Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1.

BACKGROUND: Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain's gC, TK, gG, gD, and gE genes. RESULTS: The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/µL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28. CONCLUSIONS: The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Novel herpesvirus discovered in walrus liver.

A walrus (Odobenus rosmarus) born in an aquarium and hand-reared in Japan died at the age of 11 months. The affected animal showed fever and anorexia and had high levels of AST and ALT. Necropsy showed multiple necroses in the liver and adrenal glands and histological examination revealed necrotic lesions of the liver and adrenal cortex, both of which contained intranuclear inclusions. Electron microscopic analysis of the liver sample showed herpesvirus-like particles. High-throughput sequencing analysis of the liver sample and phylogenetic analysis of herpesvirus polymerase genes identified a new virus, Walrus alphaherpesvirus 1 (WaHV-1), which belonged to the subfamily Alphaherpesvirinae and had high homology with Phocid alphaherpesvirus 1. Phylogenetic analysis of the UL30 gene encoding glycoprotein B revealed that WaHV-1 was closely related to a cluster of phocid herpesviruses, including one that caused high mortality rates in harbor seals during past outbreaks. The mother walrus of the dead animal showed evidence of herpesvirus infection in the past and potentially harbored WaHV-1. As a result of hand-rearing, the dead animal might have acquired WaHV-1 from its infected mother and succumbed to WaHV-1 due to lack of maternal IgG, including those that could neutralize WaHV-1.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

Bubaline alphaherpesvirus 1 induces a latent/reactivable infection in goats.

Latent infection is a common mechanism used by several alphaherpesviruses to persist in their host but it is not clear whether this mechanism is also triggered in heterologous infections. Cross-species infections have been documented repeatedly for alphaherpesviruses of ruminants, a group of closely related viruses. Herewith we report latent infection with bubaline alphaherpesvirus 1 (BuHV-1) in experimentally infected goats and subsequent virus reactivation after treatment with dexamethasone (DMS) at 10 months after infection. After DMS treatment, the virus was isolated in one such animal in the nasal swabs from day 3 to 9 post treatment and in the ocular swabs at day 6. The goat was euthanized 48 days after DMS treatment and viral DNA was detected by PCR in the trigeminal ganglia and in two cervical ganglia. Additionally, BuHV-1 DNA was detected by PCR in the trigeminal ganglia of the other 3 goats.

Detection of seroconversion to bovine herpesvirus 1 related alphaherpesvirus and bovine viral diarrhea virusin Polish free-living deer.

There are several infectious agents of domestic cattle that can also be present in free-living ruminant populations. These include bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) which are the causative agents of infectious bovine rhinotracheitis and bovine viral diarrhea, respectively. The study was conducted on serum samples from 59 red deer, 24 roe deer, and 3 fallow deer (86 in total), originating from two geographically separate areas of Poland. The samples were tested with commercially available ELISA tests for BoHV-1 and BVDV. The overall seroprevalence was 5.8% and 3.5%, respectively. All positive samples originated exclusively from red deer. Because of BoHV-1 ELISA cross reactivity with cervid herpesvirus 1 and 2 (CvHV-1 and -2) the nature of alphaherpesviruses infecting the sampled animals could not be assessed.

Infectious keratoconjunctivitis in free-ranging mule deer in Wyoming: a retrospective study and identification of a novel alphaherpesvirus.

We describe the clinicopathologic findings, relative prevalence, and pathogens associated with infectious keratoconjunctivitis in mule deer ( Odocoileus hemionus) in Wyoming. Seventeen cases with ocular lesions were identified among 1,036 mule deer postmortem submissions (1.6%) in an ~16 y period. Sixteen cases were observed in winter and most were in male (15 cases) and juvenile (13 cases) deer. Blindness was the most commonly reported clinical sign (10 cases). A herpesvirus was detected only in the 4 cases of bilateral necrotizing bulbar conjunctivitis. Phylogenetic analysis of glycoprotein amino acid sequences consistently identified this virus as a novel alphaherpesvirus. In 2 of these herpesvirus-positive cases, Actinomyces sp. and Moraxella ovis were also identified. Trueperella pyogenes was identified in 4 cases of unilateral ulcerative keratitis, keratoconjunctivitis, and panophthalmitis. M. ovis was cultured from 3 cases of bilateral conjunctivitis and keratoconjunctivitis. In the remaining cases, isolates included Moraxella bovis (1 case), Staphylococcus sp. and Streptococcus sp. (2), Flavobacterium sp. and Pseudomonas sp. (2), Escherichia coli and Enterobacter sp. (1), and bovine viral diarrhea virus 1 (1). No pathogens were identified in 2 cases. The relative prevalence of keratoconjunctivitis in mule deer in Wyoming appears to be low, and this disease is most commonly associated with infection by a novel alphaherpesvirus, T. pyogenes, and M. ovis.

Human herpesvirus portal proteins: Structure, function, and antiviral prospects.

Herpesviruses (Herpesvirales) and tailed bacteriophages (Caudovirales) package their dsDNA genomes through an evolutionarily conserved mechanism. Much is known about the biochemistry and structural biology of phage portal proteins and the DNA encapsidation (viral genome cleavage and packaging) process. Although not at the same level of detail, studies on HSV-1, CMV, VZV, and HHV-8 have revealed important information on the function and structure of herpesvirus portal proteins. During dsDNA phage and herpesviral genome replication, concatamers of viral dsDNA are cleaved into single length units by a virus-encoded terminase and packaged into preformed procapsids through a channel located at a single capsid vertex (portal). Oligomeric portals are formed by the interaction of identical portal protein monomers. Comparing portal protein primary aa sequences between phage and herpesviruses reveals little to no sequence similarity. In contrast, the secondary and tertiary structures of known portals are remarkable. In all cases, function is highly conserved in that portals are essential for DNA packaging and also play a role in releasing viral genomic DNA during infection. Preclinical studies have described small molecules that target the HSV-1 and VZV portals and prevent viral replication by inhibiting encapsidation. This review summarizes what is known concerning the structure and function of herpesvirus portal proteins primarily based on their conserved bacteriophage counterparts and the potential to develop novel portal-specific DNA encapsidation inhibitors.

Sequence of the ateline alphaherpesvirus 1 (HVA1) genome.

Here, we report the genome sequence of a spider monkey alphaherpesvirus (ateline alphaherpesvirus 1, HVA1) and compare it with that of other primate alphaherpesviruses. The HVA1 genome is 147,346 bp long and contains 67 predicted ORFs. The genetic layout of the HVA1 genome is similar to that of the squirrel monkey alphaherpesvirus (saimirine alphaherpesvirus 1, HVS1) in that it lacks inverted repeat regions flanking the unique long region and homologues of the UL43, UL49.5, US8.5 and US10-12 genes. Unlike HVS1, HVA1 also lacks a homologue of the RL1 (γ34.5) gene and a replication origin near the end of the genome. Consistent with previous phylogenetic analyses, all predicted proteins of HVA1 are most closely related to those of HVS1.

Discovery of herpesviruses in Canadian wildlife.

Herpesviruses (HVs) have a wide range of hosts in the animal kingdom. The result of infection with HVs can vary from asymptomatic to fatal diseases depending on subtype, strain, and host. To date, little is known about HVs naturally circulating in wildlife species and the impact of these viruses on other species. In our study, we used genetic and comparative approaches to increase our understanding of circulating HVs in Canadian wildlife. Using nested polymerase chain reaction targeting a conserved region of the HV DNA polymerase gene, we analyzed material derived from wildlife of western and northern Canada collected between February 2009 and Sept 2014. For classification of new virus sequences, we compared our viral sequences with published sequences in GenBank to identify conserved residues and motifs that are unique to each subfamily, alongside phylogenetic analysis. All alphaherpesviruses shared a conserved tryptophan (W856) and tyrosine (Y880), betaherpesviruses all shared a serine (S836), and gammaherpesviruses had a conserved glutamic acid (E835). Most of our wildlife HV sequences grouped together with HVs from taxonomically related host species. From Martes americana, we detected previously uncharacterized alpha- and beta-herpesviruses.

The Genome of a Tortoise Herpesvirus (Testudinid Herpesvirus 3) Has a Novel Structure and Contains a Large Region That Is Not Required for Replication In Vitro or Virulence In Vivo.

UNLABELLED: Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution assays, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step toward characterizing TeHV-3 and developing prophylactic methods against it. IMPORTANCE: Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome and used this information to take steps toward developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are that (i) TeHV-3 has a novel genome structure, (ii) its closest relative is a turtle herpesvirus, (iii) it contains interleukin-10 and semaphorin genes (the first time these have been reported in an alphaherpesvirus), (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo, and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.

Orofacial viral infections--an update for clinicians.

UNLABELLED: Orofacial viral infections may be less common but appear in different clinical forms. Often these infections get initially treated by antibiotics which obviously will have limited or no effect. The authors review the current concepts of orofacial viral infections, causative agents, their classification and clinical manifestations and a basis for treatment. CLINICAL RELEVANCE: Most viral infections do not require any specific treatment except in patients who are immunosuppressed or immunodeficient. Appropriate diagnosis and timely management of orofacial viral lesions are important irrespective of whether it is localized or a manifestation of a systemic infection.

Isolation and characterization of a novel alphaherpesvirus in fruit bats.

UNLABELLED: Bats are known to harbor emerging RNA viruses. Recent studies have used high-throughput sequencing technology to identify various virus species, including DNA viruses that are harbored by bats; however, little is known about the nature of these potentially novel viruses. Here, we report the characterization of a novel herpesvirus isolated from an Indonesian pteropodid bat. The virus, tentatively named fruit bat alphaherpesvirus 1 (FBAHV1), has a double-stranded DNA genome of 149,459 bp. The phylogenetic analyses suggested that FBAHV1 is phylogenetically grouped with simplexviruses within the subfamily Alphaherpesvirinae. Inoculation of FBAHV1 into laboratory mice caused a lethal infection. Virus infection was observed in lung, liver, and brain tissue. Serological and PCR screening revealed that fruit bats infected with FBAHV1 or its related virus are widely distributed in Indonesia. The identification of FBAHV1 makes a considerable contribution to our understanding of simplexviruses associated with bats. IMPORTANCE: Bats are known to harbor emerging viruses, such as lyssaviruses, henipaviruses, severe acute respiratory syndrome-like coronaviruses, and filoviruses. Although alphaherpesviruses are disseminated in humans and other animals, there is little information about their distribution in bats. Here, we isolated a previously unknown alphaherpesvirus from an Indonesian fruit bat. Genome sequence analysis suggested that the virus is a member of the genus Simplexvirus within the subfamily Alphaherpesvirinae, which also includes common human viruses, such as herpes simplex virus 1 and herpes simplex virus 2. FBAHV1 is the first bat-derived alphaherpesvirus whose complete genome has been sequenced.

First report of isolation and molecular characterization of bubaline herpesvirus 1 (BuHV1) from Argentinean water buffaloes.

Herpesviruses have mainly co-evolved with their hosts for millions of years. However, bovine herpesvirus 1 (BoHV1) and related ruminant alphaherpesviruses have been reported to cross the species barrier. Bubaline herpesvirus 1 (BuHV1) is an alphaherpesvirus closely related to BoHV1 and BoHV5. According to the serological cross-relationships between ruminant alphaherpesviruses, several surveys have studied the occurrence of BoHV1-related virus infection in wild and domestic ruminant species. Recent studies in Argentina showed an increase in serological prevalence against BoHV1 related viruses in water buffaloes (Bubalus bubalis) population. The aim of this study was to investigate the presence of related ruminant alphaherpesvirus in the Argentinean water buffalo population. BuHV1 was successfully isolated from 5 out of 225 buffaloes analyzed. One isolate was obtained from nasal secretions, and the others were from vaginal swabs. The buffaloes belonged to four different farms located in northeastern Argentina. The isolates were characterized as alphaherpesvirus by direct immunofluorescence using FITC-anti-BoHV1 IgG. Restriction analysis performed with BamHI and BstEII on the complete genome showed differences between the isolates and those from BoHV1 and BoHV5 subtypes. Phylogenetic analysis on both UL27 and US6 showed similarity in tree topology. While three of the isolates grouped together with sequences of BoHV5, two other isolates clustered separately. Genetic analysis of eight concatenated sequences from all isolates and references strains showed high nucleotide sequence identity between BuHV1 and BoHV5. While three of the isolates clustered together with the BoHV5 reference strain, the last two isolates were closely related to an Australian BuHV1 strain. To our knowledge, this is the first report on the isolation and molecular characterization of BuHV1 in South America. Phylogenetic analysis suggested that two different BuHV1 lineages circulate in the Argentinean water buffalo population.

Ocular and neural distribution of feline herpesvirus-1 during active and latent experimental infection in cats.

BACKGROUND: Herpes simplex virus 1 (HSV-1) and varicella zoster virus (VZV) cause extensive intra-ocular and neural infections in humans and are closely related to Felid herpes virus 1 (FeHV-1). We report the extent of intra-ocular replication and the extent and morphological aspects of neural replication during the acute and latent phases of FeHV-1 infection. Juvenile, SPF cats were inoculated with FeHV-1. Additional cats were used as negative controls. Cats were euthanized on days 6, 10, and 30 post-inoculation. RESULTS: FeHV-1 was isolated from the conjunctiva, cornea, uveal tract, retina, optic nerve, ciliary ganglion (CG), pterygopalatine ganglion (PTPG), trigeminal ganglion (TG), brainstem, visual cortex, cerebellum, and olfactory bulb of infected cats during the acute phase, but not the cranial cervical ganglion (CCG) and optic chiasm. Viral DNA was detected in all tissues during acute infection by a real-time quantitative PCR assay. On day 30, viral DNA was detected in all TG, all CCG, and 2 PTPG. Histologically mild inflammation and ganglion cell loss were noted within the TG during acute, but not latent infection. Using linear regression, a strong correlation existed between clinical score and day 30 viral DNA copy number within the TG. CONCLUSIONS: The correlation between clinical score and day 30 viral DNA copy number suggests the severity of the acute clinical infection is related to the quantity of latent viral DNA. The histologic response was similar to that seen during HSV-1 or VZV infection. To the author's knowledge this is the first report of FeHV-1 infection involving intraocular structures and autonomic ganglia.

Isolation and characterization of a novel herpesvirus from a free-ranging eastern grey kangaroo (Macropus giganteus).

We isolated a macropodid herpesvirus from a free-ranging eastern grey kangaroo (Macropus giganteous) displaying clinical signs of respiratory disease and possibly neurologic disease. Sequence analysis of the herpesvirus glycoprotein G (gG) and glycoprotein B (gB) genes revealed that the virus was an alphaherpesvirus most closely related to macropodid herpesvirus 2 (MaHV-2) with 82.7% gG and 94.6% gB amino acid sequence identity. Serologic analyses showed similar cross-neutralization patterns to those of MaHV-2. The two viruses had different growth characteristics in cell culture. Most notably, this virus formed significantly larger plaques and extensive syncytia when compared with MaHV-2. No syncytia were observed for MaHV-2. Restriction endonuclease analysis of whole viral genomes demonstrated distinct restriction endonuclease cleavage patterns for all three macropodid herpesviruses. These studies suggest that a distinct macropodid alphaherpesvirus may be capable of infecting and causing disease in eastern grey kangaroos.

Isolation of a novel herpesvirus from a Pacific white-sided dolphin.

During establishment of primary cell culture from the kidney of a dead Pacific white-sided dolphin (Lagenorhynchus obliquidens), a cytopathic effect was observed. Polymerase chain reaction with a set of herpesvirus consensus primers yielded a fragment of the expected size. Nucleotide sequencing of the product indicated that the isolated virus was closely related to an alphaherpesvirus detected in a bottlenose dolphin in the United States, but the sequence identity at the protein level was low (86.6 %). Phylogenetic analysis of the encoded sequence confirmed that the new isolate belonged to the subfamily Alphaherpesvirinae and clustered together with other cetacean alphaherpesviruses. The complete gene encoding glycoprotein B (2,757 bp) was amplified from the novel isolate; the encoded protein was compared with the corresponding protein of other herpesviruses, revealing that this virus belongs to the genus Varicellovirus. Taken together, these results suggest that this virus corresponds to a novel herpesvirus capable of infecting Pacific white-sided dolphins.

Association of age and gender with alphaherpesvirus infections of the central nervous system in the immunocompetent host.

BACKGROUND: The alphaherpesviruses Varicella-zoster virus (VZV) and human herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) can cause severe infections of the central nervous system (CNS). OBJECTIVES: To analyze whether age and gender of immunocompetent individuals are associated with the incidence of herpesvirus CNS diseases. STUDY DESIGN: A total of 241 patients with virologically confirmed HSV-1, HSV-2 or VZV-infection of the CNS (excluding neonatal infection and varicella), diagnosed at the Department of Virology, Medical University Vienna, from 2001 to 2009 were analyzed retrospectively. The relative incidence of disease was evaluated statistically with respect to gender and age. RESULTS: The relative incidence of VZV CNS disease increased with age (p<0.0001), and nonlinear age dependences were observed for HSV-1 (p=0.005) and HSV-2 disease (p=0.002). These effects were influenced significantly by the patient's gender in VZV (p=0.0003) and HSV-1 disease (p=0.008). Overall, 50.7% of VZV infections in males, but only 23.5% of those in females, occurred before age 45, and 28.9% of HSV-1 infections in males and 8.8% of those in females occurred before age 30. Women represented 71.9% of HSV-2 CNS infections (p=0.02). CONCLUSIONS: The patient's gender is clearly associated with the incidence of CNS disease caused by VZV, HSV-1 and HSV-2, and its influence varies over one's lifetime.

Molecular identification of novel alpha- and gammaherpesviruses from cetaceans stranded on Japanese coasts.

Herpesviral infections have been documented in some cetaceans; however, they have not yet been identified in species in the western North Pacific. In the present study, 178 tissue samples from 76 stranded cetacean individuals were tested for the presence of herpesviruses. Herpesvirus genomic DNA fragments surrounding the DNA polymerase gene were amplified in samples from four individuals. TA cloning and direct sequencing of these DNA fragments revealed the presence of two novel alphaherpesviruses, and two novel gammaherpesviruses in the four cetacean individuals. The alphaherpesviruses were associated with the lung tissue of a false killer whale (Pseudorca crassidens), and with the mucus of a melon-headed whale (Peponocephala electra). The gammaherpesviruses were found in the lymph tissues of a Stejneger's beaked whale (Mesoplodon stejnegeri) and a sperm whale (Physeter macrocephalus). The phylogenetic tree using amino acid sequences of the DNA polymerase gene supported the inclusion of the novel viruses identified here in a single monophyletic group containing alphaherpesviruses from other Atlantic cetacean species. Conversely, the novel gammaherpesviruses formed an independent clade distant from other known cetacean gammaherpesviruses.

The alphaherpesvirus US3/ORF66 protein kinases direct phosphorylation of the nuclear matrix protein matrin 3.

The protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro, suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.

Phylogenetic analysis of marine mammal herpesviruses.

Five novel DNA-dependent DNA polymerase (Dpol) herpesviral sequences were generated using nested consensus polymerase chain reaction (PCR) in clinical samples from a harbor seal (Phoca vitulina), bottlenose dolphin (Tursiops truncatus), orca (Orcinus orca), California sea lion (Zalophus californianus), and a Phocid herpesvirus 2 (PhHV-2) isolate from a harbor seal (used as positive control). These novel sequences and other representative herpesvirus sequences were included in Bayesian and Maximum Likelihood analyses to illustrate the phylogeny of herpesviruses amongst the marine mammal host species and in comparison to those of other animals. All 19 novel and known marine mammal herpesviruses included in the analyses aligned with members of the Alphaherpesvirinae or Gammaherpesvirinae subfamilies. The novel harbor seal herpesvirus clustered with members of the Macavirus genus, subfamily Gammaherpesvirinae. The novel bottlenose dolphin herpesvirus clustered together in a monophyletic group with another delphinid alphaherpesvirus but could not be associated with an established genus. The orca herpesvirus also clustered with a delphinid alphaherpesvirus and formed a separate clade. The sea lion herpesvirus clustered with PhHV-2. PhHV-1 clustered with varicelloviruses and PhHV-2 clustered strongly in the Gammaherpesvirinae genus Percavirus. All cetacean gammaherpesviruses formed a monophyletic clade and could not be associated with an established gammaherpesviral genus.