Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.     

Ocular herpes:the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus(HSV), varicella zoster virus(VZV), and cytomegalovirus(CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.     

Ocular herpes: the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.     

Three Novel Herpesviruses of Endangered Clemmys and Glyptemys Turtles

The rich diversity of the world’s reptiles is at risk due to significant population declines of broad taxonomic and geographic scope. Significant factors attributed to these declines include habitat loss, pollution, unsustainable collection and infectious disease. To investigate the presence and significance of a potential pathogen on populations of critically endangered bog turtles (Glyptemys muhlenbergii) as well sympatric endangered wood (G. insculpta) and endangered spotted (Clemmys guttata) turtles in the northeastern United States, choanal and cloacal swabs collected from 230 turtles from 19 sites in 5 states were screened for herpesvirus by polymerase chain reaction. We found a high incidence of herpesvirus infection in bog turtles (51.5%; 105/204) and smaller numbers of positive wood (5) and spotted (1) turtles. Sequence and phylogenetic analysis revealed three previously uncharacterized alphaherpesviruses. Glyptemys herpesvirus 1 was the predominant herpesvirus detected and was found exclusively in bog turtles in all states sampled. Glyptemys herpesvirus 2 was found only in wood turtles. Emydid herpesvirus 2 was found in a small number of bog turtles and a single spotted turtle from one state. Based on these findings, Glyptemys herpesvirus 1 appears to be a common infection in the study population, whereas Glyptemys herpesvirus 2 and Emydid herpesvirus 2 were not as frequently detected. Emydid herpesvirus 2 was the only virus detected in more than one species. Herpesviruses are most often associated with subclinical or mild infections in their natural hosts, and no sampled turtles showed overt signs of disease at sampling. However, infection of host-adapted viruses in closely related species can result in significant disease. The pathogenic potential of these viruses, particularly Emydid herpesvirus 2, in sympatric chelonians warrants additional study in order to better understand the relationship of these viruses with their endangered hosts.     

Association of herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida.

Sea turtle fibropapillomatosis (FP) is a disease marked by proliferation of benign but debilitating cutaneous fibropapillomas and occasional visceral fibromas. Transmission experiments have implicated a chloroform-sensitive transforming agent present in filtered cell-free tumor homogenates in the etiology of FP. In this study, consensus primer PCR methodology was used to test the association of a chelonian herpesvirus with fibropapillomatosis. Fibropapilloma and skin samples were obtained from 17 green and 2 loggerhead turtles affected with FP stranded along the Florida coastline. Ninety-three cutaneous and visceral tumors from the 19 turtles, and 33 skin samples from 16 of the turtles, were tested. All turtles affected with FP had herpesvirus associated with their tumors as detected by PCR. Ninety-six percent (89/93) of the tumors, but only 9% (3/33) of the skin samples, from affected turtles contained detectable herpesvirus. The skin samples that contained herpesvirus were all within 2 cm of a fibropapilloma. Also, 1 of 11 scar tissue samples from sites where fibropapillomas had been removed 2 to 51 wk earlier from 5 green turtles contained detectable herpesvirus. None of 18 normal skin samples from 2 green and 2 loggerhead turtles stranded without FP contained herpesvirus. The data indicated that herpesvirus was detectable only within or close to tumors. To determine if the same virus infected both turtle species, partial nucleotide sequences of the herpesvirus DNA polymerase gene were determined from 6 loggerhead and 2 green turtle samples. The sequences predicted that herpesvirus of loggerhead turtles differed from those of green turtles by only 1 of 60 amino acids in the sequence examined, indicating that a chelonian herpesvirus exhibiting minor intratypic variation was the only herpesvirus present in tumors of both green and loggerhead turtles. The FP-associated herpesvirus resisted cultivation on chelonian cell lines which support the replication of other chelonian herpesviruses. These results lead to the conclusion that a chelonian herpesvirus is regularly associated with fibropapillomatosis and is not merely an incidental finding in affected turtles.     

Detection of a Novel Bovine Lymphotropic Herpesvirus

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.     

Isolation and characterization of a new virus from owl monkeys: herpesvirus aotus type 3

Owl monkey kidney cell cultures yielded a viral agent 32 days after initiation of the culture. The virus was identified as a herpesvirus by physico-chemical, cultural, and morphological features. Serologically this herpesvirus was found to be unrelated to other members of this family, as well as to Herpesvirus aotus type 1 and 2. Based on these findings the name Herpesvirus aotus type 3 is suggested for this herpesvirus.     

Isolation of a herpesvirus from an American kestrel with inclusion body disease.

A herpesvirus was isolated from the liver of a captive-bred American kestrel (Falco sparverius) which had died of inclusion body disease. Initial isolation was achieved in chicken embryo fibroblasts after three blind passages. Cell-adapted virus produced a distinct rounding of CEF cells within 24 to 48 h. Biologic and serologic tests suggested that the kestrel virus is similar to falcon herpesvirus and pigeon herpesvirus and is at least partially related to owl herpesvirus. However, serologic tests indicated that the kestrel herpesvirus is neither related to infectious laryngotracheitis virus nor to a herpesvirus from a psittacine bird; (Eupsittula canicularis) with Pacheco's parrot disease. This is the first report on the recovery of a herpesvirus similar to falcon herpesvirus from an American kestrel with naturally-occurring inclusion body disease, and on the serologic comparison between falcon herpesvirus and a psittacine herpesvirus.     

Detection of herpesvirus DNA in the serum of immunocompetent children

The DNA of herpesviruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus-6 (HHV6), and human herpesvirus-7 (HHV7) has been detected in the serum of patients with primary infection or with immunosuppression. However, it is unknown how frequently herpesvirus DNA can be detected in the serum of immunocompetent children, or whether the detection of herpesvirus DNA indicates an active infection or virus-related diseases. Using a real-time polymerase chain reaction assay, attempts were made to detect herpesvirus DNA in the serum of 176 ambulatory children who visited a hospital for various reasons. EBV was detected in 4 (2.2%), HHV6 in 4 (2.2%), and HHV7 in 2 (1.1%) of 176 children, but CMV was not detected. Of the 10 positive patients, only 4 were considered, by virtue of clinical and serological characteristics, to have primary infections. The other 4 positive patients had other infections, such as mycoplasma and salmonella. Although herpesvirus DNA could be detected in the serum of immunocompetent children, there was not always a relationship between clinical manifestations and the detection of virus DNA. When herpesvirus DNA is detected in the serum, a careful interpretation is necessary to diagnose a primary infection or a virus-associated disease.     

Arrangement of repetitive sequences in the genome of herpesvirus Sylvilagus.

Herpesvirus sylvilagus is a lymphotropic (type gamma) herpesvirus of cottontail rabbits (Sylvilagus floridanus). Analysis of virion DNA of herpesvirus sylvilagus has revealed that the genome consists of one stretch of about 120 kilobase pairs of internal, unique DNA flanked by a variable number of 553-base-pair tandem repeats. The G + C content of the repetitive DNA is extremely high (83%), as determined by sequencing. The organization of the herpesvirus sylvilagus genome is, therefore, similar to that of the primate lymphotropic viruses herpesvirus saimiri and herpesvirus ateles.     

Tyrosine phosphorylation of the Tio oncoprotein is essential for transformation of primary human T cells

Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.     

DNA of herpesvirus pan, a third member of the Epstein-Barr virus-Herpesvirus papio group

The DNA of herpesvirus pan, a primate B-lymphotropic herpesvirus, shares about 40% well-conserved sequence relatedness with Epstein-Barr virus (EBV) and herpesvirus papio DNAs. Labeled cloned fragments from the EBV recombinant DNA library were cross hybridized to blots of EcoRI, XbaI, and BamHI restriction endonuclease fragments of herpesvirus pan DNA to identify and map homologous sequences in the herpesvirus pan genome. Regions of colinear homology were demonstrated between 6 x 10(6) daltons and 108 x 10(6) daltons in the DNAs. The structural organization of herpesvirus pan DNA was similar to the format of Epstein-Barr virus and herpesvirus papio DNAs. The DNA consists of two domains of largely unique sequence complexity, a segment US of 9 x 10(6) daltons and a segment UL of 88 x 10(6) daltons. US and UL are separated by a variable number of tandem repetitions of a sequence IR (2 x 10(6) daltons). There was homology between DNA which mapped at 26 to 28 x 10(6) daltons and 93 to 95 x 10(6) daltons in UL. The terminal reiteration component, TR, of herpesvirus pan DNA and sequences which mapped to the left of 6 x 10(6) daltons and to the right of 108 x 10(6) daltons had no detectable homology with the corresponding regions of Epstein-Barr virus DNA.     

Herpesvirus ateles Tio can replace herpesvirus saimiri StpC and Tip oncoproteins in growth transformation of monkey and human T cells

Herpesvirus saimiri group C strains are capable of transforming human and simian T-lymphocyte populations to permanent antigen-independent growth. Two viral oncoproteins, StpC and Tip, that are encoded by a single bicistronic mRNA, act in concert to mediate this phenotype. A closely related New World monkey herpesvirus, herpesvirus ateles, transcribes a single spliced mRNA at an equivalent genome locus. The encoded protein, Tio, has sequence homologies to both StpC and Tip. We inserted the tio sequence of herpesvirus ateles strain 73 into a recombinant herpesvirus saimiri C488 lacking its own stpC/tip oncogene. Simian as well as human T lymphocytes were growth transformed by the chimeric Tio-expressing viruses. Thus, a single herpesvirus protein appears to be responsible for the oncogenic effects of herpesvirus ateles.     

Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.     

Virologic investigations of free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest,Poland

We conducted virologic investigations on postmortem specimens from 261 free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest, Poland collected between 1990 and 2000. Fifty-four of 94 males had balanoposthitis; none of the 167 female bison examined had reproductive tract lesions. Peripheral blood, swabs, and various tissues were analyzed for bovine viruses as well as for viral DNA by bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 4 (BoHV-4) specific polymerase chain reaction (PCR) technique. An infectious bovine rhinotracheitis like BoHV-1 strain was isolated from the spleen of a female bison calf and additionally was detected by nested PCR from splenic tissue. None of the bison had significant antibody titers against BoHV-1, bovine herpesvirus 2, BoHV-4, caprine herpesvirus 1, cervid herpesvirus 1, or bovine viral diarrhea (BVD) virus-1. However, low antibody titers in two animals indicate that this European bison population has been exposed to BVD virus or BVD-like viruses and BoHV-2.     

Thermostabilization and thermosensitization of herpesvirus

Wallis, Craig (Baylor University College of Medicine, Houston, Tex.), and Joseph L. Melnick. Thermostabilization and thermosensitization of herpesvirus. J. Bacteriol. 90:1632-1637. 1965.-Herpesvirus, long considered as one of the most thermolabile of viruses, was stabilized by 1 m Na(2)SO(4) or Na(2)HPO(4) so that it withstood heating at 50 C, but the virus was not protected by 1 m MgCl(2), MgSO(4), or KH(2)PO(4), or 2 m KCl or NaCl; 1 m Na(2)SO(4) also stabilized herpesvirus at 25 and 37 C. In contrast, herpesvirus was made extremely thermosensitive in the presence of isotonic salt concentrations or of isotonic tris(hydroxymethyl)aminomethane buffer, especially at pH 7.2 or above. Partially purified virus was relatively thermostable when suspended in distilled water at pH 7.2, but in Earle's salt solution the virus immediately became thermosensitive. As found in tissue culture harvests, herpesvirus was thermolabile, but the virus was rendered stable at 50 C by simple dilution in distilled water. Protection by proteins or amino acids, generally accepted as virus-stabilizing agents, did not seem to be the result of a direct effect upon herpesvirus. The present data suggest that the added proteins counteract in part thermosensitizing effects of the salts contained in the virus harvest.     

Detection of four lymphotropic herpesviruses in Hungarian patients with multiple myeloma and lymphoma

It has been suggested that human herpesvirus 8 (HHV-8), also known as KSHV (Kaposi's sarcoma-associated human herpesvirus), might possess a promoting effect in the development and progression of monoclonal gammopathies. In this study, the presence of Epstein-Barr virus (EBV), human cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human herpesvirus 8 (HHV-8) were tested in patients with multiple myeloma (MM) using both serologic and nucleic acid amplification techniques. The transient reactivation or continuous presence of EBV, CMV, HHV-6 and HHV-8 could be detected in, respectively, 36, eight, 13 and 29 of 69 MM patients; nine, one, four and six of 16 monoclonal gammopathy of unknown significance patients; and seven, four, zero and five of 10 Waldenstr?m's macroglobulinemia patients. The total number of MM patients was 95. HHV-8 PCR-positivity was significantly more frequent in the MM group than in the control group of patients with non-Hodgkin's lymphoma (NHL). However, serologic testing did not reveal significant differences between the two patient groups. The number of MM patients with concomitant herpesvirus infections as detected by PCR was as follows: 15 double, seven triple and two quadruple virus nucleic acid positive. In 13/95 MM patients, the simultaneous presence of acute EBV infection and HHV-8 PCR-positivity was detected compared with none of the control group (P=0.009). These results indicate that in addition to HHV-8, the transitional reactivation of EBV may also play a role in the pathogenesis of MM.     

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.     

The size and conformation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) DNA in infected cells and virions.

The genome of a novel human herpesvirus has been detected in specimens of Kaposi's sarcoma (KS) and in several AIDS-related lymphoproliferative disorders. Here we examine the size and genomic conformation of the DNA of this virus (known as KS-associated herpesvirus or human herpesvirus 8) in latently and lytically infected cells and in virions. Pulsed-field gel electrophoresis of viral DNA shows that the viral genome is similar in size to those of other gammaherpesviruses (160 to 170 kb). As with Epstein-Barr virus, KS-associated herpesvirus DNA is stably maintained in latently infected B cells as episomal monomer circles and induction from latency is associated with the selective accumulation of linear genomic forms.     

A herpesvirus of rhesus monkeys related to the human Kaposi's sarcoma-associated herpesvirus.

A herpesvirus that is related to but distinct from the Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) was isolated from rhesus monkeys. The sequence of 10.6 kbp from virion DNA revealed the presence of an interleukin-6 homolog similar to what is present in KSHV and a closer relatedness of the DNA polymerase and glycoprotein B reading frames to those of KSHV than to those of any other herpesvirus. This rhesus monkey herpesvirus replicated lytically and to high titers in cultured rhesus monkey fibroblasts. Antibody testing revealed a high prevalence for at least 10 years in our rhesus monkey colony and a high prevalence in two other colonies that were tested. Thus, rhesus monkeys naturally harbor a virus related to KSHV, which we have called RRV, for rhesus monkey rhadinovirus.