Late development of pustular, erosive lesions in the muzzle of calves inoculated with Pseudocowpox virus.

We studied the pathogenesis of Pseudocowpox virus (PCPV), a zoonotic parapoxvirus associated with mucocutaneous lesions in cattle. Inoculation of calves with PCPV isolate SD 76-65 intranasally (n = 6) or transdermally in the muzzle (n = 2) resulted in virus replication and shedding up to day 13 post-infection (pi). No local or systemic signs were observed in inoculated calves up to day 20pi, when the clinical monitoring was discontinued. However, from days 28-34 pi, seven (7/8) inoculated calves underwent an asynchronous clinical course characterized by development of a few (one or two) to countless papulo-pustular, erosive-fibrinous and scabby lesions in the muzzle, in some cases extending to the lips and gingiva. In some animals, the lesions coalesced, forming extensive fibrinotic/necrotic and scabby plaques covering almost entirely the muzzle. The clinical course lasted 8-15 days and spontaneously subsided after day 42pi. Infectious virus and/or viral DNA were detected in swabs collected from lesions of 5/8 animals between days 34 and 42pi. Histological examination of fragments collected from the muzzle lesions of two affected calves (day 36pi) revealed marked epidermal hyperplasia and severe orthokeratotic and parakeratotic hyperkeratosis, covered by thick scabs. The epidermis showed multifocal areas of keratinocyte coalescing necrosis and mild multifocal vacuolar degeneration. Sera of inoculated calves at 50pi showed partial virus neutralization at low dilutions, demonstrating seroconversion. The delayed and severe clinical course associated with virus persistence in lesions are novel findings and contribute for the understanding of PCPV pathogenesis.

An extensive field study reveals the circulation of new genetic variants of subtype 1a of bovine viral diarrhea virus in Uruguay.

Bovine viral diarrhea virus (BVDV) is a major pathogen worldwide, causing significant economic losses to the livestock sector. In Uruguay, BVDV seroprevalence at the farm level is >80%. In this work, 2546 serum, blood or tissue samples collected from animals suspected of being affected by BVD between 2015 and 2017 were analyzed by reverse transcription PCR and sequencing. Analysis of the BVDV genomic regions 5'UTR/Npro, Npro and E2 revealed that BVDV-1a, 1i and 2b circulate in the country, with BVDV-1a being the most prevalent subtype. Population dynamics studies revealed that BVDV-1a has been circulating in our herds since ~1990. This subtype began to spread and evolve, accumulating point mutations at a rate of 3.48 × 10-3 substitutions/site/year, acquiring specific genetic characteristics that gave rise to two local genetic lineages of BVDV-1a. These lineages are divergent from those circulating worldwide, as well as the vaccine strain currently used in Uruguay. The most notable differences between field and vaccine strains were found in the E2 glycoprotein, suggesting that the amino acid substitutions could result in failure of cross-protection/neutralization after vaccination. This is the first study that compares Uruguayan BVDV field and vaccine strains with other BVDV strains from throughout the world. The results obtained in this study will be very useful for developing a suitable immunization program for BVDV in Uruguay by identifying local field strains as candidates for vaccine development.

Respiratory signs, fever and lymphopenia in calves inoculated with Brazilian HoBi-like pestiviruses.

Hobi-like viruses (HobiPeV) comprise a novel, recently classified species of bovine pestiviruses, originally identified in commercial fetal bovine serum of Brazilian origin and, subsequently, isolated from diseased animals in several countries. Although frequently isolated from clinical cases, most HobiPeV isolates failed to reproduce overt disease in cattle upon experimental inoculation. Herein, we describe the outcome of experimental infection of four to six months-old seronegative calves with two Brazilian HobiPeV isolates. Calves inoculated intranasally with isolate SV478/07 developed viremia between days 2 and 9 post-inoculation (pi) and shed virus in nasal secretions up to day 11pi. These animals presented hyperthermia (day 7 to 10-11 pi) and lymphopenia from days 4 to 8pi. Clinically, all four calves developed varied degrees of apathy, anorexia, mild to moderate respiratory signs (nasal secretion, hyperemia), ocular discharge and pasty diarrhea in the days following virus inoculation. In contrast, calves inoculated with isolate SV757/15 presented only hyperthermia (days 3 to 10-11 pi) and lymphopenia (days 4-8 pi), without other apparent clinical signs. In these animals, viremia was detected up to day 9 pi and virus shedding in nasal secretions lasted up to day 12-14 pi. Both groups seroconverted to the inoculated viruses, developing virus neutralizing (VN) titers from 320 to 5120 at day 28pi. These results extend previous findings that experimental infections of calves with HobiPeV are predominantly mild, yet they also indicate that field isolates may differ in their ability to cause disease in susceptible animals.

Generation of calves persistently infected with HoBi-like pestivirus and comparison of methods for detection of these persistent infections.

The identification and elimination of persistently infected (PI) cattle are the most effective measures for controlling bovine pestiviruses, including bovine viral diarrhea virus (BVDV) and the emerging HoBi-like viruses. Here, colostrum-deprived calves persistently infected with HoBi-like pestivirus (HoBi-like PI calves) were generated and sampled (serum, buffy coat, and ear notches) on the day of birth (DOB) and weekly for 5 consecutive weeks. The samples were subjected to diagnostic tests for BVDV--two reverse transcriptase PCR (RT-PCR) assays, two commercial real-time RT quantitative PCR (RT-qPCR), two antigen capture enzyme-linked immunosorbent assays (ACE), and immunohistochemistry (IHC)--and to HoBi-like virus-specific RT-PCR and RT-qPCR assays. The rate of false negatives varied among the calves. The HoBi-like virus-specific RT-PCR detected HoBi-like virus in 83%, 75%, and 87% of the serum, buffy coat, and ear notch samples, respectively, while the HoBi-like RT-qPCR detected the virus in 83%, 96%, and 62%, respectively. In comparison, the BVDV RT-PCR test had a higher rate of false negatives in all tissue types, especially for the ear notch samples (missing detection in at least 68% of the samples). The commercial BVDV RT-qPCRs and IHC detected 100% of the ear notch samples as positive. While ACE based on the BVDV glycoprotein E(rns) detected infection in at least 87% of ear notches, no infections were detected using NS3-based ACE. The BVDV RT-qPCR, ACE, and IHC yielded higher levels of detection than the HoBi-like virus-specific assays, although the lack of differentiation between BVDV and HoBi-like viruses would make these tests of limited use for the control and/or surveillance of persistent HoBi-like virus infection. An improvement in HoBi-like virus tests is required before a reliable HoBi-like PI surveillance program can be designed.

Effects of inactivated parapoxvirus ovis on cellular and humoral events of the innate immune response in mice.

The immunostimulating properties of inactivated parapoxvirus ovis (iPPVO) have long been demonstrated in vivo and in vitro, yet the biological and molecular mechanisms involved remain largely unknown. We herein report that intraperitoneal inoculation of iPPVO in mice results in stimulation of several events of the innate immune response. Increased interferon I (IFN-I) activity was demonstrated in sera of mice treated with iPPVO at 6 and 12 h post-inoculation (hpi), and enhanced expression of IFN-γ (15-fold increase) and IL-12 (6-fold) mRNA was detected in the spleen of treated mice at 24 and 48 hpi, respectively. A significant increase in neutrophil activity (p<0.01) was observed at 6 hpi in the blood of iPPVO treated mice. In addition, increased phagocytic activity by peritoneal macrophages of iPPVO-treated mice (p<0.01) was detected in vivo (from 24 to 72 hpi) and in vitro (12 to 96 hpi). Bactericidal activity of sera mice treated with iPPVO against Escherichia coli was also increased (p<0.05) at 24 and 72 hpi. Taken together, these results demonstrate that iPPVO administration leads to a transient stimulation of selected innate immune mechanisms, likely contributing to the immunostimulant effects observed against viral and bacterial infections in vivo.

Bovine herpesvirus type 5 infection regulates Bax/BCL-2 ratio.

Bovine herpesvirus 5 (BoHV-5) is an α-herpesvirus that causes neurological disease in young cattle and is also occasionally involved in reproductive disorders. Although there have been many studies of the apoptotic pathways induced by viruses belonging to the family Herpesviridae, there is little information about the intrinsic programmed cell death pathway in host-BoHV-5 interactions. We found that BoHV-5 is able to replicate in both mesenchymal and epithelial cell lines, provoking cytopathology that is characterized by cellular swelling and cell fusion. Viral antigens were detected in infected cells by immunofluorescence assay at 48 to 96 h post-infection (p.i.). At 48 to 72 h p.i., anti-apoptotic BCL-2 antigens were found at higher levels than Bax antigens; the latter is considered a pro-apoptotic protein. Infected cells had increased BCL-2 phenotype cells from 48 to 96 h p.i., based on flow cytometric analysis. At 48 to 96 h p.i., Bax mRNA was not expressed in any of the infected cell monolayers. In contrast, BCL-2 mRNA was found at high levels at all p.i. in both types of cells. BoHV-5 replication apparently modulates BCL-2 expression and gene transcription, enhancing production of virus progeny.

Epidemiological analysis over two years of SARS-CoV-2 circulation in southern Brazil: old and novel predictors of COVID-19 outcome.

The overwhelming majority of SARS-CoV-2 epidemiological studies cover a narrow time period, making general knowledge about the COVID-19 pandemic difficult. To assess COVID-19-related host aspects in the overall pandemic, we analyzed COVID-19 cases during the first two years of SARS-CoV-2 circulation in southern Brazil. Herein, 390 patients admitted in 2020-2022 to a Brazilian public referral hospital were allocated into two groups according to the COVID-19 outcome: hospital discharge (n=237) or death (n=153). In the univariate analysis, several variables, including sociodemographic, clinical and laboratory aspects (primary data), were significantly different between the analyzed groups. In multivariate logistic regression, eight of these factors remained associated with the COVID-19 outcome. In particular, we report oxygen supplementation and the need for hemodialysis as predictors of hospital discharge and death from COVID-19, respectively. To the best of our knowledge, none of these findings have been previously reported in the Brazilian or world population. In conclusion, our results contribute to current knowledge by demonstrating that factors described at different times may remain associated with COVID-19 over the pandemic and by identifying novel predictors of COVID-19 outcome.

A nuclear inhibitor of NF-kappaB encoded by a poxvirus.

Poxviruses have evolved various strategies to inhibit cytoplasmic events leading to activation of the nuclear factor κB (NF-κB) signaling pathway, with individual viruses often encoding multiple NF-κB inhibitors. Here, the novel orf virus (ORFV)-encoded protein ORFV002 was shown to inhibit nuclear events regulating NF-κB transcriptional activity. ORFV002 expression in cell cultures significantly decreased wild-type-virus-, tumor necrosis factor alpha (TNF-α)-, and lipopolysaccharide (LPS)-induced NF-κB-mediated gene expression. Expression of ORFV002 in cells, while not affecting phosphorylation or nuclear translocation of NF-κB-p65, markedly decreased TNF-α- and wild-type-virus-induced acetylation of NF-κB-p65, a p300-mediated nuclear modification of NF-κB-p65 that regulates its transactivating activity. ORFV002 was shown to colocalize and interact with NF-κB-p65, and expression of ORFV002 in cell cultures resulted in a reduced interaction of NF-κB-p65 with p300, suggesting that ORFV002 interferes with NF-κB-p65/p300 association. Deletion of ORFV002 from the OV-IA82 genome had no significant effect on ORFV pathogenesis in sheep, indicating that ORFV002 is nonessential for virus virulence in the natural host. This represents the first description of a nuclear inhibitor of NF-κB encoded by a poxvirus.

Orf virus ORFV121 encodes a novel inhibitor of NF-kappaB that contributes to virus virulence.

Orf virus (ORFV), the type member of the genus Parapoxvirus of the Poxviridae, has evolved novel strategies (proteins and/or mechanisms of action) to modulate host cell responses regulated by the nuclear factor-κB (NF-κB) signaling pathway. Here, we present data indicating that ORFV ORFV121, a gene unique to parapoxviruses, encodes a novel viral NF-κB inhibitor that binds to and inhibits the phosphorylation and nuclear translocation of NF-κB-p65. The infection of cells with an ORFV121 deletion mutant virus (OV-IA82Δ121) resulted in increased NF-κB-mediated gene transcription, and the expression of ORFV121 in cell cultures significantly suppressed NF-κB-regulated reporter gene expression. ORFV ORFV121 physically interacts with NF-κB-p65 in the cell cytoplasm, thus providing a mechanism for the inhibition of NF-κB-p65 phosphorylation and nuclear translocation. Notably, the deletion of ORFV121 from the viral genome markedly decreased ORFV virulence and disease pathogenesis in sheep, indicating that ORFV121 is a virulence determinant for ORFV in the natural host.

A novel inhibitor of the NF-{kappa}B signaling pathway encoded by the parapoxvirus orf virus.

The parapoxvirus orf virus (ORFV) is a pathogen of sheep and goats that has been used as a preventive and therapeutic immunomodulatory agent in several animal species. However, the functions (genes, proteins, and mechanisms of action) evolved by ORFV to modulate and manipulate immune responses are poorly understood. Here, the novel ORFV protein ORFV024 was shown to inhibit activation of the NF-kappaB signaling pathway, an important modulator of early immune responses against viral infections. Infection of primary ovine cells with an ORFV024 deletion mutant virus resulted in a marked increase in expression of NF-kappaB-regulated chemokines and other proinflammatory host genes. Expression of ORFV024 in cell cultures significantly decreased lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB-responsive reporter gene expression. Further, ORFV024 expression decreased TNF-alpha-induced phosphorylation and nuclear translocation of NF-kappaB-p65, phosphorylation, and degradation of IkappaBalpha, and phosphorylation of IkappaB kinase (IKK) subunits IKKalpha and IKKbeta, indicating that ORFV024 functions by inhibiting activation of IKKs, the bottleneck for most NF-kappaB activating stimuli. Although ORFV024 interferes with activation of the NF-kappaB signaling pathway, its deletion from the OV-IA82 genome had no significant effect on disease severity, progression, and time to resolution in sheep, indicating that ORFV024 is not essential for virus virulence in the natural host. This represents the first description of a NF-kappaB inhibitor encoded by a parapoxvirus.

Virological and clinico-pathological features of orf virus infection in experimentally infected rabbits and mice.

Many aspects of the biology of orf virus (ORFV) infection remain poorly understood and attempts to establish animal models have yielded conflicting and non-reproducible results. We herein describe the characterization of ORFV infection and disease in rabbits and mice. A protocol of intradermal inoculation was employed to inoculate 10(8.5)TCID50/mL of ORFV strain IA-82 in the skin of ears, of the back and labial commissures. All inoculated rabbits presented a clinical course characterized by erythema, macules, papules/vesicles or pustules that eventually dried originating scabs. Local signs started around days 3 and 4 post-inoculation (pi) and lasted 3-10 days. Virus was recovered from lesions between days 2 and 14pi. Histological examination of lesions revealed focal proliferative dermatitis with ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies in keratinocytes, histological hallmarks of contagious ecthyma in sheep. A similar, albeit milder clinical course occurred in 5/10 inoculated mice; virus was recovered from lesions from three animals. Inoculated lambs - used as controls - developed severe lesions of contagious ecthyma. VN tests performed at day 28pi failed to detect neutralizing antibodies in all inoculated animals. In contrast, convalescent rabbit sera were positive by ELISA at dilutions from 100 to 400. These results show that rabbits are susceptible to ORFV infection and thus may be used to study selected aspects of ORFV biology.

Genetic identification of a rabies virus from an insectivorous bat in an urban area of Rio Grande do Sul, Brazil.

In Latin America, hematophagous bats are the main reservoirs of rabies virus (RABV) to livestock, to other mammals and, occasionally, to human. Nonetheless, reports of exposure of human and pets to RABV upon aggression by non-hematophagous bats are increasing, possibly facilitated by the synanthropic habits of these bats. We, herein, report the detection and genetic identification of a RABV recovered from an insectivorous bat found sick in a student housing building at the Federal University of Santa Maria, Southern Brazil. Taxonomic characterization identified the captured bat as a member of the genus Nyctinomops, family Molossidae, the group of insectivorous bats. Brain fragments of the bat were positive for RABV antigens by fluorescent antibody test (FAT) and for sequences of the nucleoprotein (N) gene by RT-PCR. The N amplicon was submitted to nucleotide sequencing and analysis, showing that the consensus sequences (SV 33/19) had high identity with RABV sequences of insectivorous bats deposited in GenBank. At phylogenetic tree, the N gene sequences of SV 33/19 clustered with RABV recovered from Nyctinomops laticaudatus, Molossus molossus, and Tadarida lauticaudata bats, and a part of RABV variant 3, 4, and 6, that correspond to Desmodus rotundus, Tadarida brasiliensis, and Lasiurus cinereus, respectively. Although no direct human or domestic animal exposure has been reported, this case strengthens the need for a continuous rabies vaccination in pets in the surrounding areas, since non-hematophagous bats may serve as source of infection for these animals. These findings also call attention for continuous monitoring of populations of synanthropic bats to avoid/prevent human exposure.

Immunohistochemical Detection of Intralesional Antigens of Ovine Gammaherpesvirus-2 in Cattle with Sheep-associated Malignant Catarrhal Fever.

Sheep-associated malignant catarrhal fever (SA-MCF) is a severe lymphoproliferative disease of ruminants caused by ovine gammaherpesvirus-2 (OvHV-2). Since the initial identification of SA-MCF there has been extensive research related to the pathogenesis of OvHV-2, based primarily on serological and molecular assays associated with typical histopathological findings. The monoclonal antibody (MAb-15A) binds to a common epitope in MCF viruses and is used frequently in serological investigations. However, the utilization of this antibody to detect antigens of OvHV-2 in tissues has not been examined. Accordingly, this study standardized an immunohistochemical assay using MAb-15A to identify antigens of OvHV-2 in tissues of cattle (n = 5) with SA-MCF. All animals developed acute neurological signs, without ocular and nasal manifestations, and had nucleic acids of OvHV-2 in brain tissue detected by polymerase chain reaction. The principal histopathological findings were lymphocytic nephritis (n = 5), widespread arterial proliferation and vasculitis (n = 5), lymphocytic portal hepatitis (n = 3), non-suppurative meningoencephalitis (n = 2) and atrophic enteritis with cryptal necrosis and dilation (n = 2). Intralesional intracytoplasmic antigens of OvHV-2 were identified within multiple epithelial cells of the kidneys of all animals, the intestines of animals with and without atrophic enteritis, and within epithelial cells of bile ducts in animals with lymphocytic hepatitis. Additionally, there was positive intracytoplasmic immunoreactivity within histiocytes and lymphocytes in several tissues. These findings suggest that the MAb-15A detects antigens of OvHV-2 within epithelial cells and leucocytes in several organs. Moreover, this assay would contribute significantly towards understanding of the pathogenesis of SA-MCF and may be used for retrospective studies. Additionally, angiopathy in SA-MCF may be a progressive lesion, which may terminate in luminal occlusion and probably occurs irrespectively of the eye and head form of MCF.

Bovine herpesvirus 5 induces an overproduction of nitric oxide in the brain of rabbits that correlates with virus dissemination and precedes the development of neurological signs.

We herein report an investigation of nitric oxide (NO) levels, a candidate molecule for neuronal toxicity and dysfunction, in the brain of rabbits during experimental neurological infection by bovine herpesvirus 5 (BoHV-5). Spectrophotometry for NO products (NO(2) and NO(3)) revealed that NO levels were significantly increased (F(4, 40) = 3.33; P <.02) in several regions of the brain of rabbits with neurological disease, correlating with moderate to high BoHV-5 titers. Immunohistochemistry of brain regions revealed a group of cells with neuronal and astrocyte morphology expressing the enzyme inducible NO synthase (iNOS) close to virus antigen-positive neurons. In addition, the investigation of nitric oxide levels between 2 and 6 days post infection (d.p.i.) revealed an initial increase in NO levels in the olfactory bulb and cortex (OB/OC) and anterior cortex (AC) at day 3 p.i., correlating with the initial detection of virus. As the infection proceeded, increased NO levels-and infectivity-were progressively being detected in the OB/CO and AC at day 4 p.i. (F(12, 128) = 2.82; P <.003); at day 5 p.i. in several brain regions (P <.003 in the OB/OC); and at day 6 p.i. in all regions (P <.003) but the thalamus. These results show that BoHV-5 replication in the brain of rabbits induces an overproduction of NO. The increase in NO levels in early infection correlated spatially and temporally with virus dissemination within the brain and preceded the development of neurological signs. Thus, the overproduction of NO in the brain of BoHV-5-infected rabbits may be a component of the pathogenesis of BoHV-5-induced neurological disease.

A genetic profile of bovine pestiviruses circulating in Brazil (1998-2018).

The pestiviruses bovine viral diarrhea virus 1 (BVDV-1), 2 (BVDV-2), and HoBi-like (HoBiPeV) are endemic among Brazilian cattle, the world's largest commercial bovine herd. In the last two decades (1998-2018) over 300 bovine pestiviruses have been partially or fully sequenced in Brazil, including viruses from different regions, different epidemiological backgrounds, and associated with diverse clinical presentations. Phylogenetic analysis of these viruses demonstrated a predominance of BVDV-1 (54.4%), with subgenotypes -1a (33.9% of total) and -1b (16.3%) being more frequent and subgenotypes -1d, -1e, and -1i at very low frequencies. The overall BVDV-2 frequency was 25.7% but it varied largely by region, reaching up to 48% in Southern states. BVDV-2b was the predominant subgenotype (84.8% of BVDV-2), followed by BVDV-2a (8.86%). HoBiPeV accounted for 19.9% (61/307) of the genotyped viruses and were detected at high frequency in cattle from Northeastern states. These findings demonstrate a unique mix of pestivirus species and subgenotypes, unlike that seen in Europe or North America. The design of effective diagnostic tools, vaccines, and control programs for limiting bovine pestivirus infections in Brazil must take into consideration this unique mix of viruses. This article provides a critical review of two decades of genetic identification of pestiviruses in Brazil.

Molecular and antigenic characterization of Brazilian bovine herpesvirus type 1 isolates recovered from the brain of cattle with neurological disease.

Bovine herpesviruses type 1 and 5 (BoHV-1 and BoHV-5) are closely related yet differ markedly in their neuropathogenic potential. BoHV-1 isolates have been associated with respiratory and genital disease whereas BoHV-5 has been consistently isolated from neurological infection. We report the characterization of five Brazilian BoHV-1 isolates associated with neurological disease, an unusual finding. All five viruses were isolated from the brain of cattle presenting neurological disease, yet prominent histological encephalitis was not observed in three cases. The isolated viruses were identified as BoHV-1 by a glycoprotein C gene-based PCR able to differentiate BoHV-1 from BoHV-5. The identity of the isolates was confirmed by nucleotide sequencing of the amplicons and by restriction analysis of PCR products from another gC region. Monoclonal antibody binding and cross-neutralization assays with BoHV-1 and BoHV-5 antisera showed a typical BoHV-1 antigenic profile. Lastly, inoculation of rabbits with these five BoHV-1 isolates did not result in neurological disease, contrasting with fatal meningoencephalitis produced by BoHV-5. Thus, the involvement of BoHV-1 in neurological disease of cattle is more frequent than previously reported, indicating the need for fast and precise means of differentiating it from BoHV-5. Likewise, the potential role of BoHV-1 in neurological infection in cattle should be further investigated.

Acute and latent infection by bovine herpesvirus type 5 in experimentally infected goats.

The ability of alphaherpesviruses to infect different ruminant species may have important implications for control/eradication efforts. Serological data indicate that goats may be naturally infected with bovine herpesviruses. To investigate the susceptibility of goats to bovine herpesvirus-5 (BoHV-5), 3-4-month-old kids were inoculated intranasally with each of three Brazilian BoHV-5 isolates (G1, n=8; G2, n=5; G3, n=5). The acute infection was characterized by virus shedding in nasal secretions for up to 14 days (titers up to 10(5.97)TCID(50)/mL), mild respiratory signs and conjunctivitis. All animals seroconverted to BoHV-5, developing virus neutralizing (VN) titers from 4 to 32 to the homologous viruses. At day 60 post inoculation (pi), two animals from each group were euthanized for tissue collection and the remaining goats were submitted to dexamethasone administration (0.4 mg kg(-1) for 5 days). Dexamethasone treatment resulted in virus reactivation in 9 out of 12 animals, as ascertained by virus shedding and/or by increase in VN titers. Virus shedding was detected in 8/12 animals and lasted from 1 to 9 days. Latent viral DNA was detected by PCR in the olfactory bulb and/or trigeminal ganglia of 6/6 goats euthanized at day 60 pi and in 12/12 animals euthanized 40 days post-dexamethasone. These results show that young goats are susceptible to BoHV-5 and may shed virus upon reactivation of latent infection. Thus, it is reasonable to expect that goats raised in close contact with cattle in areas where BoHV-5 is endemic may be infected and therefore should be considered potential reservoirs of the virus.

Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy.

Antigenic diversity of Brazilian isolates of HoBi-like pestiviruses.

Hobi-like viruses comprise an unclassified group of bovine pestiviruses related to bovine viral diarrhea virus 1 (BVDV-1) and 2 (BVDV-2). These viruses were originally identified in fetal bovine serum from Brazilian origin and, subsequently, isolated from diseased animals in several countries. Herein we performed an antigenic characterization of eight Brazilian HoBi-like viruses isolated from persistently infected (PI) animals and from gastroenteric disease (2007-2015). Phylogenetic analysis based on the 5' unstranslated region (UTR) clustered these viruses with other HoBi-like viruses from European and Asiatic origin. Monoclonal antibody (MAb) binding indicated variability in the Hobi-like virus glycoprotein E2 and significant differences from the homologous BVDV-1 and BVDV-2 glycoprotein. Analysis of antigenic relatedness based on virus-neutralizing titers using virus-specific antisera revealed that HoBi-like viruses are antigenically very different from BVDV-1 and, to a lesser extent, from BVDV-2. Cross-neutralizing assays between pairs of HoBi-like viruses and their respective antisera indicated the existence of antigenic variability among these viruses, even for viruses isolated from the same herd in different occasions. Moreover, the identification of a HoBi-like isolate with low antigenic similarity with the other isolates indicates the potential existence of antigenic subgroups among HoBi-like virus isolates. Finally, sera of lambs immunized with commercial BVDV vaccines showed low or undetectable neutralizing activity against HoBi-like isolates. These results indicate significant antigenic differences between BVDV genotypes and Brazilian HoBi-like viruses and the existence of antigenic variability within this atypical group of pestiviruses. These findings extend the knowledge about the antigenic diversity of HoBi-like viruses and reinforce the need for their inclusion in current BVDV vaccines.

Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014.

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, border disease virus (BDV), and an atypical pestivirus named HoBi-like pestivirus. In this study, eighty-nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5' untranslated regions (5'UTR), N-terminal autoprotease (Npro ) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV-1, 33.7% as BVDV-2 and 12.4% as HoBi-like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV-1a (35.9%), BVDV-2b (31.4%), BVDV-1b (10.1%), BVDV-1d (6.7%), BVDV-2c (2.2%) and BVDV-1e (1.1%). BVDV-2c and BVDV-1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.