Complete Genome Sequence of Equine Herpesvirus Type 9

Equine herpesvirus type 9 (EHV-9), which we isolated from a case of epizootic encephalitis in a herd of Thomson''s gazelles (Gazella thomsoni) in 1993, has been known to cause fatal encephalitis in Thomson''s gazelle, giraffe, and polar bear in natural infections. Our previous report indicated that EHV-9 was similar to the equine pathogen equine herpesvirus type 1 (EHV-1), which mainly causes abortion, respiratory infection, and equine herpesvirus myeloencephalopathy. We determined the genome sequence of EHV-9. The genome has a length of 148,371 bp and all 80 of the open reading frames (ORFs) found in the genome of EHV-1. The nucleotide sequences of the ORFs in EHV-9 were 86 to 95% identical to those in EHV-1. The whole genome sequence should help to reveal the neuropathogenicity of EHV-9.     

Outbreak of human herpesvirus type 1 infection in nonhuman primates (Callithrix penincillata)

Human herpesvirus type 1 (HHV-1) is widely dispersed among the human population. Although infection is often asymptomatic in humans, nonhuman primates develop a severe and often fatal infection. In August 2006, 13 black-tufted marmosets (Callithrix penincillata) from a group of 14 presented with clinical apathy, anorexia, and ataxia. Physical examination revealed conjunctivitis, erosive or ulcerative lesions on the skin, and swollen lymph nodes. Of the 14 animals captured, 10 died. Grossly, ulcers and erosions were observed on the skin of face, nasal planum, lips, and oral mucosa. Histologically, superficial vesicular and erosive stomatitis with associated basophilic intranuclear inclusion bodies in the squamous epithelium were observed. Swabs from oral lesions and tissue samples from necropsied animals were positive for HHV-1 by nested polymerase chain reaction for eight animals.     

Expression of equine herpesvirus 1 glycoprotein D by using a recombinant baculovirus.

Glycoprotein D (gD) of equine herpesvirus 1 (EHV-1) was expressed at the surface of insect cells infected by a recombinant baculovirus. EHV-1 gD was detected as multiple forms (56, 52, and 48 kDa) from 18 to 96 h postinfection. Laboratory animals inoculated with the recombinant EHV-1 gD developed neutralizing antibody responses against both EHV-1 and EHV-4.     

Comprehensive Serology Based on a Peptide ELISA to Assess the Prevalence of Closely Related Equine Herpesviruses in Zoo and Wild Animals

Equine herpesvirus type 1 (EHV-1) causes respiratory disorders and abortion in equids while EHV-1 regularly causes equine herpesvirus myeloencephalopathy (EHM), a stroke-like syndrome following endothelial cell infection in horses. Both EHV-1 and EHV-9 infections of non-definitive hosts often result in neuronal infection and high case fatality rates. Hence, EHV-1 and EHV-9 are somewhat unusual herpesviruses and lack strict host specificity, and the true extent of their host ranges have remained unclear. In order to determine the seroprevalence of EHV-1 and EHV-9, a sensitive and specific peptide-based ELISA was developed and applied to 428 sera from captive and wild animals representing 30 species in 12 families and five orders. Members of the Equidae, Rhinocerotidae and Bovidae were serologically positive for EHV-1 and EHV-9. The prevalence of EHV-1 in the sampled wild zebra populations was significantly higher than in zoos suggesting captivity may reduce exposure to EHV-1. Furthermore, the seroprevalence for EHV-1 was significantly higher than for EHV-9 in zebras. In contrast, EHV-9 antibody prevalence was high in captive and wild African rhinoceros species suggesting that they may serve as a reservoir or natural host for EHV-9. Thus, EHV-1 and EHV-9 have a broad host range favoring African herbivores and may have acquired novel natural hosts in ecosystems where wild equids are common and are in close contact with other perissodactyls.     

Fatal herpesvirus tamarinus infection in cotton-topped marmosets (Saguinus oedipus).

Fatal herpesvirus tamarinus infection was observed in cotton-topped marmosets (Saguinus oedipus) imported from South America via the United States on August 26, 1976. In addition to the lesions hitherto reported in herpesvirus tamarinus infection, severe degenerative and necrotic changes of ganglion cells were recognized with intranuclear inclusion bodies in the plexus of the digestive tract and the sympathetic nerves and their ganglions in the abdominal cavity. Inflammatory or regressive changes were also noted in the central nervous system. A large number of basophilic or eosinophilic intranuclear inclusion bodies frequently recognized in multinucleated giant cells were observed in various organs and tissues, and they showed different shapes at the electron microscopic level. Morphological findings indicated that herpesvirus tamarinus infection seemed to be similar to herpes simplex virus infection in man. The findings of the susceptibility of a variety of cell cultures to the virus isolate serologically identified as herpesvirus tamarinus and physicochemical characteristics of the virus isolate were in general agreement with the findings of herpesvirus tamarinus already reported by previous workers.     

Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction.

Although both equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) can be associated with respiratory disease, epizootics caused by EHV-1 are much more serious because the virus can cause abortions and paralysis. It is, therefore, important to identify the type of EHV involved in an outbreak by a test that is quick, sensitive, and reliable. We have adapted the polymerase chain reaction (PCR) to detect and distinguish between EHV-1 and EHV-4 in the same reaction. Primers for PCR were designed from the sequences of the glycoprotein B genes of EHV-1 and EHV-4. The PCR products derived from EHV-1 and EHV-4 were 135 and 326 base pairs, respectively, and could be readily separated by electrophoresis. The identity of the PCR products was confirmed by determining their nucleotide sequence, which agreed with the published sequence of the gB genes. The test could be performed directly on virus pelleted from small volumes (300 microL) of medium in which nasal swabs were transported and did not rely on the presence of infectious virus. The PCR was unaffected by conditions that reduced the infectivity of a virus preparation by 99%. The PCR detected EHV-4 in 5 of 10 nasal mucous samples taken from an outbreak of respiratory disease in race horses. Virus isolation in indicator cells was successful in detecting virus in four of the five samples positive by PCR.     

Evidence of respiratory tract infection induced by equine herpesvirus, type 2, in the horse.

Five horses were experimentally exposed to equine herpesvirus 2 strain LK. Two young foals developed chronic pharyngitis (98 and 232 days, respectively). Growth characteristics, cytopathic effects (CPE), inclusion body formation, ether sensitivity, and immunofluorescent analysis indicated that the virus recovered from infected animals was a herpesvirus serologically identical with, or at least antigenically related to EHV-2 strain LK. No significant complement-fixing (CF) or virus-neutralizing (VN) antibody responses were observed in adult horses while both foals demonstrated a rise in CF antibody titer. One of the two foals demonstrated a rise in VN antibody only. The results suggest that EHV-2 virus induced chronic pharyngitis, primarily the result of lymphoid proliferation, with no overt clinical signs.     

Pathogenesis of guinea pig adenovirus infection

BACKGROUND AND PURPOSE: The existence of guinea pig adenovirus (GPAdV) has been suspected on the basis of histopathologic findings, but the virus has not yet been isolated. In susceptible animals, it may cause severe bronchopneumonia and death. Adenovirus-like inclusion bodies have been observed in the lungs of animals with clinical disease. Prevalence of the infection is unknown. Recently, a polymerase chain reaction (PCR) assay was described that was able to selectively detect GPAdV. METHODS: To investigate the pathogenesis of GPAdV, we inoculated eight guinea pigs with GPAdV; eight control animals were sham inoculated. The PCR assay was used to trace the infection. In a second experiment, transmission of GPAdV from an experimentally infected animal to five immune-naive cohorts was examined. RESULTS: None of the infected animals developed clinical disease. The GPAdV could be detected by PCR analysis of nasal-swab specimens on days 6 through 15 after infection. Infective virus could be recovered from the nasal mucosa during this period (as determined by inoculation of immune-naive animals). The virus was transmitted from an experimentally infected animal to two of five immune-naive cage mates. CONCLUSION: The GPAdV may cause transient subclinical upper respiratory tract infection that may descend to the lungs.     

Neurovirulence of mumps virus: intraspinal inoculation test in marmosets.

An intraspinal inoculation test of mumps virus using marmosets was performed in order to develop a neurovirulence test of mumps vaccines. In the group inoculated with non-neurovirulent Jeryl Lynn vaccine strain at 10(2.0) pfu/dose, there were only minimal histopathological changes in 3 of the 5 marmosets. In contrast, all marmosets inoculated with neurovirulent Urabe and NK-M46 vaccine strains developed extensive encephalitis and meningitis. Thus, this marmoset model, which can distinguish between non-neurovirulent and neurovirulent vaccine strains, is useful for evaluating neurovirulence of vaccine strains and elucidating the molecular pathogenesis of mumps.     

Isolation of a cytomegalovirus from salivary glands of white-lipped marmosets (Saguinus fuscicollis).

Minced salivary glands from seven white-lipped marmosets (Saguinus fuscicollis and Saguinus nigricollis) and one cotton-topped marmoset (Saguinus oedipus) were cocultivated with marmoset cell cultures. A viral agent, designated SSG, was isolated from two Saguinus fuscicollis. Slowly progressing foci of rounded, vacuolated, refractile cells were first observed at 40-43 days incubation. Electron microscopy revealed intranuclear herpesvirus nucleocapsids and intracytoplasmic and extracellular enveloped particles. Infected cells stained with hematoxylin and eosin contained eosinophilic intranuclear and cytoplasmic inclusion bodies. SSG could be passaged in cell cultures only using viable whole cells; infectious cell-free virus was not detected in either culture supernatants or cell lysates. SSG replicated in marmoset fibroblastic but not in marmoset epithelioid or human fibroblastic cell cultures. Plasma antibodies to SSG were detected by indirect immunofluorescence assays in 16 of 56 (28.6%) adult wild-caught marmosets but were absent in 40 colony-born, hand-reared marmosets. Antigenic cross-reactivity of SSG with a rhesus monkey (Macaca mulatta) cytomegalovirus (bidirectional) and with a human cytomegalovirus (unidirectional) was also demonstrated by indirect immunofluorescence assays. SSG was identified as a herpesvirus by morphology and was classified as a cytomegalovirus by its site of isolation, biologic properties in vitro, and antigenic characteristics.     

Aerosolized Rift Valley Fever Virus Causes Fatal Encephalitis in African Green Monkeys and Common Marmosets

Rift Valley fever (RVF) is a veterinary and human disease in Africa and the Middle East. The causative agent, RVF virus (RVFV), can be naturally transmitted by mosquito, direct contact, or aerosol. We sought to develop a nonhuman primate (NHP) model of severe RVF in humans to better understand the pathogenesis of RVF and to use for evaluation of medical countermeasures. NHP from four different species were exposed to aerosols containing RVFV. Both cynomolgus and rhesus macaques developed mild fevers after inhalation of RVFV, but no other clinical signs were noted and no macaque succumbed to RVFV infection. In contrast, both marmosets and African green monkeys (AGM) proved susceptible to aerosolized RVF virus. Fever onset was earlier with the marmosets and had a biphasic pattern similar to what has been reported in humans. Beginning around day 8 to day 10 postexposure, clinical signs consistent with encephalitis were noted in both AGM and marmosets; animals of both species succumbed between days 9 and 11 postexposure. Marmosets were susceptible to lower doses of RVFV than AGM. Histological examination confirmed viral meningoencephalitis in both species. Hematological analyses indicated a drop in platelet counts in both AGM and marmosets suggestive of thrombosis, as well as leukocytosis that consisted mostly of granulocytes. Both AGM and marmosets would serve as useful models of aerosol infection with RVFV.     

Expression of L1 and N-CAM cell adhesion molecules during development of the mouse olfactory system

The expression of the neural adhesion molecules L1 and N-CAM has been studied in the embryonic and early postnatal olfactory system of the mouse in order to gain insight into the function of these molecules during development of a neural structure which retains neuronal turnover capacities throughout adulthood. N-CAM was slightly expressed and L1 was not significantly expressed in the olfactory placode on Embryonic Day 9, the earliest stage tested. Rather, N-CAM was strongly expressed in the mesenchyme underlying the olfactory placode. In the developing nasal pit, L1 and N-CAM were detectable in the developing olfactory epithelium, but not in regions developing into the respiratory epithelium. At early developmental stages, expression of the so-called embryonic form of N-CAM (E-N-CAM) coincides with the expression of N-CAM, whereas at later developmental stages and in the adult it is restricted to a smaller number of sensory cell bodies and axons, suggesting that the less adhesive embryonic form is characteristic of morphogenetically dynamic neuronal structures. Moreover, E-N-CAM is highly expressed at contact sites between olfactory axons and their target cells in the glomeruli of the olfactory bulb. L1 and N-CAM 180, the component of N-CAM that accumulates at cell contacts by interaction with the cytoskeleton are detectable as early as the first axons extend toward the primordial olfactory bulb. L1 remains prominent throughout development on axonal processes, both at contacts with other axons and with ensheathing cells. Contrary to N-CAM 180 which remains detectable on differentiating sensory neuronal cell bodies, L1 is only transiently expressed on these and is no longer detectable on primary olfactory neuronal cell bodies in the adult. Furthermore, whereas throughout development L1 has a molecular form similar to that seen in other parts of the developing and adult central nervous systems, N-CAM and, in particular, N-CAM 180 retain their highly sialylated form at least partially throughout all ages studied. These observations suggest that E-N-CAM and N-CAM 180 are characteristic of developmentally active structures and L1 may not only be involved in neurite outgrowth, but also in stabilization of contacts among fasciculating axons and between axons and ensheathing cells, as it has previously been found in the developing peripheral nervous system.     

Antibodies against equine herpesviruses and equine arteritis virus in Burchell's zebras (Equus burchelli ) from the Serengeti ecosystem

A total of 51 sera from a migratory population of Burchell's zebras (Equus burchelli) were collected in the Serengeti National Park (Tanzania) between 1999 and 2001 to assess levels of exposure to equine herpesvirus types 1, 2, 4, 9 (EHV-1, -2, -4, -9), EHV-1 zebra isolate T965, and equine arteritis virus (EAV). Using virus-specific neutralizing antibody tests, seroprevalence was high for EHV-9 (60% of 45), moderate for EAV (24% of 51), and lower for the EHV-1-related zebra isolate (17% of 41), EHV-1 (14% of 49), and EHV-4 (2% of 50). No evidence for exposure to EHV-2 was found (0% of 51). The high level of exposure to EHV-9 is interesting because evidence of infection with this virus has not been previously described in any wild equine population. Although the epidemiology of EHV-9 in Burchell's zebras is presently unknown, our results suggest that in East Africa, this species may be a natural host of EHV-9, a neuropathogenic virus that was only recently isolated from captive Thomson's gazelles (Gazella thomsoni) in Japan. There is currently no evidence that EHV-9 induced mortality in Burchell's zebras in the Serengeti, but because of the reported virulence of this virus for more susceptible species such as Thomson's gazelles, viral transmission from infected zebras to ungulates may result in mortality.     

Oncogenic transformation by by equine herpesviruses. II. Coestablishment of persistent infection and oncogenic transformation of hamster embryo cells by equine herpesvirus type 1 preparations enriched for defective interfering particles.

Infection of permissive hamster embryo cells with virus preparations enriched for defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) resulted in persistent infection and oncogenic transformation. Six cell lines, designated DI-5 to -10, exhibited biological properties (immortality, increased saturation density, growth in soft agar, etc.) inherent to transformed cells, but 2 to 18% of the total cells in these cell lines were shown to release virus as judged by electron microscope studies and infectious center assays. The released virus was shown to be standard EHV-1 and not to contain DI particles as determined by density measurements of the viral DNA in the analytical ultracentrifuge and by interference assays using the released virus. Tumorigenicity studies revealed that inoculation of these persistently infected cells into newborn LSH inbred hamsters resulted in a lethal, fulminating hepatitis, whereas inoculation into older immunocompetent hamsters (+4 weeks) led to the development of metastatic fibrous sarcomas. Tumor cell lines (DI-5T to -10T) established from these sarcomas were shown to be transplantable and virus nonproducers. Hybridization analyses of cellular DNAs from DI transformed and tumor cell lines using 32P-labeled genomic EHV-1 DNA as probes indicated that the whole virus genome was detectable in multiple copies (23 to 45) in the transformed cells and that DNA sequences representing only 43.5 to 56.6% of the virus genome were present in amounts of 2 to 4 copies per cell in the DI tumor cells. Expression of these viral DNA sequences as demonstrated by the detection of virus-neutralizing antibodies, 50% neutralizing dose titers ranging from 1:50 to 1:1,000, in the sera of animals inoculated with either the virus-producing transformed cells or the virus-nonproducing tumor cells. Further, EHV-1-specific proteins were detected in the membrane and the perinuclear region of bothDI transformed and tumor cells by indirect immunofluorescent assays using antisera against EHV-1 structural antigens, EHV-1 nonstructural antigens, or preparations of EHV-1 DI particles. The roles of DI particles in mediating persistent infection and cellular transformation are discussed.     

Molecular characterization of equine herpesvirus 1 (EHV-1) isolated from cattle indicating no specific mutations associated with the interspecies transmission

Interspecies trasmission of equine herpesvirus 1 (EHV-1) from horse to cattle was shown by Crandell et al. (1988). Specific mutations related to the transmission were studied by comparison of five EHV-1 isolates in cattle (BH1247, 3M20-3, G118, G1753, and 9BSV4) using polymerase chain reaction and restriction fragment length polymorphism analysis with added sequencing. G118 and 3M20-3 were the genome type EHV-1 P, while G1753 was the genome type EHV-1 B. BH1247 and 9BSV4 might be other genome types. We could not identify specific mutations related to the interspecies transmission.     

Complete genomic sequence of an equine herpesvirus type 8 Wh strain isolated from China

A new strain of equine herpesvirus type 8 (EHV-8), Wh, has been isolated from horses in China, and its complete genome has been sequenced and analyzed. The result indicates that the new strain has the same constitution and arrangement of open read frames as EHV-1 and EHV-9. This work is the first announced complete genome sequence of EHV-8.     

Experimental infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the marmoset, Callithrix penicillata (Primates:Callithricidae)

Fourteen marmosets (Callithrix penicillata) were inoculated intradermally with promastigotes and/or amastigotes of Leishmania (Viannia) braziliensis (L. (V) b.) strains MHOM/BR/83/LTB-300 MHOM/BR/85/LTB-12 MHOM/BR/81/LTB-179 and MHOM/BR/82/LTB-250. The evolution of subsequent lesions was studied for 15 to 75 weeks post-inoculation (PI). All but 3 of the L. (V) b. injected marmosets developed a cutaneous lesion at the point of inoculation after 3 to 9 weeks, characterized by the appearance of subcutaneous nodules containing parasites. Parasites were isolated by culture (Difco Blood Agar) from all 11 positive animals. The maximum size of the lesions was variable and ranged between 37 mm2 to 107 mm2. Ulceration of primary nodules became evident after 3 to 12 weeks in all infected marmosets, but was faster and larger in 5 of the 11 animals. The active lesions persisted in 9 out of 11 Callithrix until the end of the observation period, which varied from 15-75 weeks. In 3 animals spontaneous healing of their lesions (13 to 25 weeks, PI) was observed but with cryptic parasitism. In another 2 infected animals there was regression followed by reactivation of the cutaneous lesions. The appearance of smaller satellite lesions adjacent to primary ones, as well as metastatic lesions to the ear lobes, were documented in 2 animals. Promastigotes of L. (Leishmania) amazonensis (L. (L) a.) MHOM/BR/77/LTB-16 were inoculated in 1 marmoset. This animal remained chronically infected for 6 months and the lesion developed in a similar manner to L. (V) b. infected marmosets. No significant differences in clinical and parasitological behaviour were observed between promastigote or amastigote derived infections of the 2 species.(ABSTRACT TRUNCATED AT 250 WORDS)     

A point mutation in a herpesvirus polymerase determines neuropathogenicity

Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission.     

A Novel Nonhuman Primate Model for Influenza Transmission

Studies of influenza transmission are necessary to predict the pandemic potential of emerging influenza viruses. Currently, both ferrets and guinea pigs are used in such studies, but these species are distantly related to humans. Nonhuman primates (NHP) share a close phylogenetic relationship with humans and may provide an enhanced means to model the virological and immunological events in influenza virus transmission. Here, for the first time, it was demonstrated that a human influenza virus isolate can productively infect and be transmitted between common marmosets (Callithrix jacchus), a New World monkey species. We inoculated four marmosets with the 2009 pandemic virus A/California/07/2009 (H1N1pdm) and housed each together with a naïve cage mate. We collected bronchoalveolar lavage and nasal wash samples from all animals at regular intervals for three weeks post-inoculation to track virus replication and sequence evolution. The unadapted 2009 H1N1pdm virus replicated to high titers in all four index animals by 1 day post-infection. Infected animals seroconverted and presented human-like symptoms including sneezing, nasal discharge, labored breathing, and lung damage. Transmission occurred in one cohabitating pair. Deep sequencing detected relatively few genetic changes in H1N1pdm viruses replicating in any infected animal. Together our data suggest that human H1N1pdm viruses require little adaptation to replicate and cause disease in marmosets, and that these viruses can be transmitted between animals. Marmosets may therefore be a viable model for studying influenza virus transmission.     

Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.