Genomic evolution of bovine viral diarrhea virus based on complete genome and individual gene analyses.

Bovine viral diarrhea virus (BVDV) genome consists of a single-stranded, positive-sense RNA with high genetic diversity. In the last years, significant progress has been achieved in BVDV knowledge evolution through phylodynamic analysis based on the partial 5'UTR sequences, whereas a few studies have used other genes or the complete coding sequence (CDS). However, no research has evaluated and compared BVDV evolutionary history based on the complete genome (CG), CDS, and individual genes. In this study, phylodynamic analyses were carried out with BVDV-1 (Pestivirus A) and BVDV-2 (Pestivirus B) CG sequences available on the GenBank database and each genomic region: CDS, UTRs, and individual genes. In comparison to the CG, the estimations for both BVDV species varied according to the dataset used, pointing out the importance of considering the analyzed genomic region when concluding. This study may provide new insight into BVDV evolution history while highlighting the need to increase the available BVDV CG sequences to perform more comprehensive phylodynamic studies in the future.

Temporal and geographic dynamics of bovine viral diarrhea virus in American countries.

Bovine viral diarrhea virus (BVDV) is a worldwide distributed pathogen of livestock classified into three species, BVDV-1 (Pestivirus A), BVDV-2 (Pestivirus B), and HoBi-like pestivirus (HoBiPeV; Pestivirus H). Despite being considered endemic in several regions of the Americas, the spatiotemporal distribution of BVDV is scarcely known. This study aimed to reconstruct the population dynamics of BVDV in American countries. The analyses performed with the partial 5´UTR gene showed that BVDV-1 and -2 would have started their diversification in the 1670s and 1790s in the United States, whereas HoBiPeV probably emerged in the 1980s in Brazil. No evident geographic clustering was observed in the Bayesian trees, which may indicate that multiple introductions events would have occurred following the first introduction. This study provides new insights into BVDV dynamics, although further analyses including sequences from other American countries and continents will help to expand the knowledge of BVDV evolution and transmission.

Spatial-temporal trends and economic losses associated with bovine abortifacients in central Argentina.

The aims of this work are, firstly, to provide the geolocalization of cases of bovine abortion with definitive diagnosis and, secondly, to estimate the economic losses due to the most frequent abortifacients diagnosed agents in cattle in Buenos Aires province, Argentina. The total beef and dairy cattle population at risk of abortion is 8,358,186 and 538,076, respectively. In beef cattle, the overall risk of abortion was estimated at 4.5% for all pregnancies, where 27.9% are due to Campylobacter fetus, Neospora caninum, Leptospira spp., Brucella abortus, and bovine viral diarrhea virus with economic losses of US$ 440 per abortion, being the annual loss to the beef industry of US$ 50,144,101. In dairy cattle, there was an 8.0% risk of suffering abortion, 26.1% produced by the same abortigenic agents. The economic losses were estimated at US$ 1,415 per abortion, which equals a total loss of US$ 17,298,498 for the dairy industry in the region. The results of this study show that infectious causes are highly prevalent in Buenos Aires province, and they caused severe economic impacts in the dairy and beef industries. Furthermore, changes in temporal trends of infectious abortion occurrence were detected, probably related to the inclusion of molecular diagnostic techniques with more sensitivity or different epidemiological or husbandry conditions in the region analyzed.

Genomic diversity and phylodynamic of bovine viral diarrhea virus in Argentina.

Bovine viral diarrhea virus (BVDV) is an important pathogen of ruminants worldwide and is characterized by high genetic diversity and a wide range of clinical presentations. In Argentina, several studies have evaluated the genetic diversity of BVDV but no phylodynamic study has been published yet. In this study, a comprehensive compilation and update of Argentinean BVDV sequences were performed, and the evolutionary history of BVDV was characterized by phylodynamic analyses based on the 5´UTR. Although BVDV-1b and BVDV-1a were the most frequent subtypes, novel subtypes for Argentina, 1e and 1i, were identified. The phylodynamic analysis suggested that BVDV started its diversification in the mid-1650s with an exponential increase in viral diversity since the late 1990s, possibly related to the livestock expansion and intensification in the country. Evolutionary rate in the 5´UTR was faster for BVDV-1a than for BVDV-1b, and both subtypes presented an endemic nature according to the demographic reconstructions. The current study contributes to clarify the evolutionary history of BVDV in the main cattle region of the country and provides useful information about the epidemiology and future development of diagnostic and control tools in Argentina.

Gene expression and in vitro replication of bovine gammaherpesvirus type 4.

In vitro cell cultures are widely used models for dissecting cellular and molecular mechanisms that lead to certain physiological conditions and diseases. The pathogenesis of BoHV-4 in the bovine reproductive tract has been studied by conducting tests on primary cultures. However, many questions remain to be answered about the role of BoHV-4 in endometrial cells. The aim of this study was to compare the replication and gene expression of BoHV-4 in cell lines and bovine reproductive tract primary cells as an in vitro model for the study of this virus. We demonstrated that BoHV-4 strains differ in their in vitro growth kinetics and gene expression but have the same cell type preference. Our results demonstrate that BoHV-4 replicates preferentially in bovine endometrial cells (BEC). However, its replication capacity extends to various cell types, since all cells that were tested were permissive to BoHV-4 infection. The highest virus titers were obtained in BEC cells. Nevertheless, virus replication efficiency could not be fully predicted from the mRNA expression profiles. This implies that there are multiple cell-type-dependent factors and strain properties that determine the level of BoHV-4 replication. The results of this study provide relevant information about the in vitro behavior of two field isolates of BoHV-4 in different cell cultures. These findings may be useful for the design of future in vitro experiments to obtain reliable results not only about the pathogenic role of BoHV-4 in the bovine female reproductive tract but also in the development of efficient antiviral strategies.

Frequency of bovine viral diarrhea virus (BVDV) in Argentinean bovine herds and comparison of diagnostic tests for BVDV detection in bovine serum samples: a preliminary study.

Bovine viral diarrhea (BVD) is a major worldwide disease with negative economic impact on cattle production. Successful control programs of BVD require the identification and culling of persistently infected (PI) animals with bovine viral diarrhea virus (BVDV). A variety of diagnostic tests are available to detect BVDV, but no comparison has been performed among those tests in Argentina. Sera collected from 2864 cattle, belonging to 55 herds from three Argentinean provinces, were analyzed by nested RT-PCR (RT-nPCR) to detect BVDV for diagnostic purposes. Additionally, this study evaluated the agreement of the RT-nPCR along with virus isolation, antigen-capture ELISA, and real-time RT-PCR for BVDV detection in archived bovine serum samples (n = 90). The RT-nPCR was useful for BVDV detection in pooled and individual serum samples. BVDV was detected in 1% (29/2864) of the cattle and in 20% (11/55) of the herds. The proportion of BVDV-positive sera was not statistically different among the tests. In addition, comparisons showed high agreement levels, with the highest values between both RT-PCR protocols. The frequency of BVDV infection at individual and herd level was lower than the reported values worldwide. Since follow-up testing was not performed, the frequency of PI cattle was unknown. Also, this study demonstrated that the four diagnostic tests can be used reliably for BVDV identification in individual serum samples. Further epidemiologically designed studies that address prevalence, risk factors, and economic impact of BVDV in Argentina will be necessary to implement effective control programs.

Detection methods and characterization of bovine viral diarrhea virus in aborted fetuses and neonatal calves over a 22-year period.

Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22 years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5'UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.

Characterization of the first bovine gammaherpesvirus 4 strain isolated from an aborted bovine fetus in Argentina.

Bovine herpesvirus 4 (BoHV-4) is increasingly believed to be responsible for several disorders of the bovine reproductive tract. The first characterization of BoHV-4 in Argentina was from samples from an aborted fetus. Argentinean isolates are highly diverse and are phylogenetically grouped in three genotypes. In this study, we describe the isolation of BoHV-4 from a bovine fetus with a gestational age of 8 months and without macroscopic lesions. Genetic analyses revealed that the isolated strain belongs to genotype 2. This is the first report on the presence of infectious BoHV-4 in tissues from an aborted bovine fetus.

Analysis of the transcripts encoding for antigenic proteins of bovine gammaherpesvirus 4.

The major glycoproteins of bovine gammaherpesvirus 4 (BoHV-4) are gB, gH, gM, gL, and gp180 with gB, gH, and gp180 being the most glycosylated. These glycoproteins participate in cell binding while some act as neutralization targets. Glycosylation of these envelope proteins may be involved in virion protection against neutralization by antibodies. In infected cattle, BoHV-4 induces an immune response characterized by low neutralizing antibody levels or an absence of such antibodies. Therefore, virus seroneutralization in vitro cannot always be easily demonstrated. The aim of this study was to evaluate the neutralizing capacity of 2 Argentine BoHV-4 strains and to associate those findings with the gene expression profiles of the major envelope glycoproteins. Expression of genes coding for the envelope glycoproteins occurred earlier in cells infected with isolate 10/154 than in cells infected with strain 07/435, demonstrating a distinct difference between the strains. Differences in serological response can be attributed to differences in the expression of antigenic proteins or to post-translational modifications that mask neutralizing epitopes. Strain 07/435 induced significantly high titers of neutralizing antibodies in several animal species in addition to bovines. The most relevant serological differences were observed in adult animals. This is the first comprehensive analysis of the expression kinetics of genes coding for BoHV-4 glycoproteins in 2 Argentine strains (genotypes 1 and 2). The results further elucidate the BoHV-4 life cycle and may also help determine the genetic variability of the strains circulating in Argentina.

Bovine herpesvirus type 5 replication and induction of apoptosis in vitro and in the trigeminal ganglion of experimentally-infected cattle.

Bovine herpesvirus (BoHV) types 1 and 5 are neuroinvasive. Cases of BoHV-1-induced encephalitis are not as frequent as those caused by BoHV-5. In this study, the capability of BoHV-5 to induce apoptosis in cell cultures and in the trigeminal ganglion during acute infection of experimentally-infected cattle was analyzed. Apoptotic changes in cell cultures agree with the ability of the viral strains to replicate in each cell line. Marked differences were observed between the in vitro induction of apoptosis by BoHV-1Cooper and BoHV-5 97/613 strains. Apoptotic neurons were clearly evident in the trigeminal ganglion of BoHV-1-infected calves. For BoHV-5 a fewer number of positive neurons was observed. There is an association between the magnitude of bovine herpesviruses replication and the induction of apoptosis in trigeminal ganglion. These findings suggest that the induction of apoptosis and the innate immune response orchestrate the final outcome of alpha herpesviruses infection of the bovine nervous system.

Dual natural infection with bovine viral diarrhea virus -1 and -2 in a stillborn calf: tissue distribution and molecular characterization.

Dual infections with both bovine viral diarrhea virus (BVDV)-1 and -2 seem to be unusual. The aim of this study was to describe an infection with both BVDV genotypes in a stillborn calf. Virus isolation and phylogenetic analyses of the 5´UTR and NS5B regions confirmed the presence of BVDV-1b and -2b in spleen and lung, whereas BVDV-2b was also detected in brain, heart, liver, kidney and, fluid of cavities. These results confirm that dual infections with both BVDV-1 and BVDV-2 species can occur naturally and their tissue distribution can be different.

Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells.

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization.

Herpesvirus bovino 4 (BoHV-4): aspectos generales de su biología y situación en la República Argentina / Bovine herpesvirus 4 (BoHV-4): general aspects of the biology and status in Argentina

El herpesvirus bovino 4 [Bovine herpesvirus 4 (BoHV-4)] ha sido aislado de bovinos con infecciones respiratorias, vulvovaginitis, mastitis, abortos, endometritis y de animales aparentemente sanos en diferentes partes del mundo. Si bien no se ha reconocido como agente causal de una entidad patológica en particular, se asocia principalmente con infecciones del tracto reproductivo de los bovinos. Este virus puede infectar un amplio rango de especies tanto in vivo como in vitro. Los primeros aislamientos dieron origen a dos grupos de cepas prototipo: el grupo americano tipo DN599 y el grupo europeo tipo Movar. En Argentina, el BoHV-4 fue aislado y caracterizado en el año 2007; este aislamiento se obtuvo de muestras de mucus cérvico-vaginal de vacas que abortaron. Hasta el momento se han registrado más de 40 aislamientos, provenientes principalmente de hembras bovinas que han abortado

Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle with respiratory infections, vulvovaginitis, mastitis, abortions, endometritis and from apparently healthy animals throughout the world. Although it has not yet been established as causal agent of a specific disease entity, it is primarily associated with reproductive disorders of cattle. This virus can infect a wide range of species, either in vivo or in vitro. Two groups of prototype strains were originated from the first isolates: the DN599-type strains (American group) and the Movar-type strains (European group). In Argentina, BoHV-4 was isolated and characterized in 2007 from vaginal discharge samples taken from cows that had aborted. So far, more than 40 isolates, mainly associated with aborting bovine females have been registered in our country

[Bovine herpesvirus 4 (BoHV-4): general aspects of the biology and status in Argentina]. / Herpesvirus bovino 4 (BoHV-4): aspectos generales de su biología y situación en la República Argentina.

Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle with respiratory infections, vulvovaginitis, mastitis, abortions, endometritis and from apparently healthy animals throughout the world. Although it has not yet been established as causal agent of a specific disease entity, it is primarily associated with reproductive disorders of cattle. This virus can infect a wide range of species, either in vivo or in vitro. Two groups of prototype strains were originated from the first isolates: the DN599-type strains (American group) and the Movar-type strains (European group). In Argentina, BoHV-4 was isolated and characterized in 2007 from vaginal discharge samples taken from cows that had aborted. So far, more than 40 isolates, mainly associated with aborting bovine females have been registered in our country.

A Neospora caninum vaccine using recombinant proteins fails to prevent foetal infection in pregnant cattle after experimental intravenous challenge.

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P<0.001). Levels of antibodies against rNcGRA7 were statistical higher in group A animals when compared with groups B and C at weeks 5 and 16 (P>0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P>0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation.