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Elephant endotheliotropic herpesviruses (EEHVs) are the cause of acute hemorrhagic disease in endangered Asian and African elephants. In the present study, we report the incidence of EEHV infection and associated mortality in the captive elephant of Assam, India. Our result showed the gross morphology and histopathological changes of EEHV infection in the elephant. Moreover, the phylogenetic analysis of the polymerase, helicase, and GPCR genes from the infected tissue samples suggested the presence of EEHV1A virus.

A high morbidity, high mortality disease process caused flock deaths in an Indian ringneck parrot (Psittacula krameri) aviary flock in Victoria, Australia. Affected birds were either found dead with no prior signs of illness, or showed clinical evidence of respiratory tract disease, with snicking, sneezing and dyspnoea present in affected birds. Necropsy examinations performed on representative birds, followed by cytological and histopathological examination, demonstrated lesions consistent with a herpesvirus bronchointerstitial pneumonia. Transmission electron microscopy analysis of lung tissue demonstrated typical herpesvirus virions measuring approximately 220 nm in diameter. Next generation sequencing of genomic DNA from lung tissue revealed a highly divergent novel Psittacid alphaherpesvirus of the genus Iltovirus. Iltoviruses have been previously reported to cause respiratory disease in a variety of avian species, but molecular characterisation of the viruses implicated has been lacking. This study presents the genome sequence of a novel avian herpesvirus species designated Psittacid alphaherpesvirus-5 (PsHV-5), providing an insight into the evolutionary relationships of the alphaherpesviruses.

Fibropapillomatosis (FP), a neoplastic disease characterized by the formation of multiple tumors affecting different species of sea turtles and, most often, the green turtle (Chelonia mydas), is considered one of the major threats to the survival of this species. Recent studies indicate that Chelonid herpesvirus (ChHV5) is the etiological agent of this disease, though its association with anthropogenically altered environments and the immune status of these animals also appears to contribute to disease expression and tumor formation. In this study, tumor biopsy and secretions from green turtles captured off the coast of São Paulo State, Brazil, were used in histological and molecular analyses to detect and characterize circulating ChHV5. In 40.9% of cases, the tumor histopathological findings revealed focal ballooning degeneration with intranuclear inclusion bodies, results which are suggestive of viral infection. ChHV5 was detected using polymerase chain reaction (PCR) on the animals' skin, ocular tumor biopsies, and ocular and oral secretions. The analysis of the detected ChHV5 sequences revealed two distinct genetic sequences together. Phylogenetic analysis indicated that Brazilian samples were similar to ChHV5 samples described for the Atlantic phylogeographic group and are therefore part of the same clade as the Gulf of Guinea and Puerto Rico samples. This similarity suggests a possible flow of the virus between these three regions.

Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100μM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models.

Feline vaccination guidelines recommend less frequent boosters for the core vaccines (rhinotracheitis, calicivirosis and infectious panleucopenia). Most guidelines recommend boosters at 3-yearly intervals after a basic vaccination including primary vaccination and revaccination one year later. The objective of this study was to assess the duration of immunity induced by PUREVAX(®) RCPCh FeLV, a non-adjuvanted vaccine against feline rhinotracheitis, calicivirosis, infectious panleucopenia, chlamydiosis and leukemia. After primary vaccination followed by revaccination one year later with a vaccine formulated at minimum dose, the cats were kept in a confined environment and challenged 3 years later with a virulent heterologous strain of feline calicivirus (FCV) and subsequently a virulent strain of feline herpesvirus (FHV). Clinical signs and viral excretion were recorded for two weeks after each viral inoculation. Contemporary unvaccinated cats and new animals added at the time of challenge were used as controls. The vaccination regimen induced a stable and long-lasting humoral response. Vaccination resulted in a significant reduction in the severity of the disease after FHV challenge and in the frequency of cats showing a severe calicivirosis (defined as a combination of systemic clinical symptoms and oronasal ulcers). As opposed to the significant reduction of excretion observed a few weeks after primo-vaccination or even one year after vaccination for FCV, viral shedding was not reduced 3 years after revaccination. This study showed that primary vaccination and revaccination one year later with PUREVAX(®) RCPCh FeLV was able to induce 3-year duration of immunity against FCV and FHV. The results and conclusion of this study are consistent with current vaccination guidelines and will allow the veterinarian to adapt the vaccination regimen to the way of life of the cat.

Highlights • Sequestration of cholesterol from the cell membrane inhibits CyHV-3 entry. • CyHV-3 egress requires cholesterol. • Lipid composition of the CyHV-3 envelope is similar to that of CCB lipid rafts., The Cyprinus herpesvirus 3 (CyHV-3) is a member of the new Alloherpesviridae virus family in the Herpesvirales order. CyHV-3 has been implicated in a large number of disease outbreaks in carp populations causing up to 100% mortality. The aim of this study was to investigate the requirement of cholesterol-rich lipid rafts in CyHV-3 entry and replication in carp cells. Plasma membrane cholesterol was depleted from common carp brain (CCB) cells with methyl-β-cyclodextrin (MβCD). Treated and non-treated cells were infected with CyHV-3 and virus binding and infection parameters were assessed using RT-qPCR, immunocytochemistry and virus titration. The effect of cholesterol reduction severely stunted virus entry in vitro, however after cholesterol replenishment virus entry and subsequent replication rates were similar to the control infection. Furthermore, cholesterol depletion did not significantly influence virus binding and the subsequent post-entry replication stage, however had an impact on virus egress. Comparative analysis of the lipid compositions of CyHV-3 and CCB membrane fractions revealed strong similarities between the lipid composition of the CyHV-3 and CCB lipid rafts. The results presented here show that cholesterol-rich lipid rafts are important for the CyHV-3 replication cycle especially during entry and egress.

Herpesviruses are significant pathogens of chelonians which most commonly cause upper respiratory tract disease and necrotizing stomatitis. Herpesvirus infection was identified in two populations of captive Eastern box turtles (Terrapene carolina carolina) using histopathology and polymerase chain reaction (PCR) with DNA sequencing. Necrotizing lesions with eosinophilic to amphophilic intranuclear inclusion bodies were identified in the tissues of one hatch-year individual in January 2013, which was herpesvirus positive by PCR. A separate captive group of adults had an observed herpesvirus prevalence of 58% using PCR in July 2011. In these cases, a novel herpesvirus, Terrapene herpesvirus 1 (TerHV1), was identified and serves as the first herpesvirus sequenced in the genus Terrapene. Similar to the other herpesviruses of the Order Testudines, TerHV1 clusters with the genus Scutavirus of the subfamily Alphaherpesvirinae.

Herpesviruses are ubiquitous in animals and cause economic losses concomitant with many diseases. Most of the domestic animal herpesviruses are within the subfamily Alphaherpesvirinae, which includes human herpes simplex virus 1 (HSV-1). Suppression of HSV-1 replication has been reported with α-hydroxytropolones (αHTs), aromatic ring compounds that have broad bioactivity due to potent chelating activity. It is postulated that αHTs inhibit enzymes within the nucleotidyltransferase superfamily (NTS). These enzymes require divalent cations for nucleic acid cleavage activity. Potential targets include the nuclease component of the herpesvirus terminase (pUL15C), a highly conserved NTS-like enzyme that cleaves viral DNA into genomic lengths prior to packaging into capsids. Inhibition of pUL15C activity in biochemical assays by various αHTs previously revealed a spectrum of potencies. Interestingly, the most potent anti-pUL15C αHT inhibited HSV-1 replication to a limited extent in cell culture. The aim of this study was to evaluate three different αHT molecules with varying biochemical anti-pUL15C activity for a capacity to inhibit replication of veterinary herpesviruses (BoHV-1, EHV-1, and FHV-1) and HSV-1. Given the known discordant potencies between anti-pUL15C and HSV-1 replication inhibition, a second objective was to elucidate the mechanism of action of these compounds. The results show that αHTs broadly inhibit herpesviruses, with similar inhibitory effect against HSV-1, BoHV-1, EHV-1, and FHV-1. Based on immunoblotting, Southern blotting, and real-time qPCR, the compounds were found to specifically inhibit viral DNA replication. Thus, αHTs represent a new class of broadly active anti-herpesviral compounds with potential veterinary applications.

Cyprinid herpesvirus 2 (CyHV-2) is known as the causative agent of herpesviral haematopoietic necrosis (HVHN) of goldfish (Carassius auratus). Recently, the virus has also been detected from Prussian carp (C. gibelio) and crucian carp (C. carassius) from European and Asian countries. To analyze the risk of spreading to new host species, the susceptibility of other fish species to the virus is essential. In this study experimental infections of indigenous Cyprininae species in Japan were performed by immersion in and intraperitoneal injection of a CyHV-2 isolate. Although Edonishiki, a variety of goldfish, immersed with the virus showed a cumulative mortality of 90%, no mortality was observed in ginbuna C. auratus langsdorfii, nagabuna C. auratus buergeri, nigorobuna C. auratus grandoculis and common carp Cyprinus carpio. Cumulative mortality was 100, 20 and 10% in intraperitoneally injected Edonishiki, ginbuna and nagabuna, respectively. Furthermore all Edonishiki immersed with the virus died. However, even after stimuli of sudden temperature changes, the immersed ginbuna and nagabuna did not die. Moreover no mortality was observed in co-reared Ranchu, another variety of goldfish, with immersed ginbuna and nagabuna although all three Ranchu co-reared with immersed Edonishiki died. CyHV-2 DNA was detected and the virus was re-isolated from all dead fish. Moreover CyHV-2 DNA was detected from some of the surviving Carassius spp. These results revealed that susceptibility of Japanese indigenous Cyprininae fish species to CyHV-2 is much lower than for goldfish. In addition, ability of replication of CyHV-2 might be different among Carassius fish species.

An epizootic with severe mortality has emerged in cultured gibel carp, Carassius auratus gibelio, in China since 2009, and caused huge economic loss. The signs and epidemiology background of the disease were investigated. Parasite examination, bacteria and virus isolation were carried out for pathogen isolation. The causative pathogen was obtained and identified as Cyprinid herpesvirus 2 (CyHV-2) by experimental infection, electron microscopy, cell culture, PCR assay and sequence alignment, designated as CyHV-2-JSSY. Experimental infection proved the high virulence of CyHV-2-JSSY to healthy gibel carp. Electron microscopy revealed that the viral nucleocapsid was hexagonal in shape measuring 110-120 nm in diameter with a 170-200 nm envelope. The virus caused significant CPE in Koi-Fin cells at the early passages, but not beyond the fifth passages. Sequence alignment of the partial viral helicase gene (JX566884) showed that it shared 99-100% identity to the published sequences of other CyHV-2 isolates. This study represented the first isolation and identification of CyHV-2 in cultured gibel carp in China and laid a foundation for the further studies of the disease.

Phocine herpesvirus 1 (PhHV-1) infections in seals are associated with disease and sometimes high mortality, primarily in young animals. PhHV-1 has been detected in seals from European waters as well as in waters on both coasts of North America. Serological surveys of various pinniped species have indicated a wide geographical distribution of PhHV-1. A quantitative and sensitive real-time PCR assay targeting the gene encoding glycoprotein B of PhHV-1 was developed for detection of PhHV-1 in ocular and nasal swab samples from wild harbor seals (Phoca vitulina) from Svalbard (Norway). PhHV-1 DNA was detected in samples from 6 (8%) seals collected in 2009 and 2 (3%) in 2010; all had herpesvirus DNA in the ocular swab sample, whereas only one of these animals also had herpesvirus DNA in the nasal swab sample. Four PCR positive animals were approximately 1 year of age and four were pups of the year. Serum samples obtained in 1998 (n=59), 1999 (n=74), 2000 (n=81), 2009 (n=69) and 2010 (n=83) were tested for anti-PhHV-1 antibodies in an indirect ELISA. The PhHV-1 seroprevalence in the population remained high throughout this period, varying from 77 to 100% between years. No eye disease was observed in this harbor seal population, but the ELISA and PCR findings reported here suggest that PhHV-1 is endemic in this globally northernmost harbor seal colony, and that the virus is shed on the mucosa of the eye and nose.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly infectious causative agent to common carp and koi worldwide. The virus is mainly consisted of European and Asian genotype isolates. To date, no European genotype CyHV-3 has been found emerging in the East and Southeast Asian regions. In late March 2011, an outbreak of CyHV-3 disease occurred in Guangzhou City, Guangdong Province, China, resulting in the deaths of approximately 200 large-sized adult koi within four weeks. One moribund koi was sampled for CyHV-3 isolation. Thus, a CyHV-3 was isolated in KCF-1 cells and designated as KHV-GZ11. Abundant mature or immature virions in infected KCF-1 cells were observed under a transmission electron micrograph. In addition, intra-nuclear inclusion body-like structures with masses of virions were also observed. Based on the TK and ORF136H genes, the sequence analyses revealed that KHV-GZ11 is a distinct European genotype of CyHV-3. Moreover, the infectivity experiment showed that KHV-GZ11 was highly virulent to koi. In summary, we are the first to confirm the emergence of fatal European genotype CyHV-3/KHV in East and Southeast Asia. Our study will provide new insight to explore the virus origin and epidemiology, as well as its pathogenicity.

Highlights • Vaccination induces a pharyngeal antibody response in shorthorn zebu cross (SZC). • Direct challenge with the AlHV-1 virus is effective at inducing MCF in SZC. • Attenuated AlHV–1 + Emulsigen® vaccine efficacy in SZC calculated to be 50%. • Bacterial flagellin is not a good adjuvant as inclusion reduced antibody response. • We provide evidence that non-fatal AlHV-1 infections occur in SZC., Malignant catarrhal fever (MCF) is a fatal disease of cattle that, in East Africa, follows contact with wildebeest excreting alcelaphine herpesvirus 1 (AlHV-1). Recently an attenuated vaccine (atAlHV-1) was tested under experimental challenge on Friesian-Holstein (FH) cattle and gave a vaccine efficacy (VE) of approximately 90%. However testing under field conditions on an East African breed, the shorthorn zebu cross (SZC), gave a VE of 56% suggesting that FH and SZC cattle may respond differently to the vaccine. To investigate, a challenge trial was carried out using SZC. Additionally three adjuvant combinations were tested: (i) Emulsigen®, (ii) bacterial flagellin (FliC) and (iii) Emulsigen® + bacterial flagellin. We report 100% seroconversion in all immunized cattle. The group inoculated with atAlHV-1 + Emulsigen® had significantly higher antibody titres than groups inoculated with FliC, the smallest number of animals that became infected and the fewest fatalities, suggesting this was the most effective combination. A larger study is required to more accurately determine the protective effect of this regime in SZC. There was an apparent inhibition of the antibody response in cattle inoculated with atAlHV-1 + FliC, suggesting FliC might induce an immune suppressive mechanism. The VE in SZC (50–60%) was less than that in FH (80–90%). We speculate that this might be due to increased risk of disease in vaccinated SZC (suggesting that the vaccine may be less effective at stimulating an appropriate immune response in this breed) and/or increased survival in unvaccinated SZC (suggesting that these cattle may have a degree of prior immunity against infection with AlHV-1).

Malignant catarrhal fever (MCF), a frequently fatal herpesviral disease primarily of ruminant species, has been sporadically reported in pigs. All cases of naturally occurring porcine MCF reported to date have been linked to ovine herpesvirus 2 (OvHV-2), a gammaherpesvirus in the genus Macavirus carried by sheep. Experimental induction of MCF by aerosolization of the virus in nasal secretions collected from infected sheep has been successful in bison, cattle and rabbits. The goals of this study were to determine the susceptibility of pigs to MCF following experimental intranasal inoculation of OvHV-2, and to characterize the disease. Twelve pigs in four groups were nebulized with 105, 106, 107, or 108 DNA copies of OvHV-2 from sheep nasal secretions. Three control pigs were nebulized with nasal secretions from uninfected sheep. Three additional pigs were inoculated intravenously with 107 DNA copies of OvHV-2 to evaluate this route of infection with cell-free virus. Seven of twelve intranasally challenged pigs became infected with OvHV-2. Five of these seven, all in higher dose groups, developed MCF. Lesions resembled those reported in natural cases of porcine MCF. The most striking and consistent histological lesions were in trachea, lung, kidney and brain. These comprised mucopurulent tracheitis, interstitial pneumonia, necrotizing arteritis–periarteritis, and nonpurulent meningoencephalitis. No infection was established in the intravenously challenged or control groups. The study showed that MCF can be experimentally induced in pigs by aerosol challenge using sheep nasal secretions containing OvHV-2. Domestic pigs are a natural clinically susceptible host for sheep-associated MCF. They represent a useful, cost-effective model for MCF research.

Cyprinid herpesvirus 3 (CyHV-3) is a lethal DNA virus that infects common carp and koi. It has caused outbreak of the disease within both aquaculture and natural environmental ecosystems. However, there is not enough understanding of the distribution of CyHV-3 in the natural environments, partly because there is no suitable quantification method. In this study, we tested CyHV-3 extraction methods from sediment and then compared its abundance between sediment and water using real-time PCR. Sediment samples were taken from lake and pond, and total viral DNA was extracted using the viral elution method recommended by the US Environmental Protection Agency (manual method), as well as a commercial DNA extraction kit for soil (commercial kit method) before PCR detection. 7 of 12 (58%) and 5 of 10 (50%) sediment samples showed PCR positive signal for CyHV-3 DNA using the manual method and the commercial kit, respectively, and consistent results were obtained from the samples using the manual method between two independent primer sets. The quantification of CyHV-3 DNA in natural sediment using the manual method and external standard virus revealed that its concentration was 1.2 × 104 to 3.3 × 105 copies DNA/kg. The concentration in sediments was 46–1238 times higher than that in water from the same location, suggesting that sediment could act as a reservoir for CyHV-3 in natural freshwater environments. This is the first report of the existence of CyHV-3 in the sediment of a natural lake or pond.

Psittacid herpesvirus (PsHV) was isolated from 41 birds kept in captivity in Belo Horizonte, Minas Gerais/Brazil using chicken embryo fibroblasts (CEF) cell cultures. For this study, leukocytes or cloacal swabs of live birds were used. Also, portions of liver, spleen or kidney from birds collected at necropsy were utilized for these tests. PCR tests confirmed the presence of PsHV in 100% of samples. Thirty-three of the PCR products were sequenced and the results disclosed a 99% and 100% identity when compared with other sequences PsHV-1 (AY372243.1 and AF261756.1), previously deposited in GenBank. In addition, histopathology was performed and 19 of the 29 birds contained random multifocal lymphoplasmacytic hepatitis with necrotic foci, suggestive of viral infection. Three samples were examined by electron microscopy to visualize the viral particles obtained from cell culture. The viral structures measured 269 nm in average, had envelopes with an icosahedral capsid and tegument, consistent with herpesvirus. Thus, a total of 41 isolates were obtained from PsHV cell cultivation in CEF, confirming the circulation of the virus between parrots kept in captivity in Belo Horizonte, and affirming the importance of further studies in this area.

Malignant catarrhal fever (MCF) is a frequently fatal lymphoproliferative disease syndrome primarily of ruminant species, caused by gammaherpesviruses in the genus Macavirus. Ovine herpesvirus 2 (OvHV-2), carried by sheep, causes sheep-associated MCF worldwide, while Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest, causes wildebeest-associated MCF, mainly in Africa. Diseases in rabbits can be induced by both viruses, which are clinically and pathologically similar; however, recent studies revealed different expression of viral genes associated with latency or lytic replication during clinical disease between the two viruses. In this study, we further characterized experimentally induced MCF in rabbits by nebulization with OvHV-2 from sheep nasal secretions to elucidate the course of viral replication, along with in vivo incorporation of 5- Bromo-2 0-Deoxyuridine (BrdU), to evaluate lymphoproliferation. All six rabbits nebulized with OvHV-2 developed MCF between 24 and 29 days post infection. OvHV-2 DNA levels in peripheral blood leukocytes (PBL) remained undetectable during the incubation period and increased dramatically a few days before onset of clinical signs. During the clinical stage, we found that predominantly lytic gene expression was detected in PBL and tissues, and both T and B cells were proliferating. The data showed that the viral gene expression profile and lymphoproliferation in rabbits with OvHV-2 induced MCF were different from that in rabbits with AlHV-1 induced MCF, suggesting that OvHV-2 and AlHV-1 may play a different role in MCF pathogenesis. Published by Elsevier B.V.

In a zoological collection, four black bears (Ursus americanus) died from neurological disease within six months. Independently in a geographically different zoo, two Thomson's gazelles (Eudorcas thomsoni) and 18 guinea pigs (Cavia porcellus f. dom.) suffered from neurological disorders. In addition, guinea pigs showed abortions and stillbirths. All affected animals displayed a non suppurative meningoencephalitis with intranuclear inclusion bodies. Immunohistology demonstrated equine herpes virus antigen and ultrastructurally herpes viral particles were detected. Virus isolation and molecular analysis identified neurotropic equine herpesvirus (EHV) 1 strains in both epizootics. There is serological evidence of a possible virus transmission from other equids to the affected animals. Cross-species transmission of EHV-1 should be considered in the management of captive wild equids and ungulates, particularly with respect to fatal disease in irreplaceable species.

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9(-)), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10(7.69) TCID(50)/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10(9.25) TCID(50) of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11-13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9(-) recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves.

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease of some members of the order Artiodactyla. OvHV-2 is carried as a lifelong subclinical infection in sheep (Ovis aries). To date OvHV-2 has not been propagated in vitro and this has hampered studies of viral pathogenesis and efforts to develop a vaccine to protect animals from SA-MCF. Lytic OvHV-2 replication occurs in the lungs of experimentally infected sheep at early times post-inoculation (PI) and in the nasal cavities of naturally infected sheep during virus shedding episodes. Identification of specific cell types supporting lytic virus replication in vivo provides information that can be used in the development of an in vitro propagation system for the virus. Using fluorescence immunohistochemical techniques, we identified lytically infected alveolar epithelial cells in the lungs of sheep early during infection. Lytically infected epithelial cells were also detected in samples of nasal secretions collected from naturally infected sheep during episodes of virus shedding. This is the first reported identification in the natural reservoir species of specific cell types that support OvHV-2 lytic replication in vivo.

Herpesviruses are highly host specific and share a long synchronous evolution with their hosts. Only in rare cases, species barriers fall and allow animal to human or human to animal transmission. Among the zoonotic herpesviruses, Cercopithecine herpesvirus 1 is the most significant and can be transmitted from macaques to human. Conversely, Human herpesvirus 1 is capable of causing severe disease in primates. Besides those two examples, there are several herpesviruses with a certainly limited or only suspected ability to cross species barriers. Those include Saimiriine herpesvirus 2, Phocid herpesvirus 2, Equid herpesvirus 1, Epstein-Barr Virus, Marek's disease virus, and Pseudorabies virus. Concerning xenotransplantations, porcine gammaherpesviruses must be considered as a zoonotic threat.

Infection with Equid Herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism (A 2254 /G 2254 ) in the genome region of the open reading frame 30 (ORF30), which results in an amino acid variation (N 752 /D 752 ) of the EHV-1 DNA polymerase, is significantly associated with the neuropathogenic potential of naturally occurring strains. In order to estimate the prevalence of the EHV-1 neuropathogenic genotype in our country, we analyzed the ORF30 genome region of Argentinean EHV-1 isolates. The study was carried out by real time allelic discrimination PCR in 90 equine EHV-1-positive samples, being 89 from 54 cases of abortion outbreaks (two of which were in association with neurological disease) and one from the respiratory tract of a healthy horse in training. Our results indicate that 7% (4/54) of the abortion outbreaks studied were induced by the neuropathogenic (G 2254 ) genotype of EHV-1 and 50% (2/4) of them were associated with simultaneous neurological disease. This information emphasizes the necessity to extreme the hygienic and preventive measures to diminish EHV-1 infections and consequently reduce the risk of epizootic neurological disease as has been recently observed in other countries.

The objectives of this study were to estimate the prevalence and the potential role of equine herpesviruses (EHVs) detection in both bronchoalveolar lavage (BAL) and tracheal wash (TW). The population included a control group (CTL; 37 TW and 25 BAL) and a pathological group (PAT; 259 TW and 387 BAL), including horses either suffering from respiratory diseases including syndrome of tracheal inflammation, inflammatory airway disease, recurrent airway obstruction, or submitted to respiratory investigation because of exercise intolerance or poor performance. Each respiratory liquid was submitted to a standardised cytological analysis, mentioning the morphological abnormalities of exfoliated epithelial cells (ECAb) and ciliocytophthoria (CCPh) as markers of potential viral infection, as well as PCR assays including a consensus PCR and virus-specific PCR for both equine alphaherpesviruses (EHV-1; EHV-4) and gammaherpesviruses (EHV-2; EHV-5). The EHV infections were more prevalent in the TW of PAT group (P = 0.004), with the highest prevalence being for EHV-2 (P = 0.006). The EHV detection in BALs was not significantly different between groups. The EHVs detection in TW was correlated to the polymorphonuclear neutrophil (PMN) counts in the respiratory liquid but not with CCPh or ECAb. CCPh or ECAb were associated with both consensus PCR and EHV-2 and EHV-5 virus-type PCR in the BAL only. The significant detection of EHVs in the TW of PAT group in association with the PMN increased counts could lead to further investigations about their putative role in equine syndrome of tracheal inflammation.

Based on small scale studies or on little sensitive serological tests, bovines in the south of the state of Rio Grande do Sul, Brazil, are known to be infected with either bovine herpesvirus 1 (BoHV-1) or 5 (BoHV-5). However, whether the prevalence of each of these viruses is high or low is currently still unknown. In order to determine the extent of BoHV (-1 and/or -5) infections in bovines in this region of Brazil, 200 bovines were studied for the presence of BoHV DNA. To this end, first a quantitative PCR was developed that amplified BoHV-1 DNA as well as BoHV-5 DNA. Using this PCR the number of BoHV genomes normally present in latently infected ganglia of naturally infected bovines was estimated. The new PCR was sensitive enough to detect most BoHV DNA in infected ganglia. The results of this first PCR showed that at least 87% of the bovines in the south of Rio Grande do Sul were (latently) infected with either BoHV-1 or BoHV-5. To determine the prevalence of BoHV-1 and BoHV-5 separately, two type-specific PCRs - one for each virus - were developed that used the products of the first PCR as a template. The results of these type-specific PCRs showed that 82.8% of the BoHV positive population was (latently) infected with BoHV-1, 93.1% with BoHV-5 and 75.9% with both BoHV-1 and BoHV-5. This is the first time that such a high frequency of co-infection of BoHV-1 and BoHV-5 in bovines has been demonstrated.

Infections with feline herpesvirus type 1 (FeHV-1) are frequently associated with recurrent ocular disease, which may occur even in vaccinated cats. The underlying pathogenesis is poorly understood. Specifically, the role of circulating, superinfecting virus strains is unknown. To begin addressing this complex question, we reconstituted a marker-tagged mutant FeHV-1 from a bacterial artificial chromosome (BAC) harboring the FeHV-1 genome. This mutant was deleted for the glycoprotein G gene (ΔgG) but carried instead a gene encoding the green fluorescent protein (GFP). Nine latently with wild-type (wt) FeHV-1-infected cats were superinfected with this mutant and monitored for clinical, virological, and immunological parameters. While the mutant virus replicated locally, induced a rise in neutralizing antibody titers, and stimulated the interferon system, no evidence for ocular illness or reactivation of the underlying wtFeHV-1-infection was detected. However, cyclophosphamide–dexamethasone (C–D) treatment, applied 16 months after the superinfection, was able to reactivate wtFeHV-1. Reactivation was accompanied by recrudescence of ocular disease signs. In contrast, reactivation of the superinfecting mutant virus was not detected. Since kittens are normally infected with wtFeHV-1 prior to the first immunization, the data described in this study may be valuable for designing future live attenuated FeHV-1 vaccines.

Equids are commonly infected by herpesviruses, but isolation of herpesviruses from mules has apparently not been previously reported. Furthermore, the genomic relationships among the various equid herpesviruses are poorly characterized. We describe the isolation and preliminary characterization of a mule gammaherpesvirus tentatively identified as asinine herpesvirus-2 (AHV-2; also designated equid herpesvirus-7 (EHV-7)) from the nasal secretions (NS) of a healthy mule in northern California. The virus was initially identified by transmission electron microscopic examination of lysates of cell culture inoculated with NS collected from the mule. A 913 nucleotide sequence of the DNA polymerase gene was amplified using degenerate primers, and comparison of this sequence with those of various other herpesviruses showed that the mule herpesvirus was most closely related to EHV-2 (AHV-2 sequences were not available for comparison). The sequence of a shorter portion (166 nucleotides) of the mule herpesvirus DNA polymerase gene was identical to that of the published sequence of an asinine gammaherpesvirus, previously designated as AHV-4-3 (AY054992). AHV-2 was detected by real-time polymerase chain reaction assay in the NS of approximately 8% of a cohort of 114 healthy mules and 13 donkeys.

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).

Herpesviruses are associated with lung–eye–trachea disease and gray patch disease in maricultured green turtles ( Chelonia mydas ) and with fibropapillomatosis in wild sea turtles of several species. With the exception fibropapillomatosis, no other diseases of wild sea turtles of any species have been associated with herpesviral infection. In the present study, six necropsied Atlantic loggerhead sea turtles ( Caretta caretta ) had gross and histological evidence of viral infection, including oral, respiratory, cutaneous, and genital lesions characterized by necrosis, ulceration, syncytial cell formation, and intranuclear inclusion bodies. Nested polymerase chain reaction targeting a conserved region of the herpesvirus DNA-dependent-DNA polymerase gene yielded two unique herpesviral sequences referred to as loggerhead genital-respiratory herpesvirus and loggerhead orocutaneous herpesvirus. Phylogenetic analyses indicate that these viruses are related to and are monophyletic with other chelonian herpesviruses within the subfamily α-herpesvirinae . We propose the genus Chelonivirus for this monophyletic group of chelonian herpesviruses.

The major role of EHV-1 in equine abortion is widely reported in the literature but the contribution of EHV-2, EHV-3, EHV-4 or EHV-5 remains less well documented. The objective of this study is to evaluate the contribution of these five different EHVs to equine abortion in a variety of biological tissues using a consensus polymerase chain reaction (PCR). The test was validated for specificity and sensitivity in horses before screening specimens from 407 foetuses, stillbirths and premature foals collected over a 2.5-year interval. Positive results obtained with this assay were compared to other EHV type-specific PCR or by sequencing. EHV-1 was identified as the major cause of abortion in French mares (59/407 cases). However, there was evidence to suggest some variation in the potential of EHV-1 strains to induce abortion. Indeed, DNA samples from EHV-2 (in three cases) and EHV-5 (in one case) inferred a role of these viruses in abortion. The presence of viral DNA from EHV-3 or EHV-4 strains was not detected in the specimens studied. The data obtained suggest that the consensus herpesvirus PCR is an efficient screening tool. In association with a specific PCR, the test provides a rapid identification of the type of herpesvirus involved in abortion and is useful for routine diagnostic tests as it allows the identification of herpesviruses other than the EHV-1 strain.

A novel herpesvirus was detected in a captive mob of eastern grey kangaroos (Macropus giganteus) during diagnostic workup for individuals with ulcerative cloacitis. Virus was initially detected in tissues using a consensus herpesvirus PCR. No viral inclusions or particles had been evident in routine histologic or transmission electron microscopic sections of cloacal lesions. Virus was isolated from samples and transmission electron microscopy of the resulting isolates confirmed that the virus was morphologically consistent with a herpesvirus. Nucleotide sequencing of the PCR product from tissue samples and from the isolates revealed that the virus was in the subfamily Gammaherpesvirinae and was distinct from other known herpesviruses. The correlation between the lesions and the novel virus remains unknown. Two herpesviruses, both in the subfamily Alphaherpesvirinae, have previously been described in macropods and are known to cause systemic clinical disease. This is the first reported gammaherpesvirus within the order Marsupialia, and may provide valuable information regarding the evolution and phylogeny of this virus family. Based on current herpesvirus nomenclature convention, the authors propose the novel herpesvirus be named Macropodid herpesvirus 3 (MaHV-3).

This is the first description of malignant catarrhal fever-like lesions associated with ovine herpesvirus-2 (OvHV-2) infection in goats with central nervous symptoms. The diagnosis was based on typical histological lesions characterized by systemic lymphohistiocytic and fibrinoid vasculitis and confirmed by polymerase chain reaction and subsequent phylogenetic analysis of the detected OvHV-2 sequences.

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB?) labelled with 6-carboxy-fluorescein (FAM?) and VIC® for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.

A nested multiplex PCR was developed as a rapid (

Vaccination with "imperfect" vaccines that prevent disease but not infection is strongly implicated in the observed increased virulence of Marek's disease virus (MDV) over the past six decades. The current "gold standard" vaccine, Rispens CVI988 (Rispens), has maintained efficacy despite use for five decades, raising the question of whether it too favours higher virulence MDVs. To investigate this, we studied the kinetics of Rispens CVI988 (Rispens) and two MDV strains of different virulence in 236 commercial ISA Brown chickens vaccinated with Rispens at hatch and challenged with vMDV isolate MPF57 or vvMDV isolate FT158 on day five. Each treatment was replicated in two isolators and from 7 to 56 days post infection (dpi) peripheral blood leucocytes (PBL), feather and dust samples were collected and subjected to differential quantitative PCR (qPCR). Rispens vaccination significantly reduced challenge MDV viral load in a sample-dependant manner with evidence of a differentially greater inhibitory effect on the less virulent MDV. Similarly, challenge with the more virulent MDV reduced the Rispens viral load in PBL. Rispens virus load displayed a distinctive pattern of viral load that was similar in PBL and feathers, but different in dust. The clear effects of vaccination and challenge evident in PBL and feather samples were less clearly reflected in dust samples. The data are consistent with the Rispens vaccine reducing replication of lesser virulent MDVs to a greater extent like the HVT vaccine. Likely reasons for the persistent efficacy of Rispens vaccine are discussed.

As part of a larger investigation into three emerging disease syndromes highlighted by conjunctivitis and pharyngitis, systemic lymphoid proliferation and encephalitis, and lymphonodular skin infiltrates in farmed saltwater crocodiles (Crocodylus porosus) and one emerging syndrome of systemic lymphoid proliferation in captive freshwater crocodiles (Crocodylus johnstoni), cytopathic effects (CPE), including syncytial cell formation, were observed in primary crocodile cell lines exposed to clarified tissue homogenates from affected crocodiles. Ten cell cultures with CPE were then screened for herpesviruses using two broadly-reactive herpesvirus PCRs. Amplicons were obtained from 9 of 10 cell cultures and were sequenced. Three novel herpesviruses were discovered and the phylogenetic analysis of these viruses showed there was a 63% Bayesian posterior probability value supporting these viruses clustering with the subfamily Alphaherpesvirinae, and 100% posterior probability of clustering with a clade containing the Alphaherpesvirinae and other unassigned reptile herpesviruses. It is proposed that they are named Crocodyline herpesvirus (CrHV) 1, 2 and 3. CrHV1 and 2 were only isolated from saltwater crocodiles and CrHV3 was only isolated from freshwater crocodiles. A duplex PCR was designed that was able to detect these herpesviruses in formalin-fixed paraffin-embedded tissues, a sample type that neither of the broadly-reactive PCRs was able to detect these herpesviruses in. This work describes the isolation, molecular detection and phylogeny of these novel herpesviruses but the association that they have with the emerging disease syndromes requires further investigation.

Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID₅₀/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future.

Three species of porcine lymphotropic herpesviruses (PLHVs) have been described but there are few reports on the distribution and prevalence of these viruses in domestic pigs. We aimed to determine the PLHV status of Irish commercial pig herds, and to this end spleens taken from 110 healthy adult pigs sourced from 22 geographically distributed farms in Ireland were analysed for PLHV DNA using novel species-specific polymerase chain reaction assays. We now report that PLHV infection is widespread in the Irish domestic pig population and that PLHV-1 infections are most common (74% of all animals tested), followed by PLHV-3 and PLHV-2 (45% and 21%, respectively) and that infections with multiple PLHV species were frequently detected. As the PLHVs are lymphotrophic agents, we also investigated if co-infection with PLHVs was linked to the development of porcine circovirus-2 (PCV2)-associated postweaning mutlisystemic wasting syndrome (PMWS), a disease characterised in part by histopathological lesions in lymphoid tissues. We examined the PLHV infection status of young animals on two farms that were experiencing outbreaks of PMWS. Overall the findings are further evidence of the widespread prevalence of PLHVs in domestic pigs and are a first indication that co-infection with PCV2 and PLHVs does not lead to the development of PMWS in the absence of other cofactors.

Studies were conducted to evaluate the feasibility of using canine herpesvirus (CHV) as a vaccine vector for bait-delivered oral vaccination of wild foxes. To test the viability of CHV in baits, CHV was freeze-dried, incorporated into different baits, stored, and the remaining viral infectivity tested in cell culture after varying periods of time at different storage temperatures. Experimental baits (mouse carcasses) and commercial baits (FOXOFF and PROBAIT) were prepared with either liquid or freeze-dried CHV and tested in two fox trials for their capacity to induce CHV-specific antibodies following oral baiting. Freeze-drying and storage temperatures below 0 degrees C had a stabilizing effect to virus infectivity. When stored at -20 degrees C, freeze-dried CHV retained its full infectivity for up to 3 months in PROBAIT baits, the remaining infectivity in FOXOFF baits was 100-fold less. Oral baiting with CHV induced antiviral serum antibodies in all vaccinated foxes (20/20). None of the vaccinated foxes became ill or shed infectious virus into the environment although viral DNA was detected in body secretions as evaluated by PCR. The results indicate that CHV can be freeze-dried and stored over extended periods of time without loosing much of its infectivity. This is the first report of CHV being used for oral bait vaccination of foxes. It appears that CHV is well suited for use as a recombinant vector for wild canids.

In vitro studies demonstrated that most equine herpesvirus 1 (EHV-1)-infected peripheral blood mononuclear cells (PBMC) do not expose viral envelope proteins on their surface. This protects them against antibody-dependent lysis. We examined whether viral envelope proteins are also undetectable on infected PBMC during cell-associated viremia. Further, surface expression of major histocompatibility complex (MHC)-I was examined, since MHC-I assists in making infected cells recognizable for cytotoxic T-lymphocytes (CTL). Four ponies, previously exposed to EHV, and two ponies that had no contact with EHV before, were inoculated with EHV-1. PBMC were collected at different time points up to 28 days post inoculation. Ninety-eight percent of the infected PBMC did not show viral envelope proteins on their surface. Moreover, infected PBMC without surface expression only produced immediate early and, at least, one early protein, ICP22, but not late envelope proteins gB and gM. This indicates that surface expression of viral envelope proteins is absent, simply because the PBMC are in an early phase of infection. The percentage of infected PBMC showing surface expression of MHC-I was similar as observed in non-infected PBMC from the same ponies (80-100%). Therefore, inefficient recognition of EHV-1-infected PBMC by CTLs does not arise from absent surface expression of MHC-I.

Herpes virions are complex particles that consist of more than 30 different virally encoded proteins. The molecular basis of how this complicated structure is assembled is only recently beginning to emerge. After replication in the host cell nucleus viral DNA is incorporated into preformed capsids which leave the nucleus by budding at the inner nuclear membrane resulting in the formation of primary enveloped virions in the perinuclear space. The primary envelope then fuses with the outer leaflet of the nuclear membrane, thereby releasing nucleocapsids into the cytoplasm. Final envelopment including the acquisition of more than 15 tegument and more than 10 envelope (glyco)proteins occurs by budding into Golgi-derived vesicles. Mature virions are released after fusion of the vesicle membrane with the plasma membrane of the cell. Thus, herpesvirus morphogenesis requires a sequence of envelopment--de-envelopment--re-envelopment processes which are distinct not only in the subcellular compartments in which they occur but also in the viral proteins involved. This review summarizes recent advances in our understanding of the complex protein-protein interactions involved in herpesvirus assembly and egress.

Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections such as infectious rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV, balanoposthitis (IBP), and abortion. Despite of a pronounced immune response, the virus is never eliminated from an infected host but establishes life-long latency and may be reactivated at intervals. Europe has a long history of fighting against BoHV-1 infections, yet, only a small number of countries has achieved IBR-eradication. Therefore, it seemed appropriate to review the reasoning pro and contra such a task. Clearly, the goal can indeed be achieved as has been demonstrated by a number of European countries. However, detection and stamping out of seemingly healthy virus carriers is inevitable in the process. Unfortunately, the use of vaccines is only of temporary and limited value. Therefore, there are numerous considerations to be put forward against such plans, including the high costs, the great risks, and the unsatisfactory quality of tools. If either control or eradication of IBR is nonetheless a goal, then better vaccines are needed as well as better companion tests. Moreover, better tools for the characterization of viral isolates are required. Collaborative actions to gather viral strains from as many countries as possible for inclusion into a newly created clustering library would be most advantageous.

Gammaherpesviruses are members of an emerging subfamily among the Herpesviridae. Two genera are discriminated: (i) lymphocryptovirus, including its type species Epstein-Barr virus (EBV), and (ii) rhadinovirus, including viruses of interest for medicine, veterinary medicine, and biomedical research, i.e. alcelaphine herpesvirus 1, bovine herpesvirus 4, equine herpesvirus 2, human herpesvirus 8, mouse herpesvirus 68, and ovine herpesvirus 2 (OvHV-2). The perception that these viruses have a narrow host range is misleading, since they cover a surprisingly wide host range, both on the cellular and the organism's level. For example, the natural range of OvHV-2 infection extends over a common animal order. While the host range determinants of EBV are well known, the corresponding features of the rhadinoviruses need still to be defined. Similarly, the gene expression patterns of the veterinary rhadinoviruses during latency require further characterization. In vivo, the gammaherpesviruses have evolved to actively protect their latently infected cells from being destroyed by immune functions of their native host. In return, those reservoir hosts have evolved to being infected and transmit the virus without overt disease symptoms. However, a balanced immune response needs to be in control over the number of infected cells. Virus excretion is usually at low level and may occur either constantly or intermittently. Animal species that are targeted by the virus but did not participate in the process of co-evolution as well as hosts with immune deficiencies are known to loose control over the amount of latently infected cells, which results in the development of lethal diseases, such as malignant catarrhal fever or Kaposi's sarcoma.

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.

The pseudorabies virus code for an ICP0 protein which is half the size of the HSV1 ICP0 protein. In this work, we made the assumption that some function might have been lost in the ICP0 from PRV. One function attributed to the ICP0 from HSV1 was the stabilization of cyclins D. We then looked at the stability of these cyclins during the lytic infection with the PRV. Our results show that cyclins D are not stabilized during infection with the PRV. These results are in accord with recent data from the literature.

A key aspect in the life cycle of alphaherpesviruses is their neurotropic behaviour. Sensory neurons of the trigeminal ganglion (TG) are important target cells for many alphaherpesviruses (including herpes simplex virus 1, pseudorabies virus (PRV), bovine herpesvirus 1) and constitute major sites for latent infections. The aim of this study was to develop an in vitro model that simulates the in vivo infection pattern of TG neurons by alphaherpesviruses. To this end, we developed a homologous in vitro two-chamber model using PRV and porcine TG neurons. TG of 4- to 6-week-old piglets were dissociated and cultured in the inner chamber of the in vitro model, which is separated from the outer chamber by a medium- and virus-impermeable silicon barrier. Outgrowth of axons from neuronal cell bodies in the inner chamber through the silicon barrier into the outer chamber could be observed after 2-3 weeks of cultivation. Subsequent addition of PRV to the outer chamber resulted in exclusive infection of the TG neurons by transport of virus through the axons, subsequently giving rise to productively infected TG neurons that transmitted virus to contacting neurons and non-neuronal cells in the inner chamber. Thus, we established a homologous in vitro model that mimics the natural route of alphaherpesvirus infection of TG neurons that can be used to study interactions between these viruses and this pathogenetically very important cell type.

Cyprinid herpesvirus-3 (CyHV-3) causes a fatal disease in carp (Cyprinus carpio) and its ornamental koi varieties which seriously affects production and trade of this fish species globally. Up to now, the pathophysiology of this disease remains unclear. Affected individuals develop most prominent lesions in gills, skin and kidney, in tissues which are involved in the osmotic regulation of freshwater teleosts. Therefore, here serum and urine electrolyte levels were examined during the course of an experimental infection of carp with CyHV-3. In infected carp an interstitial nephritis with a progressive deterioration of nephric tubules developed, which was paralleled by elevated electrolyte losses, mainly Na(+) in the urine. The urine/plasma ratio for Na(+) increased from 0.03 in uninfected carp to 0.43-0.83 in carp under CyHV-3 infection, while concentration of divalent ions were not significantly changed. These electrolyte losses could not be compensated since plasma osmolality and Na(+) concentration dropped significantly in CyHV-3 infected carp. This was most probably caused by the progressive deterioration of the branchial epithelium, which in teleosts plays a prominent role in osmoregulation, and which was seen concomitantly with decreasing electrolyte levels in the serum of carp under CyHV-3 infection. Immediately after infection with CyHV-3, by day 2 post exposure, affected carp showed severe anaemia and prominent leucocytosis indicating the development of an acute inflammation, which could intensify the observed hydro-mineral imbalances. The data presented here show that an infection with CyHV-3 induces an acute inflammation and a severe dysfunction of osmoregulation in affected carp or koi, which may lead to death in particular in the case of acute disease progression.

The equine herpesvirus 1 (EHV-1) was isolated in Argentina from an aborted equine foetus in 1979. This virus (SPv) has special restriction patterns (RP) in comparison with other Argentine isolates. In addition, SPv could be distinguished on the basis of its pathogenicity in baby mice inoculated intracerebrally. We studied the growth properties of the SPv in cell culture and its effects in a mouse respiratory and abortion model. We observed that SPv did not modify its capacity to grow in cell culture with respect to reference HH1 strain. Nevertheless, we found significant differences between the titres of the two strains at 8-14 h post-infection (PI). In this work we demonstrated that SPv showed low virulence in female at different stages of gestation, consistently, with results found in the mouse respiratory model. We considered that this low virulence of SPv could be related to its RP because the RP of HH1 strain are similar to those of the HVS25A strain and both showed effect on pregnant mice. More specific studies about genomic alterations to the SPv are necessary for identifying, more clearly, if the intra-strain variations have relation with the low virulence in the mouse respiratory and abortion model.