Isolation and in vitro characterization of a herpesvirus from ground squirrels (Citellus sp).

A viral agent was isolated from ground squirrel primary kidney cultures which presented a spontaneous cell layer alteration. The cultural, morphologic, and physico-chemical characteristics of the agent indicated that it belongs in the herpesvirus group. The isolate presented a narrow in vitro cell host range, growing best in marmoset monkey, owl monkey, rabbit, and hamster kidney cultures and poorly in Vero and dog fetal lung cells. The agent is readily released from infected cells, a fact which indicates that it is a cell-free virus. Good plaque formation was observed only in marmoset monkey kidney monolayers. The virus was not neutralized by antisera against other herpesviruses with the exception of a partial 1-way cross-neutralization between ground squirrel agent and Herpesvirus saguinus antisera. All these characteristics indicate that this virus is likely a new member of the herpesvirus family.     

Notices

A virus isolated from the lung of an aborted Hereford fetus was shown to possess the physical and chemical properties of the herpesvirus group. The virus designated bovine herpesvirus (BH-1247) was isolated in cultures of Madin-Darby bovine kidney (MDBK) and primary bovine embryonic kidney (BEK) cells. Electron microscopic studies revealed typical forms of virus particles of the herpesvirus group. The virus was sensitive to chloroform and virus replication was inhibited by the addition of 5-iododeoxyuridine into the cell culture medium. The characteristic features of the cytopathic changes were syncytial formations with intranuclear inclusion bodies. Discrete plaques were formed in MDBK cell cultures overlaid with agar. Virus growth studies in BEK cells revealed infectious virus to be cell associated and replicated at low titer. By serum neutralization tests the virus was shown to be distinct from bovine herpesviruses; infectious bovine rhinotracheitis, DN-599, Movar 33/63, bovine mammallitis, malignant catarrhal fever and feline viral rhinotracheitis, equine herpesvirus I and pseudorabies. The isolate was nonpathogenic to mice inoculated subcutaneously, intracerebrally and intraperitoneally. Virus replication was not demonstrated when inoculated on the chorioallantoic membrane of embryonated eggs.     

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.     

A novel gammaherpesvirus associated with genital lesions in a Blainville's beaked whale (Mesoplodon densirostris)

An adult male Blainville's beaked whale (Mesoplodon densirostris) was found stranded on the Atlantic coast of the USA on 28 January 2004. Necropsy revealed a focal papilloma-like penile lesion, the cells from which revealed single 4-6 microm basophilic intranuclear inclusions. Total DNA extracted from lesion material was tested using a pan-herpes-virus PCR assay that targets the DNA polymerase gene and found to be positive. When the amplified DNA fragment was cloned, sequenced, and compared to GenBank-deposited herpesvirus DNA polymerase sequences, the detected virus was determined to be a distinct member of the Gammaherpesvirinae subfamily of herpesviruses. This new virus, tentatively named Ziphiid herpesvirus type 1, was associated with but not determined to be the cause of genital disease in the Blainville's beaked whale.     

New endogenous herpesvirus of guinea pigs: biological and molecular characterization.

Two known guinea pig herpesviruses, guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV), and well characterized. A third herpesvirus (GPXV) was originally isolated from leukocytes of healthy strain 2 guinea pigs. Growth of GPXV in guinea pig embryo fibroblastic cells produced a characteristic cytopathic effect. Electron microscopy of guinea pig cells infected with GPXV revealed the morphological development of a herpesvirus. Cross-neutralization tests and immunoferritin electron microscopy demonstrated that GPXV, GPCMV, and GPHLV were serologically distinct herpeviruses of guinea pigs. To confirm the distinction between these three herpesviruses, DNA genomes were compared by CsCl equilibrium buoyant density measurements and restriction endonuclease cleavage analysis. 32P-labeled viral DNA ws obtained from nucleocapsids isolated from virus-infected cells, and the buoyant density of GPXV DNA differed from that of GPCMV and GPHLV. Cleavage of viral DNAs with restriction endonucleases followed by gel electrophoresis revealed distinct patterns for each virus.     

Two Distinct Gamma-2 Herpesviruses in African Green Monkeys: a Second Gamma-2 Herpesvirus Lineage among Old World Primates?

Primate gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesvirus [KSHV], also called human herpesvirus 8), macaques (Macaca spp.) (rhesus rhadinovirus [RRV] and retroperitoneal fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri sciureus) (herpesvirus saimiri), and spider monkeys (Ateles spp.) (herpesvirus ateles). Using serological screening and degenerate consensus primer PCR for the viral DNA polymerase gene, we have detected sequences from two distinct gamma-2 herpesviruses, termed Chlorocebus rhadinovirus 1 (ChRV1) and ChRV2, in African green monkeys. ChRV1 is more closely related to KSHV and RFHV, whereas ChRV2 is closest to RRV. Our findings suggest the existence of two distinct rhadinovirus lineages, represented by the KSHV/RFHV/ChRV1 group and the RRV/ChRV2 group, respectively, in at least two Old World monkey species. Antibodies to members of the RRV/ChRV2 lineage may cross-react in an immunofluorescence assay for early and late KSHV antigens.     

An adenovirus infection of the kidney of Franklin's ground squirrels (Spermophilus franklini) in Saskatchewan, Canada

During routine pathological studies of Franklin's ground squirrels (Spermophilus franklini) collected during a predator control program, basophilic intranuclear inclusions were found in the collecting tubule epithelium of the renal papillae in seven of 13 squirrels. This was associated with marked karyomegaly in affected cells. An inflammatory response was not seen in the adjacent tissues. Electron microscopic examination of affected cells demonstrated that the enlarged nuclei contained numerous virus-like particles. Autoculture and serial passage of renal medullary cells resulted in the isolation of virus particles producing intranuclear inclusions and cytopathic effect. The virus possessed properties typical of adenoviruses, but showed no evidence of hemagglutinating activity with a range of species of erythrocytes tested under several temperature conditions. The isolates were relatively host-cell specific; they failed to grow in hamster and rabbit kidney cell lines and in ground squirrel kidney cortical cells.     

Antigenic comparisons between herpesviruses isolated from fallow deer in Alberta and the viruses of infectious bovine rhinotracheitis, equine rhinopneumonitis and DN-599, a non-IBR bovine herpesvirus.

Antigenic comparison studies of three herpesviruses isolated from fallow deer (Dama dama) in Alberta and herpesviruses from some domestic species were carried out by the alpha serum-virus neutralization test. Complete cross neutralization was demonstrated among the deer herpesviruses and equine herpesvirus type 1.     

Distinct Roles for Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 in the Structure and Production of a Primate Gammaherpesvirus

During their progression from intranuclear capsids to mature trilaminar virions, herpesviruses incorporate an extensive array of viral as well as a smaller subset of cellular proteins. Our laboratory previously reported that rhesus monkey rhadinovirus (RRV), a close homolog of the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is comprised of at least 33 different virally encoded proteins. In the current study, we found that RRV infection activated the extracellular signal-regulated kinase (ERK) pathway and nascent virions preferentially incorporated the activated form of ERK2 (pERK2) into the tegument. This was evident even in the face of greatly diminished stores of intracellular ERK2, suggesting a clear bias toward the incorporation of pERK2 into the RRV particle. Similar to earlier findings with KSHV, activation of ERK was essential for the production of lytic viral proteins and virions. Knockdown of intracellular ERK, however, failed to inhibit virus production, likely due to maintenance of residual pools of intracellular pERK2. Paradoxically, selective knockdown of ERK1 enhanced virion production nearly 5-fold and viral titers more than 10-fold. These data are the first to implicate ERK1 as a negative regulator of lytic replication in a herpesvirus and the first to demonstrate the incorporation of an activated signaling molecule within a herpesvirus. Together, the results further our understanding of how herpesviruses interact with host cells during infection and demonstrate how this family of viruses can exploit cellular signal transduction pathways to modulate their own replication.     

Antiviral activity and pathogenetic targets for seaweed sulfated polysaccharides in herpesvirus infections

The review summarizes results of studies of effects of sulfated polysaccharides from seaweed on herpesviruses and the course of herpesvirus infections. Importance of this problem is determined by the prevalence of herpesviruses that can persist in the human body and demonstrate a high degree of immune mimicry and resistance to antiviral drugs. A wide range of physiological action of sulfated polysaccharides, receptor agonists of innate and adaptive immune cells, which possess potent antiviral, antioxidant and anti-inflammatory activities, open the possibility of their use for creation of new generation pharmacological substances and drugs with associated activity for the treatment of herpesvirus infections.     

Herpesviral inclusion body disease in owls and falcons is caused by the pigeon herpesvirus (columbid herpesvirus 1)

A herpesviral disease of Rock Pigeons (Columba livia), called "inclusion body disease" or "inclusion body hepatitis," was first described in the 1940s. The disease involves hepatic and splenic necrosis with associated intranuclear inclusion bodies and occurs primarily in young squabs. A similar herpesviral disease occurs in falcons and owls. Serologic and restriction endonuclease digestion studies indicate that herpesviruses from pigeons, falcons, and owls are very closely related and that most reported cases of disease in falcons and owls involve prior documented or possible ingestion of pigeons. These findings led to the hypothesis that an endemic herpesvirus of pigeons may be causing disease in falcons and owls. In order to test this hypothesis, we sequenced a fragment of the herpesviral DNA polymerase gene from naturally infected owls, falcons, and pigeons with inclusion body disease collected between 1991 and 2006. Sequences from all three sources were almost identical, and we therefore propose that the usual agent of inclusion body hepatitis in owls and falcons is columbid herpesvirus 1.     

Regulated Expression of a Sindbis Virus Replicon by Herpesvirus Promoters

We describe the use of herpesvirus promoters to regulate the expression of a Sindbis virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and β-galactosidase only after infection with a herpesvirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesviruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegalovirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesviruses for which a permissive cell culture system is not available.     

Synthesis of bovine growth hormone in primates by using a herpesvirus vector.

A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders.     

Genome organization of herpesvirus aotus type 2.

Herpesvirus aotus type 2, a virus commonly found in owl monkeys without overt disease, has a similar genome structure to the oncogenic herpesviruses of nonhuman primates (herpesvirus saimiri, herpesvirus ateles). Virion DNA of herpesvirus aotus type 2 (M-DNA) has an unique 110-kilobase-pair region of low G + C content (40.2%, L-DNA), inserted between stretches of repetitive H-DNA (68.7% G + C, about 41 kilobase pairs per molecule) that are variable in length. A minority of virions contain defective genomes that consist of repetitive H-DNA only. The H-DNA is composed of various types of repeat units that are related in sequence with each other. The two dominant types of repeats (2.3 and 2.7 kilobase pairs) were cloned and compared by restriction enzyme cleavages and partial nucleotide sequencing. They are homologous in at least 1.3 kilobase pairs. The two forms of repeat units are randomly arranged and oriented in tandem. Reassociation kinetics did not allow detection of sequence homologies between H- and L-DNA of herpesvirus aotus type 2 and the respective sequences of oncogenic primate herpesviruses.     

A Genomic Approach to Unravel Host-Pathogen Interaction in Chelonians: The Example of Testudinid Herpesvirus 3

We report the first de novo sequence assembly and analysis of the genome of Testudinid herpesvirus 3 (TeHV3), one of the most pathogenic chelonian herpesviruses. The genome of TeHV3 is at least 150,080 nucleotides long, is arranged in a type D configuration and comprises at least 102 open reading frames extensively co-linear with those of Human herpesvirus 1. Consistently, the phylogenetic analysis positions TeHV3 among the Alphaherpesvirinae, closely associated with Chelonid herpesvirus 5, a Scutavirus. To date, there has been limited genetic characterization of TeHVs and a resolution beyond the genotype was not feasible because of the lack of informative DNA sequences. To exemplify the potential benefits of the novel genomic information provided by this first whole genome analysis, we selected the glycoprotein B (gB) gene, for detailed comparison among different TeHV3 isolates. The rationale for selecting gB is that it encodes for a well-conserved protein among herpesviruses but is coupled with a relevant antigenicity and is consequently prone to accumulate single nucleotide polymorphisms. These features were considered critical for an ideal phylogenetic marker to investigate the potential existence of distinct TeHV3 genogroups and their associated pathology. Fifteen captive tortoises presumptively diagnosed to be infected with TeHVs or carrying compatible lesions on the basis of either the presence of intranuclear inclusions (presumptively infected) and/or diphtheronecrotic stomatitis-glossitis or pneumonia (compatible lesions) were selected for the study. Viral isolation, TeHV identification, phylogenetic analysis and pathological characterization of the associated lesions, were performed. Our results revealed 1) the existence of at least two distinct TeHV3 genogroups apparently associated with different pathologies in tortoises and 2) the first evidence for a putative homologous recombination event having occurred in a chelonian herpesvirus. This novel information is not only fundamental for the genetic characterization of this virus but is also critical to lay the groundwork for an improved understanding of host-pathogen interactions in chelonians and contribute to tortoise conservation.     

Herpesviruses and the Type III Interferon System

Type III interferons (IFNs) represent the most recently discovered group of IFNs. Together with type I IFNs (e.g. IFN-α/β), type III IFNs (IFN-λ) are produced as part of the innate immune response to virus infection, and elicit an anti-viral state by inducing expression of interferon stimulated genes (ISGs). It was initially thought that type I IFNs and type III IFNs perform largely redundant functions. However, it has become evident that type III IFNs particularly play a major role in antiviral protection of mucosal epithelial barriers, thereby serving an important role in the first-line defense against virus infection and invasion at contact areas with the outside world, versus the generally more broad, potent and systemic antiviral effects of type I IFNs. Herpesviruseses are large DNA viruses, which enter their host via mucosal surfaces and establish lifelong, latent infections. Despite the importance of mucosal epithelial cells in the pathogenesis of herpesviruses, our current knowledge on the interaction of herpesviruses with type III IFN is limited and largely restricted to studies on the alphaherpesvirus herpes simplex virus (HSV). This review summarizes the current understanding about the role of IFN-λ in the immune response against herpesvirus infections.     

Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater

Herpesviruses are associated with several diseases of marine turtles including lung-eye-trachea disease (LETD) and gray patch disease (GPD) of green turtles (Chelonia mydas) and fibropapillomatosis (FP) of green, loggerhead (Caretta caretta), and olive ridley turtles (Lepidochelys olivacea). The stability of chelonian herpesviruses in the marine environment, which may influence transmission, has not been previously studied. In these experiments, LETD-associated herpesvirus (LETV) was used as a model chelonian herpesvirus to test viral infectivity after exposure to seawater. The LETV virus preparations grown in terrapene heart (TH-1) cells were dialyzed for 24 to 120 hr against aerated artificial or natural seawater or Hank's balanced salt solution (HBBS). Fresh TH-1 cells were inoculated with dialyzed LETV, and on day 10 post-infection cells were scored for cytopathic effect. Virus samples dialyzed up to 120 hr were positive for the herpesvirus DNA polymerase gene by polymerase chain reaction. Electron microscopy revealed intact LETV nucleocapsids after exposure of LETV to artificial seawater or HBSS for 24 hr at 23 C. LETV preparations remained infectious as long as 120 hr in natural and artificial seawater at 23 C. Similar results were obtained with a second culturable chelonian herpesvirus, HV2245. LETV infectivity could not be detected after 48 hr exposure to artificial seawater at 30 C. Since LETV and HV2245 remain infectious for extended periods of time in the marine environment, it is possible that FP-associated and GPD-associated herpesviruses also may be stable. These findings are significant both for researchers studying the epidemiological association of herpesviruses with diseases of marine turtles and for individuals who handle turtles in marine turtle conservation efforts.     

Kinetics of the sodium pump in red cells of different temperature sensitivity

Ouabain-sensitive K influx into ground squirrel and guinea pig red cells was measured at 5 and 37 degrees C as a function of external K and internal Na. In both species the external K affinity increases on cooling, being three- and fivefold higher in guinea pig and ground squirrel, respectively, at 5 than at 37 degrees C. Internal Na affinity also increased on cooling, by about the same extent. The effect of internal Na on ouabain-sensitive K influx in guinea pig cells fits a cubic Michaelis-Menten-type equation, but in ground squirrel cells this was true only at high [Na]i. There was still significant ouabain-sensitive K influx at low [Na]i. Ouabain-binding experiments indicated around 800 sites/cell for guinea pig and Columbian ground squirrel erythrocytes, and 280 sites/cell for thirteen-lined ground squirrel cells. There was no significant difference in ouabain bound per cell at 37 and 5 degrees C. Calculated turnover numbers for Columbian and thirteen-lined ground squirrel and guinea pig red cell sodium pumps at 37 degrees C were about equal, being 77-100 and 100-129 s-1, respectively. At 5 degrees C red cells from ground squirrels performed significantly better, the turnover numbers being 1.0-2.3 s-1 compared with 0.42-0.47 s-1 for erythrocytes of guinea pig. The results do not accord with a hypothesis that cold-sensitive Na pumps are blocked in one predominant form.     

Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate and n-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.     

Spontaneous adenocarcinoma immunoreactive to cyclooxygenase-2 and transforming growth factor-beta1 in the buccal salivary gland of a Richardson's ground squirrel (Spermophilus richardsonii).

The ground squirrel is used as an experimental animal because of its unique biological nature. A 3-year-old female Richardson's ground squirrel developed a mass, 1.5 cm in diameter, in the buccal mucosa. The mass consisted of neoplastic epithelial cells showing acinar, ductular, intraductal papillary, solid, and lobular growth patterns; the cells were immunoreactive to cytokeratin, cyclooxygenase-2 (a marker of malignancy) and TGF-beta1. After resection, the tumor recurred with increased area having a solid or lobular pattern with little differentiation. This tumor was diagnosed as an adenocarcinoma arising from the buccal gland, the first case reported in the ground squirrel. A prominent desmoplastic reaction was present. The interstitial cells reacted to alpha-smooth muscle actin and vimentin, indicating a myofibroblastic nature, presumably induced by epithelial TGF-beta1.