Caprine Herpesvirus 1 Infection Research Articles

Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells

Caprine herpesvirus 1 (CpHV-1) is a member of the alpha subfamily of herpesviruses, which is responsible for genital lesions and latent infections in goat populations worldwide. In this study, for the first time, the transcriptome and proteomics of CpHV-1 infected Madin Darby bovine kidney (MDBK) cells were explored using RNA-Sequencing (RNA-Seq) and isobaric tags for relative and absolute quantitation-liquid chromatography tandem mass spectrometry (iTRAQ-LC-MS/MS) technology, respectively. RNA-Seq analysis revealed 81 up-regulated and 19 down-regulated differentially expressed genes (DEGs) between infected and mock-infected MDBK cells. Bioinformatics analysis revealed that most of these DEGs were mainly involved in the innate immune response, especially the interferon stimulated genes (ISGs). Gene Ontology (GO) enrichment analysis results indicated that the identified DEGs were significantly mainly enriched for response to virus, defense response to virus, response to biotic stimulus and regulation of innate immune response. Viral carcinogenesis, the RIG-I-like receptor signaling pathway, the cytosolic DNA-sensing pathway and pathways associated with several viral infections were found to be significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Eleven selected DEGs (Mx1, RSAD2, IFIT1, IFIT2, IFIT5, IFIH1, IFITM3, IRF7, IRF9, OAS1X and OAS1Y) associated with immune responses were selected, and they exhibited a concordant direction both in RNA-Seq and quantitative real-time RT-PCR analysis. Proteomic analysis also showed significant up-regulation of innate immunity-related proteins. GO analysis showed that the differentially expressed proteins were mostly enriched in defense response and response to virus, and the pathways associated with viral infection were enriched under KEGG analysis. Protein-protein interaction network analysis indicated most of the DEGs related to innate immune responses, as DDX58(RIG-I), IFIH1(MDA5), IRF7, Mx1, RSAD2, OAS1 and IFIT1, were located in the core of the network and highly connected with other DGEs. Our findings support the notion that CpHV-1 infection induced the transcription and protein expression alterations of a series of genes related to host innate immune response, which helps to elucidate the resistance of host cells to viral infection and to clarify the pathogenesis of CpHV-1.

Use of a commercial ELISA kit specific for glycoprotein E peptides to indirectly detect Caprine Herpesvirus 1 (CpHV-1) in the state of São Paulo, Brazil

ABSTRACT: Caprine herpesvirus 1 (CpHV-1) infection is associated with clinical manifestations related to animal age, with high mortality in kids and infertility in adults. Given the scarcity of research about the epidemiological situation of this infection in Brazilian flocks, we aimed to conduct a cross-sectional descriptive study to detect antibodies against CpHV-1 in goats in the state of São Paulo, Brazil. Fifty-five male and female goats — kids and adult — were assessed in this study. Blood serum was analyzed by a commercial ELISA kit to detect antibodies against CpHV-1, which had not been used in Brazil before. No animals were reactive. Brazil lacks information about CpHV-1 infection in goat flocks. Continuing the study is crucial to understand the epidemiological situation of the disease and establish protocols for infection control.

Caprine herpesvirus 1 (CpHV-1) as a potential candidate for oncolytic virotherapy

ABSTRACTCaprine Herpesvirus type 1 (CpHV-1) is a species-specific herpes virus able to induce apoptosis in several biological systems. In the present study we aimed to investigate the ability of CpHV-1 to reduce cells viability, to replicate and to cause cell death also in human cancer cell lines. We tested the CpHV-1 effects on HEL-299, Vero, MDA-MB-468, HeLa, U2OS, PC3, A549 and K562 neoplastic cell lines and on MDBK cells. Firstly, we evaluated the effect of CpHV-1 infection on cell viability by MTT assay and our data showed that CpHV-1 can induce a marked cytopathic effect (CPE) in most of cell lines tested, except for HEL-299, Vero and K562 cells. The reduction of cell viability was associated with a significant increase of viral production. We next investigated if CpHV-1 was able to induce cell death and so through western blotting analysis we evaluated cleaved caspase 3, LC3II and p62 protein levels after infection. Caspase 3 activation was detected in MDBK cells and, even if at different times p.i., also in MDA-MB-468, U2OS, and PC3 cell lines, while LC3II increase and concomitant p62 protein reduction were observed only in U2OS, and A549 cells, no significant alteration of these proteins was observed in the other cell lines tested. Finally, to confirm virus ability to trigger apoptosis we performed an Annexin-V apoptosis test after 24 h p.i.Although we need to further explore mechanisms underlying CpHV-1 treatment, this study could serve as the basis for the development of new treatment options aiming to fight several cancer types.

First Description of Infection of Caprine Herpesvirus 1 (CpHV-1) in Goats in Mainland France.

The purpose of this study was to investigate the epidemiological situation of the caprine herpesvirus 1 (CpHV-1) infection in nine districts in mainland France, mostly in the south, near Italy or Spain, where high seroprevalence has been observed. Two more central areas were also included in the study. The serosurvey was carried out in 9564 goats (275 herds) using bovine herpesvirus 1 (BoHV-1) glycoprotein B and E ELISAs. To confirm the presence of specific CpHV-1 antibodies, some of the samples were tested in neutralization assay. Results demonstrate, for the first time, CpHV-1 infection in goat herds on the French mainland. The analysis found cases of alphaherpesviruses infection in each district studied, with different levels of seroprevalence observed within each district (ranging from 0.2% to 31.56% at an individual level and from 9% to 46.2% for herd seroprevalence). Moreover, in the Alpes-Maritimes district, the seroprevalence seemed to be higher in older goats (79.45% of animals 6 years old or more) than in younger animals (40.99% of one-year-olds). This result suggests frequent virus re-excretion and circulation in herds. Results analysis also shows that the seroprevalence was higher when the herd size increased. In addition, the first French CpHV-1 strain was isolated from nasal swabs taken on an infected goat. The data reported herein demonstrate that CpHV-1 circulates in mainland France, which should henceforth be taken into consideration in cases of unexplained abortion in goats.

Caprine herpesvirus type 1 infection in goat: Not just a problem for females

Clinical, virological and serological analyses of a natural case of caprine herpesvirus 1 (CpHV-1) infection in a buck are reported. Three days after mating with a CpHV-1-infected female goat, the buck developed lesions referable to genital CpHV-1 infection. In particular, the animal suffered from typical painful ulcerative-crusted lesions associated with hyperemia, edema of the prepuce and healed completely within 15 days post-infection (p.i.). Infectious CpHV-1 was isolated from preputial swabs for 9 days p.i. while the virus was detected by real-time-PCR for 24 days p.i. Neutralizing antibodies were detected 7 days p.i. reaching maximal titers by day 14 p.i.The prolonged shedding of the virus from the preputial route may impact the genital transmission of CpHV-1 in goat flocks during mating.

Characterization of caprine herpesvirus 1 (CpHV1) glycoprotein E and glycoprotein I ectodomains expressed in mammalian cells

Caprine herpesvirus 1 (CpHV1) is a member of ruminant alphaherpesviruses antigenically related to the prototype bovine herpesvirus 1 (BoHV1). Although cross reactivity between the two viruses involves many structural glycoproteins, the use of two competitive BoHV1 ELISAs detecting anti gB and gE antibodies has been proposed for CpHV1 infection, resulting mainly in a gB+/gE- reactivity and leading to suppose that CpHV1 gE may represent an useful target for the development of specific diagnostic test. Since CpHV1 gE gene has been only partially characterized so far, in this study the genome fragment of the short unique unit (Us) encompassing gI and gE gene was amplified and sequenced. Gene fragments encoding the ectodomain of both glycoproteins were subcloned into pSECTag2/Hygro and expressed in HEK293T cells as secreted form in serum free medium. Due to the lack of specific monoclonal antibodies (Mabs), the same recombinant glycoproteins were obtained from BoHV1 and used as positive control with a panel of specific gE and gI Mabs as well as in some ELISA assays. Results clearly indicate that the ectodomain of CpHV1 gE, immobilized on solid face in an indirect ELISA format, represents a sensitive and specific marker of infection, when compared with neutralization test, with absence of very low degree of cross-reactivity with BoHV1 gE counterpart, while the use of CpHV1 gI-ELISA or a combination of gE/gI complex did not significantly improve the sensitivity of the assay. In addition, in the rare event in which cross species barrier occurs for both viruses from their natural host to other species, the use of both BoHV1 and CpHV1 gE in a comparative assay may be proposed.

Serological evidence and risk factors associated with Caprine herpesvirus 1 in dairy goat flocks in a semiarid region of northeastern Brazil

A cross-sectional study was carried out to determine the flock-level seroprevalence of Caprine herpesvirus 1 (CpHV-1) and Bovine herpesvirus 1 (BoHV-1) and 2 (BoHV-2) and risk factors associated with CpHV-1 in dairy goat flocks from a semiarid region of northeastern Brazil. A total of 1,034 serum samples from 110 flocks were collected from March 2009 through March 2010. A structured questionnaire focusing on variables related to risk factors for CpHV-1 infection was given to each farmer at the time of blood collection. Antibodies against CpHV-1, BoHV-1, and BoHV-2 were detected by neutralization tests. The flock-level prevalences of CpHV-1, BoHV-1, and BoHV-2 were 89.1% (98/110; 95% confidence interval [CI]: 81.7-94.2), 80% (88/110; 95% CI: 71.3-87), and 4.5% (5/110; 95% CI: 1.5-10.3), respectively. Frequencies of seropositive animals were 36.6% (379/1,034), 25.8% (267/1,034), and 0.6% (6/1,034) for CpHV-1, BoHV-1, and BoHV-2, respectively. The use of natural mating was identified as a risk factor associated with CpHV-1 flock-level prevalence (P = 0.001). It is suggested that adoption of veterinary services and active surveillance of the at-risk flocks in the study region should be initiated to reduce the prevalence of herpesvirus infections.

A caprine herpesvirus 1 vaccine adjuvanted with MF59™ protects against vaginal infection and interferes with the establishment of latency in goats.

The immunogenicity and the efficacy of a beta-propiolactone-inactivated caprine herpesvirus 1 (CpHV-1) vaccine adjuvanted with MF59™ were tested in goats. Following two subcutaneous immunizations, goats developed high titers of CpHV-1-specific serum and vaginal IgG and high serum virus neutralization (VN) titers. Peripheral blood mononuclear cells (PBMC) stimulated in vitro with inactivated CpHV-1 produced high levels of soluble IFN-gamma and exhibited high frequencies of IFN-gamma producing cells while soluble IL-4 was undetectable. On the other hand, control goats receiving the inactivated CpHV-1 vaccine without adjuvant produced only low serum antibody responses. A vaginal challenge with virulent CpHV-1 was performed in all vaccinated goats and in naïve goats to assess the efficacy of the two vaccines. Vaginal disease was not detected in goats vaccinated with inactivated CpHV-1 plus MF59™ and these animals had undetectable levels of infectious challenge virus in their vaginal washes. Goats vaccinated with inactivated CpHV-1 in the absence of adjuvant exhibited a less severe disease when compared to naïve goats but shed titers of challenge virus that were similar to those of naïve goats. Detection and quantitation of latent CpHV-1 DNA in sacral ganglia in challenged goats revealed that the inactivated CpHV-1 plus MF59™ vaccine was able to significantly reduce the latent viral load when compared either to the naïve goats or to the goats vaccinated with inactivated CpHV-1 in the absence of adjuvant. Thus, a vaccine composed of inactivated CpHV-1 plus MF59™ as adjuvant was strongly immunogenic and induced effective immunity against vaginal CpHV-1 infection in goats.

Detection of Caprine Herpesvirus 1–Specific Antibodies in Goat Sera Using an Enzyme-Linked Immunosorbent Assay and Serum Neutralization Test

An enzyme-linked immunosorbent assay (ELISA) was developed to detect immunoglobulin G (IgG) antibodies directed to whole Caprine herpesvirus 1 (CpHV-1). Sera from 248 goats were obtained from CpHV-1-free and CpHV-1-infected flocks and were subjected to both IgG ELISA and serum neutralization (SN) assays, with the latter considered the gold standard for the diagnosis of CpHV-1 infection. In flocks where CpHV-1 infection was detected, 57 sera were negative by the SN and the ELISA tests and 97 sera were positive with both tests. Thus, although based on biologically different principles, the ELISA was as sensitive as the SN assay in detecting seropositive animals and could be efficiently used as a faster and less expensive alternative to the SN test for the screening of many samples.

Analysis of apoptosis induced by Caprine Herpesvirus 1 in vitro

It is known that Caprine Herpesvirus 1 (CpHV-1) causes apoptosis in mitogen-stimulated as well as not stimulated caprine peripheral blood mononuclear cells (PBMC). Initial experiments in Madin Darby bovine kidney (MDBK) cells revealed that CpHV-1 infection induced apoptotic features like chromatin condensation and DNA laddering. Thus, to characterize in more detail this apoptotic process, activation of caspase-8, -9 and -3 in MDBK cells CpHV-1 infected was investigated and demonstrated. In addition, CpHV-1 infection resulted in disruption of mitochondrial membrane potential, cytochrome c release and alterations in the pro- and anti-apoptotic proteins of Bcl-2 family. Proteolytic cleavage of poly(ADP-ribose) polymerases (PARP), confirming the activation of downstream caspases, was also observed. Our data indicated that a “cross-talk” between the death-receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway occurred in CpHV-1-induced apoptosis in vitro.

Isolation of caprine herpesvirus 1 from a major outbreak of infectious pustular vulvovaginitis in goats

We describe an outbreak of infectious pustular vulvovaginitis caused by Caprine herpesvirus 1 (CpHV1) in a group of approximately 200, 8 month old virgin does that were imported to Victoria from New Zealand. CpHV1 was isolated in cell cultures from vaginal swabs from three of three affected does but not from two bucks that had been with the does. The identity of the virus as a herpesvirus was confirmed by negative stain electron microscopy. Restriction endonuclease DNA fingerprint analysis showed that the DNA fingerprints were similar, but not identical, to previously described CpHV1 isolates made in New Zealand, New South Wales, and in other parts of the world. Acute and convalescent phase sera from selected does supported the diagnosis of CpHV1 infection. It is most likely that the disease was initiated by reactivation of latent virus in at least one of four bucks that served the does, since each was positive for CpHV neutralising antibody when first tested. This is the first report of CpHV infectious pustular vulvovaginitis in goats in Victoria and to our knowledge appears to be one of the largest outbreaks recorded anywhere.

Cidofovir is effective against caprine herpesvirus 1 infection in goats

Caprine herpesvirus 1 (CpHV-1) is a virus able to cause genital infection leading to vulvovaginitis or balanoposthitis in adult goats. CpHV-1 shares several biological similarities with herpes simplex type 2 (HSV-2) infection in man, such as genital tropism, type and site of typical lesions and it might provide an animal model for studies on antiviral drugs for HSV-2 infection in man. In this view the efficacy of cidofovir (CDV) drug was tested in six goats intravaginally infected with BA.1 strain of CpHV-1. Three goats received an intravaginal application of 3 ml of a 1% CDV preparation at 4 h post infection and then every 12 h for five consecutive days. Three goats were kept as untreated controls. The goats were daily examined for clinical evidence of the infection and viral shedding. CDV was able to protect against disease progression and inhibited the onset of the local lesions due to the CpHV-1 replication. Treated animals shed virus for a shorter period (3 days less) and at lower titres than the control animals. CpHV-1 infection in goats may represent an excellent animal model for the study of novel strategies for the treatment of primary genital HSV-2 infection in man.

A live attenuated glycoprotein E negative bovine herpesvirus 1 vaccine induces a partial cross-protection against caprine herpesvirus 1 infection in goats

Taking into account the close antigenic relationship between bovine herpesvirus 1 (BoHV-1) and caprine herpesvirus 1 (CpHV-1), a live attenuated glycoprotein E (gE) negative BoHV-1 vaccine was assessed in goats with the aim to protect against CpHV-1 infection. Vaccine safety was evaluated by intranasal inoculation of two groups of goats with either a gE-negative BoHV-1 vaccine or a virulent BoHV-1. The length of viral excretion and the peak viral titre were reduced with the gE-negative vaccine. To assess the efficacy, two goats were inoculated intranasally twice 2 weeks apart with a gE-negative BoHV-1 vaccine. Four weeks later, immunised and control goats were challenged with CpHV-1. A 2 log(10) reduction in the peak viral titre was observed and the challenge virus excretion lasted 2 days more in immunised than in control goats. These data indicate the safety and the partial efficacy of a live attenuated gE-negative BoHV-1 vaccine intranasally administrated in goats.

Latency and reactivation of bovine herpesvirus 1 (BHV-1) in goats and of caprine herpesvirus 1 (CapHV-1) in calves.

Bovine Herpesvirus 1 (BHV-1) and Caprine Herpesvirus 1 (CapHV-1) are related members of the herpesvirus family. Since their natural hosts are often kept in close contact with each other, concern was raised that a reservoir might be established in the heterologous host in addition to the homologous host. To investigate this possibility, cross-infection experiments with BHV-1 in goats and CapHV-1 in calves were performed. BHV-1 infected goats developed mild disease signs during acute infection, whereas CapHV-1 infection in calves took a subclinical course. However, virus excretion and antibody production were indicative of successful cross-infection of both BHV-1 and CapHV-1. Reactivation of BHV-1 was achieved in 5 out of 8 goats as demonstrated by recurrent virus excretion and rising antibody titers. In constrast CapHV-1 in calves could not be reactivated experimentally. Nevertheless, PCR revealed that both viruses established latency in the trigeminal ganglia of the heterologous host. Based on these results we conclude that goats should indeed be regarded as a potential BHV-1 reservoir, which must be considered during IBR eradication programs.