Granulomatous mural folliculitis in 16 domestic goats: Infection with malignant catarrhal fever viruses and colocalization with ovine herpesvirus-2 using in situ hybridization.

Granulomatous mural folliculitis (GMF) is an uncommon reaction pattern occasionally observed in nonadapted ruminant hosts infected with malignant catarrhal fever viruses. This report characterizes GMF and concurrent cutaneous lesions in 16 goats with crusting dermatitis using histochemistry including hematoxylin and eosin, periodic acid-Schiff, and Grocott's methenamine silver, and immunohistochemistry for CD3, CD20, ionized calcium binding adaptor molecule 1, and cytokeratin AE1/3. Infiltrates in all 16 GMF cases consisted of macrophages and fewer T lymphocytes, and variably included eosinophils, multinucleated histiocytic giant cells, and/or neutrophils. Formalin-fixed paraffin-embedded skin and fresh skin samples from caprine GMF cases were tested using pan-herpesvirus nested conventional polymerase chain reaction (PCR) and partial sequencing, ovine herpesvirus-2 (OvHV-2) real-time PCR, and OvHV-2 colorimetric in situ hybridization (ISH). Five of 16 goats with GMF (31%) were PCR positive for malignant catarrhal fever viruses, including caprine herpesvirus 3 in 1 goat and OvHV-2 in 4 goats. Three goats also had positive intranuclear OvHV-2 hybridization signal in follicular keratinocytes, among other cell types, localized to areas of GMF. Herpesviruses were not detected in the formalin-fixed paraffin-embedded skin of 9 goats without GMF. This case series describes relatively frequent detections of malignant catarrhal fever viruses in the skin of goats with GMF, including the first report of caprine herpesvirus 3, and localizes OvHV-2 infected follicular keratinocytes within areas of GMF.

Histopathologic and molecular characterization of Erethizon dorsatum papillomavirus 1 and Erethizon dorsatum papillomavirus 2 infection in North American porcupines (Erethizon dorsatum).

Erethizon dorsatum papillomavirus 1 (EdPV1) and Erethizon dorsatum papillomavirus 2 (EdPV2) are associated with cutaneous papillomas in North American porcupines (Erethizon dorsatum). This study defined gross, histopathologic, and molecular characteristics of viral papillomas in 10 North American porcupines submitted to the New York State Animal Health Diagnostic Center. Investigation for the presence of EdPV1 and EdPV2 DNA via polymerase chain reaction (PCR) was performed in 9 of the 10 (90.0%) porcupines, and all porcupines were investigated for the detection and localization of EdPV1 and EdPV2 E6 and E7 nucleic acid via chromogenic in situ hybridization (CISH). Next-generation sequencing (NGS) was performed in 2 porcupines. Papillomas were diagnosed on the muzzle (n = 4), caudal dorsum (n = 1), upper lip (n = 1), chin (n = 1), gingiva (n = 2), and nasal planum (n = 1). Histologically, the lesions consisted of hyperplastic epidermis or epithelium with orthokeratotic keratin, prominent keratohyalin granules, and intranuclear inclusion bodies. PCR identified EdPV1 in 6 of 9 samples and EdPV2 in the remaining 3 samples. NGS resulted in 100% genome coverage of EdPV1 and 76.20% genome coverage of EdPV2 compared with GenBank reference sequences, with 99.8% sequence identity to the complete EdPV2 L1 gene of a novel subtype recently identified in France. Hybridization patterns in 9 of the 10 (90.0%) porcupines were characterized by strong nuclear signals in the superficial epidermis, with strong nuclear and punctate cytoplasmic signals in the stratum spinosum and basale. In one animal, CISH suggested dual EdPV1 and EdPV2 infection.

Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 760 million cases and over 6.8 million deaths as of March 2023. Vaccination has been the main strategy used to contain the spread of the virus and to prevent hospitalizations and deaths. Currently, two mRNA-based vaccines and one adenovirus-vectored vaccine have been approved and are available for use in the U.S. population. The versatility, low cost, and rapid production of DNA vaccines provide important advantages over other platforms. Additionally, DNA vaccines efficiently induce both B- and T-cell responses by expressing the antigen within transfected host cells, and the antigen, after being processed into peptides, can associate with MHC class I or II of antigen-presenting cells (APCs) to stimulate different T cell responses. However, the efficiency of DNA vaccination needs to be improved for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been used successfully in the field of veterinary oncology, resulting in high rates of response after electrochemotherapy. Here, we evaluate the safety, immunogenicity, and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate delivered by intramuscular injection followed by electroporation (Vet-ePorator™) in ferrets. The linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects. Additionally, the vaccine elicited neutralizing antibodies and T cell responses on day 42 post-immunization using a low dose of the linear DNA construct in a prime-boost regimen. Most importantly, vaccination significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.

Identification and genetic characterization of an isolate of bovine adenovirus 7 from the United States, a putative member of a new species in the genus Atadenovirus.

The bovine adenovirus 7 (BAdV-7) isolate SD18-74 was recovered from lung tissue of calves in South Dakota. The 30,043-nucleotide (nt) genome has the typical organization of Atadenovirus genus members. The sequence shares over 99% nt sequence identity with two Japanese BAdV-7 sequences, followed by 74.9% nt sequence identity with the ovine adenovirus 7 strain OAV287, a member of the species Ovine atadenovirus D. SD18-74 was amplified in both bovine and ovine primary nasal turbinate cells, demonstrating greater fitness in bovine cells. The genomic and biological characteristics of BAdV-7 SD18-74 support the inclusion of the members of the BAdV-7 group in a new species in the genus Atadenovirus.

Characterization of bovine ileal epithelial cell line for lectin binding, susceptibility to enteric pathogens, and TLR mediated immune responses.

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

GTPase-activating protein-binding protein 1 (G3BP1) plays an antiviral role against porcine epidemic diarrhea virus.

Porcine epidemic diarrhoea virus (PEDV) is a single-stranded, positive-sense RNA virus that belongs to the Coronaviridae. PEDV causes severe diarrhoea and dehydration in nursing piglets, which leads to significant economic losses to the swine industry worldwide. Stress granules (SGs) are sites of mRNA storage that are formed under various stress conditions including viral infections. Increasing evidence suggests that SGs function in antiviral innate immunity of host cells to limit virus replication. Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) is a key stress granule-resident protein that nucleates stress granule assembly. Depletion of G3BP1 inhibits SGs formation and overexpression of G3BP1 nucleates SGs assembly. We observed that knockdown of G3BP1 by silencing RNA significantly increased PEDV replication. Overexpression of exogenous G3BP1, on the other hand, lowered virus replication by 100-fold compared to vector control. An increase in the levels of mRNAs of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) was also observed in PEDV-infected G3BP1 depleted cells compared to PEDV-infected control cells. Taken together, our results suggest that G3BP1 plays an antiviral role and impairs PEDV replication.

Senecavirus A 3C Protease Mediates Host Cell Apoptosis Late in Infection.

Senecavirus A (SVA), an oncolytic picornavirus used for cancer treatment in humans, has recently emerged as a vesicular disease (VD)-causing agent in swine worldwide. Notably, SVA-induced VD is indistinguishable from foot-and-mouth disease (FMD) and other high-consequence VDs of pigs. Here we investigated the role of apoptosis on infection and replication of SVA. Given the critical role of the nuclear factor-kappa B (NF-κB) signaling pathway on modulation of cell death, we first assessed activation of NF-κB during SVA infection. Results here show that while early during infection SVA induces activation of NF-κB, as evidenced by nuclear translocation of NF-κB-p65 and NF-κB-mediated transcription, late in infection a cleaved product corresponding to the C-terminus of NF-κB-p65 is detected in infected cells, resulting in lower NF-κB transcriptional activity. Additionally, we assessed the potential role of SVA 3C protease (3Cpro) in SVA-induced host-cell apoptosis and cleavage of NF-κB-p65. Transient expression of SVA 3Cpro was associated with cleavage of NF-κB-p65 and Poly (ADP-ribose) polymerase (PARP), suggesting its involvement in virus-induced apoptosis. Most importantly, we showed that while cleavage of NF-κB-p65 is secondary to caspase activation, the proteolytic activity of SVA 3Cpro is essential for induction of apoptosis. Experiments using the pan-caspase inhibitor Z-VAD-FMK confirmed the relevance of late apoptosis for SVA infection, indicating that SVA induces apoptosis, presumably, as a mechanism to facilitate virus release and/or spread from infected cells. Together, these results suggest an important role of apoptosis for SVA infection biology.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV) 1-7-4-type strains in Peru.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. While PRRSV has been endemic in North America since 1989, it was not until 1999 that the virus was first described in South America. Notably, recently an increased number of PRRSV outbreaks have been reported in South American countries. However, epidemiological information related to these outbreaks is limited and the genetic characteristics of the PRRSV strains circulating in the region are poorly understood. In this study, we describe the genetic analyses of PRRSV strains associated with severe PRRS outbreaks in Peru. Samples originating from 14 farms located in two Departments in Peru (Lima and Arequipa), were subjected to RT-PCR amplification of the PRRSV ORF5 gene and sequencing followed by restriction fragment length polymorphism (RFLP) analysis. Results demonstrated the circulation of PRRSV-2 in Peru. Notably ORF5 RFLP typing revealed that 15 (75%) of the PRRSV strains detected in this study belong to the RFLP 1-7-4 type. Phylogenetic analysis showed that the Peruvian strains are closely related to the highly virulent PRRSV 1-7-4 strains that emerged in the US in 2013-2014. Results here indicate the presence of highly virulent PRRSV 1-7-4 strains in Peru and provide important information on the geographical distribution of PRRSV, confirming the recent geographical expansion of this important swine pathogen towards South America.

Detection of Fowlpox virus carrying distinct genome segments of Reticuloendotheliosis virus.

Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam's wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication.

A novel bovine papillomavirus type in the genus Dyokappapapillomavirus.

Papillomaviruses are a diverse group of viruses that are known to infect a wide range of animal species. Bovine papillomaviruses (BPVs) are divided into at least 21 genotypes (BPV1 to BPV21),  with most BPV isolates/strains described to date belonging to one of four genera, including Deltapapillomavirus, Xipapillomavirus, Epsilonpapillomavirus and Dyoxipapillomavirus. Here, we describe the identification and genetic characterization of a new BPV type in the genus Dyokappapapillomavirus. A farm in the state of New York, USA, reported chronic cases of vulvovaginitis in Holstein cows in 2016. Biopsies and/or swab samples collected from the vaginal mucosa were subjected to diagnostic investigation. Conventional diagnostic assays yielded negative results, and vaginal swab samples were subjected to viral metagenomic sequencing. Notably, BLAST searches revealed a papillomavirus genome with 7480 bp in length (67% nt sequence identity to BPV16). Additionally, phylogenetic analysis of the L1 gene of the papillomavirus identified here (tentatively named BPV22) revealed that it clusters with members of the genus Dyokappapapillomavirus. Interestingly, the recently identified BPV16, which was detected in fibropapilloma lesions in cattle also clusters within the Dyokappapapillomavirus group. Each virus, however, forms a separate branch in the phylogenetic tree. These results indicate that the putative BPV22 represents the second BPV within the genus Dyokappapapillomavirus.

Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity.

More effective vaccines are needed to control avian diseases. The use of chicken interferon gamma (chIFNγ) during vaccination is a potentially important but controversial approach that may improve the immune response to antigens. In the present study, three different systems to co-deliver chIFNγ with Newcastle disease virus (NDV) antigens were evaluated for their ability to enhance the avian immune response and their protective capacity upon challenge with virulent NDV. These systems consisted of: 1) a DNA vaccine expressing the Newcastle disease virus fusion (F) protein co-administered with a vector expressing the chIFNγ gene for in ovo and booster vaccination, 2) a recombinant Newcastle disease virus expressing the chIFNγ gene (rZJ1*L/IFNγ) used as a live vaccine delivered in ovo and into juvenile chickens, and 3) the same rZJ1*L/IFNγ virus used as an inactivated vaccine for juvenile chickens. Co-administration of chIFNγ with a DNA vaccine expressing the F protein resulted in higher levels of morbidity and mortality, and higher amounts of virulent virus shed after challenge when compared to the group that did not receive chIFNγ. The live vaccine system co-delivering chIFNγ did not enhanced post-vaccination antibody response, nor improved survival after hatch, when administered in ovo, and did not affect survival after challenge when administered to juvenile chickens. The low dose of the inactivated vaccine co-delivering active chIFNγ induced lower antibody titers than the groups that did not receive the cytokine. The high dose of this vaccine did not increase the antibody titers or antigen-specific memory response, and did not reduce the amount of challenge virus shed or mortality after challenge. In summary, regardless of the delivery system, chIFNγ, when administered simultaneously with the vaccine antigen, did not enhance Newcastle disease virus vaccine immunogenicity.

Expression of interferon gamma by a highly virulent strain of Newcastle disease virus decreases its pathogenicity in chickens.

The role of interferon gamma (IFN-γ) expression during Newcastle disease virus (NDV) infection in chickens is unknown. Infection of chickens with highly virulent NDV results in rapid death, which is preceded by increased expression of IFN-γ in target tissues. IFN-γ is a cytokine that has pleiotropic biological effects including intrinsic antiviral activity and immunomodulatory effects that may increase morbidity and mortality during infections. To better understand how IFN-γ contributes to NDV pathogenesis, the coding sequence of the chicken IFN-γ gene was inserted in the genome of the virulent NDV strain ZJ1 (rZJ1-IFNγ), and the effects of high levels of IFN-γ expression during infection were determined in vivo and in vitro. IFN-γ expression did not significantly affect NDV replication in fibroblast or in macrophage cell lines. However, it affected the pathogenesis of rZJ1-IFNγ in vivo. Relative to the virus expressing the green fluorescent protein (rZJ1-GFP) or lacking the IFN-γ insert (rZJ1-rev), expression of IFN-γ by rZJ1-IFNγ produced a marked decrease of pathogenicity in 4-week-old chickens, as evidenced by lack of mortality, decreased disease severity, virus shedding, and antigen distribution. These results suggest that early expression of IFN-γ had a significant protective role against the effects of highly virulent NDV infection in chickens, and further suggests that the level and timing of expression of this cytokine may be critical for the disease outcome. This is the first description of an in vivo attenuation of a highly virulent NDV by avian cytokines, and shows the feasibility to use NDV for cytokine delivery in chicken organs. This approach may facilitate the study of the role of other avian cytokines on the pathogenesis of NDV.

Newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range.

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are multifunctional proteins that play critical roles during infection. Here, we assessed the ability of NDV to replicate in macrophages and investigated the contribution of the F and HN proteins to NDV infection/replication in these cells. Results of our study revealed that, while presenting similar replication kinetics in a fibroblast cell line (DF1) or in primary non-adherent splenocytes, the NDV strain CA02 replicates better in macrophages (HD11 and primary adherent splenocytes) than the NDV strain Anhinga/93. Notably, exchange of the HN or both F and HN genes of NDV Anhinga/93 by the corresponding genes from NDV CA02 markedly improved the ability of the chimeric viruses to replicate in macrophages. These results indicate that the F and HN proteins are determinants of NDV macrophage host range. This represents the first description of productive NDV infection in macrophages.

Characterization of Newcastle disease viruses isolated from cormorant and gull species in the United States in 2010.

Newcastle disease virus (NDV), a member of the genus Avulavirus of the family Paramyxoviridae, is the causative agent of Newcastle disease (ND), a highly contagious disease that affects many species of birds and which frequently causes significant economic losses to the poultry industry worldwide. Virulent NDV (vNDV) is exotic in poultry in the United States; however, the virus has been frequently associated with outbreaks of ND in cormorants, which poses a significant threat to poultry species. Here, we present the characterization of 13 NDV isolates obtained from outbreaks of ND affecting cormorants and gulls in the states of Minnesota, Massachusetts, Maine, New Hampshire, and Maryland in 2010. All 2010 isolates are closely related to the viruses that caused the ND outbreaks in Minnesota in 2008, following the new evolutionary trend observed in cormorant NDV isolates since 2005. Similar to the results obtained with the 2008 isolates, the standard United States Department of Agriculture F-gene real-time reverse-transcription PCR (RRT-PCR) assay failed to detect the 2010 cormorant viruses, whereas all viruses were detected by a cormorant-specific F-gene RRT-PCR assay. Notably, NDV-positive gulls were captured on the eastern shore of Maryland, which represents a significant geographic expansion of the virus since its emergence in North America. This is the first report of vNDV originating from cormorants isolated from wild birds in Maryland and, notably, the first time that genotype V vNDV has been isolated from multiple wild bird species in the United States. These findings highlight the need for constant epidemiologic surveillance for NDV in wild bird populations and for consistent biosecurity measures to prevent the introduction of the agent into domestic poultry flocks.

Aspectos virológicos e clínico-patológicos da infecção genital aguda e latente pelo herpesvírus bovino tipo 1.2 em bezerras infectadas experimentalmente / Virological and clinico-pathological features of acute vulvovaginitis and latent infection by bovine herpesvirus 1.2 in heifers experimentally infected

A infecção genital de vacas pelo herpesvírus bovino tipo 1.2 (BoHV-1.2) pode resultar em vulvovaginite e infertilidade temporária. Após a infecção aguda, o BoHV-1 estabelece infecção latente, que pode cursar com episódios periódicos de reativação. O presente trabalho descreve os aspectos virológicos e clínico-patológicos da vulvovaginite aguda e infecção latente resultantes da inoculação de bezerras com uma amostra de BoHV-1.2 isolada de casos de balanopostite em touros. A inoculação do vírus em quatro bezerras pela via genital (10(8.1)TCID50/animal) resultou em replicação viral na mucosa genital e no desenvolvimento de vulvovaginite moderada a severa. Os animais inoculados excretaram o vírus nas secreções genitais até o dia 10 pós-inoculação (p.i.) com título máximo de 10(7.3)TCID50/mL. Foram observados congestão e edema da mucosa vulvovestibular, e formação de pequenas vesículas e pústulas. Durante a progressão clínica, as vesículas e pústulas aumentaram de tamanho e eventualmente se tornaram coalescentes e recobertas por um exsudato fino de coloração amarelada. Estes sinais foram observados a partir do dia 2 p.i. e aumentaram progressivamente de severidade até os dias 5-8 p.i. A administração de dexametasona no dia 55 p.i. resultou em excreção viral nas secreções genitais dos quatro animais por até 10 dias. A reativação da infecção latente foi acompanhada de recrudescência clínica, porém com sinais menos severos e com menor duração do que na infecção aguda. O DNA viral latente foi detectado por PCR, aos 36 dias pós-reativação (p.r.), nos seguintes tecidos: gânglio sacrais: pudendo (4/4); genitofemoral e retal caudal (3/4) e obturador (4/4) e em alguns linfonodos regionais. Estes resultados demonstram que o isolado SV-56/90 é virulento para fêmeas soronegativas, após inoculação genital, e pode ser utilizado em estudos de patogenia e de desafio vacinal.

Venereal infection of heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) may result in vulvovaginitis and transient infertility. The acute infection is followed by the establishment of latent infection which can be periodically reactivated. We herein describe the virology and clinico-pathological aspects of acute and recrudescent vulvovaginitis in heifers inoculated with a Brazilian BoHV-1.2 isolate recovered from an outbreak of balanoposthitis. Genital inoculation of isolate SV-56/90 (10(8.1)TCID50/animal) in four eight-months-old heifers resulted in efficient virus replication in the genital mucosa and the development of moderate to severe vulvovaginitis. The inoculated heifers shed virus in genital secretions in titers up to 10(7.3)TCID50/mL until day 10 pi and developed genital congestion, swelling, vesicles and pustules. The vesicles and pustules increased in size eventually coalesced and became covered with a yellowish exsudate. These signs appeared at day 2 pi, increased in severity up to days 5 - 8 pi and progressively subsided thereafter. Dexamethasone administration at day 55 pi resulted in virus shedding in vaginal secretions for up to 10 days. Virus reactivation in all animals was accompanied by clinical recrudescence of the disease, yet less severe than during acute infection. Examination of sacral ganglia and lymph nodes by PCR at day 36 post-reactivation revealed the presence of latent viral DNA in the pudendal (4/4), genito-femoral, sciatic and rectal caudal (3/4) and obturator nerve ganglia (1/4); in addition to several regional lymph nodes. These results demonstrate the virulence of isolate SV-56/90 for heifers and pave the way for its use in further pathogenesis studies and vaccine-challenge trials.

Caracterização clinicopatológica da mamilite aguda em ovelhas lactantes infectadas experimentalmente com o herpesvírus bovino 2 / Clinic and pathological characterization of acute mammillitis in lactating ewes inoculated with bovine herpesvirus 2

A mamilite causada pelo herpesvírus bovino tipo 2 (BoHV-2) possui importante repercussão sanitária em gado leiteiro de alguns países, mas a sua patogenia permanece pouco conhecida. Este trabalho descreve a reprodução e caracterização da mamilite aguda em ovelhas lactantes inoculadas com o BoHV-2 na pele do úbere e dos tetos. Cinco de oito ovelhas inoculadas desenvolveram extensas placas com necrose focal, pequenas vesículas e formação de crostas nos locais de inoculação. As lesões foram inicialmente observadas no 4º dia pós-inoculação (pi), progrediram em extensão e gravidade até os dias 7-8pi e posteriormente regrediram. O vírus foi isolado das lesões entre os dias 7 e 8 pi. Antígenos virais e partículas típicas de herpesvírus foram demonstrados por microscopia eletrônica em biópsias de lesões coletadas nos dias 5, 6 e 10pi. Os achados histológicos foram caracterizados por necrose epitelial, erosões e úlceras, com formação de células sinciciais e infiltrado inflamatório linfoplasmocitário na derme. Associadas a essas lesões observaram-se inclusões eosinofílicas intranucleares em células epiteliais, sinciciais e inflamatórias. Em um segundo experimento, 7 de 10 cordeiros inoculados na mucosa nasal desenvolveram hiperemia e descarga nasal, bem como vesículas e erosões no focinho. O vírus foi isolado das secreções nasais por até três dias e todos os cordeiros soroconverteram ao BoHV-2. Tentativas de reativar a infecção latente pela administração de dexametasona no 40º dia pi foram infrutíferas, pois não foram observados re-excreção viral, recrudescência clínica ou soroconversão. Esses resultados demonstram que ovelhas são susceptíveis à infecção experimental pelo BoHV-2 e desenvolvem lesões semelhantes às observadas em casos naturais de mamilite herpética. Além disso, esses resultados sugerem o uso dessa espécie animal como modelo para o estudo de vários aspectos da biologia da infecção pelo BoHV-2.

Mammillitis caused by bovine herpesvirus type 2 (BoHV-2) is an important disease in dairy herds yet its pathogenesis remains largely unknown. This report describes the reproduction and characterization of acute mammillitis in lactating ewes inoculated with BoHV-2 in the skin of the udder and teats. Five out of eight inoculated ewes developed large plaques, with focal necrosis, small vesicles and crust formation in the inoculated areas. The lesions were first observed on day 4 post-inoculation (pi), progressed in size and severity up to days 7-8pi and subsided progressively thereafter. Infectious virus was isolated from the lesions at days 7 and 8pi. Viral antigens and herpesvirus-like particles were demonstrated by electron microscopy in lesions examined at days 5, 6 and 10pi. Histological findings included epithelial necrosis, erosions and ulcers, and formation of syncytial cells. Intranuclear inclusions bodies in epithelial, syncytial and inflammatory cells and lymphoplasmacytic inflammatory infiltrate in the dermis were also observed. In a second experiment, seven out of ten lambs inoculated into the nostrils and muzzle developed nasal hyperemia and discharge, vesicles, and erosions in the nose. Infectious virus was isolated from lesions during up to three days and all lambs seroconverted to BoHV-2. Attempts to reactivate the latent infection by dexamethasone administration on day 40pi failed, since virus shedding, clinical recrudescence or seroconversion were not observed. The reproduction of acute infection and mammillitis resembling that occurring in cattle paves the way for the use of sheep to study several aspects of the biology of BoHV-2 infection.