The monitoring of herpesvirus infection provides useful information when assessing marine mammals' health. This paper shows the prevalence of herpesvirus infection (80.85%) in 47 cetaceans stranded ...on the coast of the Valencian Community, Spain. Of the 966 tissues evaluated, 121 tested positive when employing nested-PCR (12.53%). The largest proportion of herpesvirus-positive tissue samples was in the reproductive system, nervous system, and tegument. Herpesvirus was more prevalent in females, juveniles, and calves. More than half the DNA PCR positive tissues contained herpesvirus RNA, indicating the presence of actively replicating virus. This RNA was most frequently found in neonates. Fourteen unique sequences were identified. Most amplified sequences belonged to the
subfamily, but a greater variation was found in
sequences. This is the first report of systematic herpesvirus DNA and RNA determination in free-ranging cetaceans. Nine (19.14%) were infected with cetacean morbillivirus and all of them (100%) were coinfected with herpesvirus. Lesions similar to those caused by herpesvirus in other species were observed, mainly in the skin, upper digestive tract, genitalia, and central nervous system. Other lesions were also attributable to concomitant etiologies or were nonspecific. It is necessary to investigate the possible role of herpesvirus infection in those cases.
Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) ...neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins.
Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.
Introduction: The fourth symposium of the Colorado Alphaherpesvirus Latency Society (CALS) convened May 14-16, 2014 at the Christiania Lodge in the relaxed setting of the quiet mountain community of ...Vail, Colorado. In attendance were 65 investigators (Fig. 1) who traveled over 128,000 miles to attend the 2-day symposium consisting of 28 oral and 16 poster presentations that discussed current advances in alphaherpesvirus latency research involving herpes simplex virus types 1 and 2, varicella zoster virus, bovine herpesvirus, Marek's disease virus, and pseudorabies virus. This year's plenary talk by Michael Stark, Brigham Young University, on the developing peripheral nervous system continued the CALS theme of a major presentation from an expert outside the field of virology to help view latency in a different light. This year also brought expansion of the mission of CALS to include mentorship of postdoctoral fellows as well as a new open forum to discuss pressing topics not previously ad ...
Duck plague (DP) is an acute infectious disease in the duck industry. The duck plague virus (DPV) is the pathogen, a subfamily of
. gE is a type I membrane protein that contains three parts: an ...extracellular domain, a transmembrane domain, and a cytoplasmic domain. gE is the major virulence determinant of α-herpesvirus. However, the functions of the gE extracellular and cytoplasmic domains have not been reported in DPV. In this study, a gE extracellular domain deletion mutant and a gE cytoplasmic domain deletion mutant were constructed from DPV. Virus replication kinetics showed that the growth titers of both the gE ectodomain-deleted mutant virus and the gE cytoplasmic domain-deleted virus in DEFs were lower than that of the parental virus CHv-50. DPV CHv-gEΔET and DPV CHv-gEΔCT were continuously passed to the 20th passage in DEFs and the 10th in ducklings. The mutant virus DNA after passage was extracted for identification. The results showed that the gE ectodomain and gE cytoplasmic domain deletion mutant viruses have good genetic stability. The ducklings in each group (n=10) were inoculated with the same titers of DPV CHv-gEΔET, DPV CHv-gEΔCT, DPV CHv-ΔgE, and parental CHv-50, respectively. Clinical symptoms and serum antibody levels were detected after inoculation. The results showed that the virulence of DPV CHv-gEΔCT to ducklings was reduced compared with parental CHv-50, while the virulence of DPV CHv-gEΔET to ducklings was significantly reduced. 10
TCID
DPV CHv-gEΔET or DPV CHv-ΔgE can induce ducklings to produce DPV-specific antibodies, protect the ducklings from virulent CHv challenge. Therefore, DPV CHv-gEΔET may serve as a promising vaccine candidate to prevent and control duck plague.
An 11-day-old little blue penguin (Eudyptula minor) died unexpectedly. Prior to hatching, the egg experienced trauma and resultant defects were repaired. The chick hatched without complication and ...was clinically normal prior to death. Necropsy revealed congested lungs. Histologic examination showed moderate nonsuppurative encephalitis with focally extensive neuronal necrosis and intranuclear inclusions in neurons within necrotic foci. Herpesvirus DNA was detected in brain tissue with a generic herpesvirus polymerase chain reaction. Sanger sequencing demonstrated 100% and 98% sequence homology to sphenicid alphaherpesvirus 1 and penguin herpesvirus 2, respectively. In situ hybridization demonstrated large amounts of herpesvirus nucleic acid in intranuclear inclusions and neuronal nuclei. Combined histology, polymerase chain reaction, Sanger sequencing, and in situ hybridization results were most consistent with herpesviral encephalitis, most likely caused by sphenicid alphaherpesvirus 1. To our knowledge, this is the first report of a herpesvirus infection causing encephalitis in a penguin and the first report of herpesvirus in this species.
Duck plague virus (DPV), also known as anatid herpesvirus, is a double-stranded DNA virus and a member of α herpesvirus. DPV pUL15 is a homolog of herpes simplex virus 1 (HSV-1) pUL15, a terminase ...large subunit, and plays a key role in the cleavage and packaging of the viral concatemeric genome. However, the sequence similarity between DPV pUL15 and its homologs is low, and it is not sure if DPV pUL15 has the potential to cleave the concatemeric genome as same as its homologs. Here, we expressed the C terminal domain of DPV pUL15 to explore the nuclease function of DPV pUL15. The main results showed that (Ⅰ) DPV pUL15 C-terminal domain possesses nonspecific nuclease activity and lacks the DNA binding ability. (Ⅱ) DPV pUL15 nuclease activity needs to coordinate with divalent metal ions and tends to be more active at high temperatures. (Ⅲ) Even though the structure of DPV pUL15 nuclease domain is relatively conserved, the mutations of conserved amino acids on the nuclease domain do not significantly inhibit the nuclease activity.
•DPV pUL15 C-terminal domain possesses a nonspecific nuclease activity.•DPV pUL15 nuclease activity needs to coordinate with divalent metal ions, Mn2+ or Mg2+, and tends to be more active at high temperature.•The mutations of conserved amino acids on the nuclease domain do not significantly inhibit the nuclease activity.
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.
•Deletion of the tk gene from the CpHV-1 genome attenuates the virus in goats.•Traditional infection/transfection resulted in efficient CpHV-1 recombination.•Both parental and tk-deleted viruses ...established latency in neurons of the trigeminal ganglia.
Caprine alphaherpesvirus 1 (CpHV-1) is a pathogen associated with systemic infection and respiratory disease in kids and subclinical infection or reproductive failure and abortions in adult goats. The enzyme thymidine kinase (TK) is an important viral product involved in nucleotide synthesis. This property makes the tk gene a common target for herpesvirus attenuation. Here we deleted the tk gene of a CpHV-1 isolate and characterized the recombinant CpHV-1ΔTKin vitro and in vivo. In vitro characterization revealed that the recombinant CpHV-1ΔTK replicated to similar titers and produced plaques of similar size to the parental CpHV-1 strain in BT and CRIB cell lines. Upon intranasal inoculation of young goats, the parental virus replicated more efficiently and for a longer period than the recombinant virus. In addition, infection with the parental virus resulted in mild systemic and respiratory signs whereas the kids inoculated with the recombinant CpHV-1ΔTK virus remained healthy. Goats inoculated with the parental virus also developed higher neutralizing antibody titers when compared to CpHV-1ΔTK inoculated animals. Dexamethasone (Dx) administration on days 35–39 post-inoculation did not result in virus shedding in nasal secretions, indicating lack of reactivation from latency. However, viral DNA was detected in the trigeminal ganglia of animals euthanized at 14 days post-Dx, indicating that both viruses successfully established latent infection. Our results show that the recombinant CpHV-1ΔTK presents an attenuated phenotype when compared to the parental virus, and hence may represent a promising vaccine candidate to prevent CpHV-1 disease in goats.
Abstract A virus was found to be associated with a severe disease affecting rabbits on a farm near Anchorage, Alaska. Extracts from the skin of infected rabbits produced syncytia and cell lysis in ...cultured rabbit skin, rabbit kidney, and Vero cells. Examination of the infectious agent by electron microscopy revealed an icosahedral nucleocapsid surrounded by an envelope with a diameter of about 120 nm, suggesting that it was a herpesvirus. The viral genome was determined to be composed of double-stranded DNA of 120–130 kbp. PCR using degenerate primers to conserved herpesvirus genes was used to amplify sequences from purified viral DNA. Sequencing of these products allowed the design of specific primers so that complete sequence data for a number of genes could be determined. Analysis of these data indicated that the virus is most closely related to bovine herpesvirus 2. The next most closely related viruses are human herpesviruses 1 and 2, and a number of cercopithecine herpesviruses. Experimental exposure of domestic rabbits to the isolate resulted in severe clinical disease and necrosis in the spleen and lymph node. In addition, viral DNA was identified in a variety of tissues by PCR, consistent with a systemic infection. Taken together, these data suggest that this virus is highly pathogenic for domestic rabbits and belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplexvirus.
pUL51 is a minor tegument protein important for viral assembly and cell-to-cell spread (CCS) but dispensable for replication in cell culture of all Herpesviruses for which its role has been ...investigated. Here, we show that pUL51 is essential for the growth of Marek's disease virus, an oncogenic alphaherpesvirus of chickens that is strictly cell-associated in cell culture. MDV pUL51 localized to the Golgi apparatus of infected primary skin fibroblasts, as described for other Herpesviruses. However, the protein was also observed at the surface of lipid droplets in infected chicken keratinocytes, hinting at a possible role of this compartment for viral assembly in the unique cell type involved in MDV shedding
Deletion of the C-terminal half of pUL51 or fusion of GFP to either the N- or C-terminus were sufficient to disable the protein's essential function(s). However, a virus with a TAP domain fused at the C-terminus of pUL51 was capable of replication in cell culture, albeit with viral spread reduced by 35% and no localization to lipid droplets.
, we observed that although the replication of this virus was moderately impacted, its pathogenesis was strongly impaired. This study describes for the first time the essential role of pUL51 in the biology of a herpesvirus, its association to lipid droplets in a relevant cell type, and its unsuspected role in the pathogenesis of a herpesvirus in its natural host.
Viruses usually spread from cell to cell through two mechanisms: cell-released virus and/or cell-to-cell spread (CCS). The molecular determinants of CCS and their importance in the biology of viruses during infection of their natural host are unclear. Marek's disease virus (MDV) is a deadly and highly contagious herpesvirus of chickens that produces no cell-free particles
, and therefore, spreads only through CCS in cell culture. Here, we show that viral protein pUL51, an important factor for CCS of Herpesviruses, is essential for MDV growth
. We demonstrate that the fusion of a large tag at the C-terminus of the protein is sufficient to moderately impair viral replication
and almost completely abolish pathogenesis while only slightly reducing viral growth
. This study thus uncovers a role for pUL51 associated with virulence, linked to its C-terminal half, and possibly independent of its essential functions in CCS.
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