDV-1a (13.3%), and BVDV-2 (4.5%). Virus isolation and serological profile assessment were performed for the four HoBi-positive FBS lots. These results confirm the circulation of HoBi-like virus in some regions of the Argentinean territory, highlighting the need to review the diagnostic techniques currently used in the clinical cases suspected of BVDV and in contamination control protocols for adventitious agents in cells and biotechnological products.

In Vitro and In Vivo Characterization of a Typical and a High Pathogenic Bovine Viral Diarrhea Virus Type II Strains.

Non-cytopathic (ncp) type 2 bovine viral diarrhea virus (BVDV-2) is widely prevalent in Argentina causing high mortality rates in cattle herds. In this study, we characterized an Argentinean ncp BVDV-2 field isolate (98-124) compared to a high-virulence reference strain (NY-93), using in silico analysis, in vitro assays, and in vivo infections of colostrum-deprived calves (CDC) to compare pathogenic characters and virulence. In vitro infection of bovine peripheral blood mononuclear cells (PBMC) with BVDV 98-124 induced necrosis shortly after infection while NY-93 strain increased the apoptotic rate in infected cells. Experimental infection of CDC (n = 4 each) with these strains caused an enteric syndrome. High pyrexia was detected in both groups. Viremia and shedding were more prolonged in the CDC infected with the NY-93 strain. In addition, NY-93 infection elicited a severe lymphopenia that lasted for 14 days, whereas 98-124 strain reduced the leukocyte counts for 5 days. All infected animals had a diminished lymphoproliferation activity in response to a mitogen. Neutralizing and anti-NS3 antibodies were detected 3 weeks after infection in all infected calves. Virulence was associated with a more severe clinical score, prolonged immune-suppression, and a greater window for transmission. Studies of apoptosis/necrosis performed after in vitro PBMC infection also revealed differences between both strains that might be correlated to the in vivo pathogenesis. Our results identified 98-124 as a low-virulence strain.

Serologic evidence of HoBi-like virus circulation in Argentinean water buffalo.

HoBi-like pestiviruses (also known as bovine viral diarrhea virus 3) have been sporadically reported from naturally infected cattle in Brazil, Asia, and Europe. Although HoBi-like viruses seem to be endemic in Brazilian cattle and buffalo, they have not been studied in the other countries of South America to our knowledge. Herein we report serologic results of buffalo from 12 large farms in Argentina located near the Brazilian border. These buffalo were not vaccinated against pestiviruses. Our results indicate that HoBi-like virus may be circulating in the northeastern region of Argentina given that half of the analyzed animals showed high levels of neutralizing antibodies against the pestivirus. The HoBi-like seropositive animals were also checked for neutralizing antibodies against BVDV-1a, BVDV-1b, and BVDV-2, and in most cases these animals had low levels or no detectable antibodies against these other pestiviruses. Our study suggests a need for continued pestivirus surveillance in Argentinean cattle and buffalo.

Desarrollo de una vacuna mejorada de la subunidad del virus de la diarrea viral bovina basada en la glicoproteína E2 fusionada con un anticuerpo de cadena única que se dirige a las células presentadoras de antígenos / Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle

El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versión truncada de la E2 (tE2) se fusionó a un anticuerpo de cadena simple (APCH), que se dirige a las células presentadoras de antígeno. Se expresaron las proteínas APCH-tE2 y tE2 en el sistema de baculovirus y su inmunogenicidad fue evaluada y comparada en cobayos; la proteína APCH-tE2 fue la que indujo la mejor respuesta humoral. Por dicha razón se la evaluó en bovinos utilizando 1,5µg de antígeno. Los animales presentaron altos títulos de anticuerpos neutralizantes contra BVDV hasta un año posinmunización. Esta nueva vacuna está en proceso de escalado y se transfirió al sector privado. Actualmente se está evaluando para su registro como la primera vacuna argentina de subunidad para bovinos

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells. / Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants.

Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems. The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1 µg/g at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 µg of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection.

Comparison between hyaluronic acid and platelet-rich plasma, intra-articular infiltration in the treatment of gonarthrosis.

BACKGROUND: Arthrosis is particularly prevalent in the knee. Infiltration treatment for gonarthrosis is among the most widely used techniques in orthopaedic practice. PURPOSE: To compare the clinical response of hyaluronic acid (HA) and platelet-rich plasma (PRP) treatment in 2 groups of patients affected by gonarthrosis. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: A total of 120 patients affected by clinically and radiographically documented gonarthrosis were included in this study. The gonarthrosis was graded using the Kellgren-Lawrence radiographic classification scale. The 120 patients were randomized into 2 study groups in a 1:1 ratio: 60 patients received 4 intra-articular injections of PRP (specifically, autologous conditioned plasma [ACP], 5.5 mL), and 60 patients received 4 intra-articular injections of HA (20 mg/2 mL). An unblinded physician performed infiltration once a week for 4 weeks into the knee affected by clinically relevant gonarthrosis (in both groups). All patients were evaluated with the Western Ontario and McMaster (WOMAC) score before the infiltration and at 4, 12, and 24 weeks after the first injection. RESULTS: Treatment with a local injection of ACP had a significant effect shortly after the final infiltration and a continuously improving sustained effect up to 24 weeks (WOMAC score, 65.1 and 36.5 in the HA and ACP groups, respectively; P < .001), where the clinical outcomes were better compared with the results with HA. In the HA group, the worst results were obtained for grade III gonarthrosis, whereas the clinical results obtained in the ACP group did not show any statistically significant difference in terms of the grade of gonarthrosis. The mean WOMAC scores for grade III gonarthrosis were 74.85 in the HA group and 41.20 in the ACP group (P < .001). CONCLUSION: Treatment with ACP showed a significantly better clinical outcome than did treatment with HA, with sustained lower WOMAC scores. Treatment with HA did not seem to be effective in the patients with grade III gonarthrosis.

Safety and efficacy of an E2 glycoprotein subunit vaccine produced in mammalian cells to prevent experimental infection with bovine viral diarrhoea virus in cattle.

Bovine viral diarrhea (BVD) infection caused by bovine viral diarrhea virus (BVDV), a Pestivirus of the Flaviviridae family, is an important cause of morbidity, mortality and economical losses in cattle worldwide. E2 protein is the major glycoprotein of BVDV envelope and the main target for neutralising antibodies (NAbs). Different studies on protection against BVDV infection have focused on E2, supporting its putative use in subunit vaccines. A truncated version of type 1a BVDV E2 (tE2) expressed in mammalian cells was used to formulate an experimental oleous monovalent vaccine. Immunogenicity was studied through immunisation of guinea pigs and followed by trials in cattle. Calves of 8-12 months were vaccinated, twice with a 4 week interval, with either a tE2 subunit vaccine (n = 8), a whole virus inactivated vaccine (n = 8) or left untreated as negative control group (n = 8). Four weeks after the last immunisation the animals were experimentally challenged intranasally with a non-cythopathic BVDV strain. Following challenge, BVDV was isolated from all unvaccinated animals, while 6 out of 8 animals vaccinated with tE2 showed complete virological protection indicating that the tE2 vaccine presented a similar performance to a satisfactory whole virus inactivated vaccine.