A bovine herpesvirus type 1 mutant virus with truncated glycoprotein E cytoplasmic tail has defective anterograde neuronal transport in rabbit dorsal root ganglia primary neuronal cultures in a microfluidic chamber system.

Bovine herpesvirus type 1 (BHV-1) is an important component of the bovine respiratory disease complex (BRDC) in cattle. Following primary intranasal and ocular infection of cattle, BHV-1 establishes lifelong latent infection in trigeminal ganglia (TG). Upon reactivation from latency, the virus is transported from neuronal cell bodies in the TG to projected nerve endings in nose and cornea of latently infected cattle where the virus shedding occurs. This property of BHV-1 plays a significant role in the pathogenesis of BRDC and maintenance of BHV-1 in the cattle population. Recently, we have reported that a glycoprotein E (gE) cytoplasmic tail-truncated BHV-1 (BHV-1 gEAm453) did not reactivate from latency and was not shed in the nasal and ocular secretions of calves and rabbits. Here we describe the methods to establish rabbit primary dorsal root ganglia (DRG) neuron cultures in a microfluidic chamber system and to characterize in vitro anterograde and retrograde axonal transport properties of BHV-1 gE-deleted and BHV-1 cytoplasmic tail-truncated gEAm453 mutant viruses relative to BHV-1 gEAm453-rescued/wild-type viruses. The results clearly demonstrated that whereas the BHV-1 gE-deleted, BHV-1 gEAm453, and BHV-1 gEAm453-rescued/wild-type viruses were transported equally efficiently in the retrograde direction, only the BHV-1 gEAm453-rescued/wild-type virus was transported anterogradely. Therefore, we have concluded that sequences within the BHV-1 gE cytoplasmic tail are essential for anterograde axonal transport and that primary rabbit DRG neuronal cultures in the microfluidic chambers are suitable for BHV-1 neuronal transport studies.

A bovine herpesvirus type 1 mutant virus specifying a carboxyl-terminal truncation of glycoprotein E is defective in anterograde neuronal transport in rabbits and calves.

Bovine herpesvirus type 1 (BHV-1) is an important component of the bovine respiratory disease complex (BRDC) in cattle. The ability of BHV-1 to transport anterogradely from neuronal cell bodies in trigeminal ganglia (TG) to nerve ending in the noses and corneas of infected cattle following reactivation from latency plays a significant role in the pathogenesis of BRDC and maintenance of BHV-1 in the cattle population. We have constructed a BHV-1 bacterial artificial chromosome (BAC) clone by inserting an excisable BAC plasmid sequence in the long intergenic region between the glycoprotein B (gB) and UL26 genes. A BAC-excised, reconstituted BHV-1 containing only the 34-bp loxP sequence within the gB-UL26 intergenic region was highly infectious in calves, retained wild-type virulence properties, and reactivated from latency following treatment with dexamethasone. Using a two-step Red-mediated mutagenesis system in Escherichia coli, we constructed a gE cytoplasmic tail-truncated BHV-1 and a gE-rescued BHV-1. Following primary infection, the gE cytoplasmic tail-truncated virus was efficiently transported retrogradely from the nerve endings in the nose and eye to cell bodies in the TG of calves and rabbits. However, following dexamethasone-induced reactivation from latency, the gE mutant virus was not isolated from nasal and ocular sheddings. Reverse transcriptase PCR assays detected VP5 transcription in the TG of rabbits infected with gE-rescued and gE cytoplasmic tail-truncated viruses during primary infection and after dexamethasone treatment but not during latency. Therefore, the BHV-1gE cytoplasmic tail-truncated virus reactivated in the TG; however, it had defective anterograde transport from TG to nose and eye in calves and rabbits.

Transinhibition of herpes simplex virus replication by an inducible cell-resident gene encoding a dysfunctional VP19c capsid protein.

This study demonstrates that cells expressing a dysfunctional analog of a herpes simplex virus (HSV) capsid protein inhibits HSV replication. Vero cell lines expressing HSV-1 capsid protein VP19c/beta-galactosidase fusion proteins were constructed and tested for their kinetics of expression, intracellular location, and ability to interfere with HSV replication. Two chimeric genes were constructed for these studies. The larger chimeric gene encodes the amino terminal 327 amino acids (aa) of VP19c fused to the carboxy terminal 1026 aa of beta-galactosidase, and the shorter chimeric gene encodes VP19c aa 1-30 and 302-327 fused to the carboxy-terminal 1026 aa of beta-galactosidase. Cell lines V32G-1 and V32G-2 containing the larger and the shorter chimeric genes, respectively, were isolated after cotransfection with plasmid pSV2-neo DNA, cell selection, and limiting-dilution cloning. The chimeric VP19c/beta-galactosidase genes resident in V32G-1 and V32G-2 cell lines were induced by early gene products of superinfecting wild-type HSV-1 and HSV-2, but were not constitutively expressed. The hybrid proteins expressed in infected V32G-1 and V32G-2 cells both colocalized with infected cell protein 8 (ICP8) into virus-replicative compartments in the cell nuclei. HSV-1 and HSV-2 growth in V32G-1 cells (which express the larger chimeric gene) was significantly reduced compared to growth in V32G-2 and control Vero cells. The data suggest that the larger VP19c/beta-galactosidase hybrid protein interferes with virus capsid assembly or morphogenesis in a competitive manner. Results also demonstrate that a small portion of VP19c containing the predicted endoplasmic reticulum signal sequence for this capsid protein (aa 1-30) promotes incorporation of the VP19c/beta-galactosidase fusion proteins into nuclear viral replication compartments.

Molecular biological characterization of equine herpesvirus type 1 (EHV-1) isolates from ruminant hosts.

Isolates of equine herpesvirus type 1 (EHV-1) originating from affected antelope and cattle were compared with reference EHV-1 and EHV-4 isolates and were characterized. Based on cross-neutralization, DNA restriction profiles and blot-hybridization data these isolates could be characterized as EHV-1. One isolate (from an antelope) with a different restriction profile showed significant DNA homology with EHV-1, partial homology with EHV-4, and little or no homology with EHV-2 and HSV-1 DNAs. Blot hybridization revealed differences in DNA restriction fragments located at the termini of two isolates and size heterogeneity in the unique long/internal repeat junction fragment (UL/IR) of one isolate.

Rapid method for the identification and screening of herpesviruses by DNA fingerprinting combined with blot hybridization.

Rapid characterization of herpesvirus isolates exemplified by equine herpesviruses is described. Total DNAs were isolated from virus infected small scale cell cultures. The DNA fragments obtained after restriction enzyme digestion were separated on agarose gels, transferred and immobilized on filter membranes. A radioactively labelled probe derived from the purified DNA of an EHV-1 reference strain was used for hybridization in order to detect the restriction fragments of different EHV-1 field isolates. This method allows the typing of many isolates within a short period of time.

The renal afferent pathways in the rat: a pseudorabies virus study.

Retrograde tract tracing studies have indicated that dorsal root ganglion cells from T8 to L2 innervate the rat's left kidney. Electrophysiology studies have indicated that putative second-order sympathetic afferents are found in the dorsal horn at spinal segments T10 to L1 in laminae V-VII. Here, the spread of pseudorabies virus through renal sensory pathways was examined following 2-5 days post-infection (PI) and the virus was located immunocytochemically using a rabbit polyclonal antibody. Two days PI, dorsal root ganglion neurons (first-order sympathetic afferents) were infected with PRV. An average of 1.2, 0.8, 2.1 and 4.4% of the infected dorsal root ganglion neurons were contralateral to the injected kidney at spinal segments T10, T11, T12 and T13, respectively. Four days PI, infected neurons were detected within laminae I and II of the dorsal horn of the caudal thoracic and upper lumbar spinal cord segments. The labeling patterns in the spinal cord are consistent with previous work indicating the location of renal sympathetic sensory pathways. The nodose ganglia were labeled starting 4 days PI, suggesting the involvement of parasympathetic sensory pathways. Five days PI, infected neurons were found in the nucleus tractus solitarius. In the present study, it was unclear whether the infected neurons in the nucleus tractus solitarius are part of sympathetic or parasympathetic afferent pathways or represent a convergence of sensory information. Renal denervation prevented the spread of the virus into the dorsal root ganglia and spinal cord. Sectioning the dorsal roots from T10-L3 blocked viral spread into the spinal cord dorsal horn, but did not prevent infection of neurons in dorsal root ganglion nor did it prevent infection of putative preganglionic neurons in the intermediolateral cell column. The present results indicated that renal afferent pathways can be identified after pseudorabies virus infection of the kidney. Our results suggest that renal afferents travel in sympathetic and parasympathetic nerves and that this information may converge at the NTS.

Neural circuitry of the kidney: NO-containing neurons.

The neurons synthesizing nitric oxide (NO) that are part of the renal sympathetic pathways were located by double-staining for the neuronal isoform of nitric oxide synthase (nNOS) using immunocytochemistry to identify NO-synthesizing neurons and transneuronal tracing following infection of the left kidney with pseudorabies virus (PRV). Following kidney injection with PRV, the animals survived 4-day post-inoculation prior to sacrifice and tissue processing. PRV-infected neurons that double-stained for nNOS were found in the paraventricular hypothalamic nucleus (PVN), the raphe obscurus nucleus (ROb), the ventromedial medulla (VMM), the rostral ventrolateral medulla (rVLM) and the A5 cell group. In the thoracolumbar spinal cord, nNOS neurons co-localized with PRV-infected cells in the dorsal horn in laminae I, III-V ipsilateral to the injected kidney and in lamina X, the intermediolateral cell column, the lateral funiculus, the intercalated nucleus and the central autonomic area. We conclude that NO synthesizing cells may significantly affect renal autonomic pathways in the rat by interacting with the renal sensory and sympathomotor circuitry at multiple sites.

The estrous cycle affects pseudorabies virus (PRV) infection of the CNS.

Previous work had suggested that mucosal immunity may be affected by the stage of the estrous cycle. Here, susceptibility to a neurotropic virus infection at different stages of the estrous cycle was assessed in a rodent model after direct injection of the virus into visceral organs. In the first two experiments, female Sprague-Dawley rats were infected with pseudorabies virus (PRV, Bartha's K-strain) by injection into either the cervix or the kidney after monitoring their estrous cycle. After either 4- or 5-day survival period post-infection, the rats were euthanized by transcardially perfusion and peripheral and central nervous system tissues were removed for immunocytochemical staining. The number of infected neurons was counted in various regions. Statistical analysis revealed that: (1) the number of infected cells in the sympathetic or parasympathetic ganglion, or the dorsal root ganglia was not affected regardless of the stage of the estrous cycle after cervix injection with PRV; (2) in contrast, the number of infected neurons in the spinal cord was affected significantly by the stage of the estrous cycle during viral infection of the cervix; (3) after kidney infection, the number of infected neurons found within the spinal cord or dorsal root ganglia varied significantly across the estrous cycle. In both cases, animals infected in proestrus or estrus had fewer infected neurons than animals infected in diestrus I or diestrus II (proestrous and estrous animals had less than 20% of infected cells found in diestrus I or diestrus II rats). In the third experiment, older, persistent estrous or persistent diestrous rats were infected by kidney injection and given a 4-day survival period, prior to virus isolation from lower thoracic spinal cord. Animals in persistent estrous had significantly less virus per gram of tissue than the persistent diestrous rats. These data suggest that the CNS of animals in proestrus or estrus is less susceptible to PRV infection compared to animals in either diestrus I or diestrus II. Because estrogen replacement therapy is known to restore some immune functions during reproductive ageing, it is speculated that plasma estrogen levels modulate the central nervous system's susceptibility to viral infections.

Fine mapping of bovine herpesvirus 1 (BHV-1) glycoprotein C neutralizing epitopes by type-specific monoclonal antibodies and synthetic peptides.

Bovine herpesvirus glycoprotein C (gC) functions as a major virus attachment protein. Here, two BHV-1 gC-specific epitopes that are specified by complement-dependent neutralizing MAbs are mapped. The BHV-1 gC-specific peptides and MAbs were used to specifically localize continuous epitopes by direct binding to the MAbs and by blocking the Mab reactivity (competitive ELISA) to authentic viral antigen. The results of competitive ELISA indicated that the complement-dependent neutralizing epitopes recognized by MAbs F2 and 24 were located between BHV-1 gC amino acids (aa) 47-69 and (aa) 109-119, respectively.

Construction and characterization of an attenuated bovine herpesvirus type 1 (BHV-1) recombinant virus.

A recombinant bovine herpesvirus type 1 (BHV-1) virus (Gal-TK) has been constructed. The Gal-TK virus contains a chimeric reporter/marker gene coding for bacterial beta-galactosidase (beta-gal gene) that was inserted stably within the viral TK gene. This resulted in inactivation of the TK gene. The beta-gal gene is under the regulation of a strong, human cytomegalovirus-immediate early (HCMV-IE) promoter and is expressed as an authentic viral-coded gene. Even though the one-step growth kinetics of the recombinant and parent viruses were similar, the recombinant virus yielded less than the parent virus on Madin-Darby bovine kidney cells. After intranasal inoculation, the engineered virus was virtually avirulent for colostrum-deprived new-born calves. Similar to the parent virus, the recombinant virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was reduced significantly. The progeny viruses recovered from nasal swabs of animals inoculated with the recombinant and the Cooper strains of BHV-1 were easily distinguishable based on the beta-gal marker.

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model.

The suitability of a rabbit seizure model for studying the neuropathogenesis of bovine herpesvirus type 5 (BHV-5) encephalitis was evaluated. Intranasal administration of BHV-5 (strain TX89) together with intramuscular administration of dexamethasone produced seizures in 70% of rabbits tested and meningo-encephalitis in 100%. Infectious BHV-5 was consistently isolated from the following sites: olfactory bulb; anterior cortex, containing the frontal cortex, olfactory tract and anterior portion of the olfactory cortex; posterior cortex, containing the temporal, parietal, piriform, entorhinal and occipital cortices; amygdala; hippocampus. Less frequently, BHV-5 was isolated from the midbrain and diencephalon, the pons and medulla, the cerebellum, and the trigeminal ganglia. Rabbits similarly infected with the Cooper strain of bovine herpesvirus type 1 showed no neurological signs or meningo-encephalitis, and virus was not recovered from the brain. The brains of BHV-5-infected rabbits showed neuronal degeneration, leptomeningitis, gliosis and perivascular cuffing, predominantly in the olfactory cortex (piriform and entorhinal cortices), amygdala and hippocampus. Mild lymphocytic meningitis was seen in the olfactory bulb and focal lymphocytic infiltration was sometimes present in the medulla and cerebellum. BHV-5, specific antigens and nucleic acids were detected in the olfactory cortex, amygdala and hippocampus by immunohistochemical methods and in-situ hybridization. The results suggested that, after intranasal BHV-5 inoculation, the virus spread to the central nervous system via the olfactory and trigeminal pathways. The olfactory pathway was more susceptible than the trigeminal pathway to neuropathogenic effects.

Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant.

OBJECTIVE: To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1). PROCEDURE: The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests. RESULTS AND CONCLUSIONS: The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is immunogenic for calves and confers protection upon homologous challenge and BoHV-1 challenge.

A recombinant bovine herpesvirus 5 lacking thymidine kinase and glycoprotein E genes (BoHV-5gEΔTKΔ) was evaluated as a live experimental vaccine. In a first experiment, ten-months-old calves were vaccinated intramuscularly (n=9) or remained as controls (n=8) and 42 days later were challenged with BoHV-5 or BoHV-1 intranasally. The four control calves challenged with BoHV-5 developed severe depression and neurological signs and were euthanized in extremis at days 13 and 14 pos-infection (pi); the five vaccinated animals challenged with BoHV-5 remained healthy. The titers of virus shedding were reduced (p<0.01) from days 3 to 7 post-infection (pi) in vaccinated animals. Control and vaccinated calves challenged with BoHV-1 presented mild transient respiratory signs; yet the magnitude of virus shedding was reduced (p<0.05) in vaccinated animals (days 5, 9 and 11pi). In a second experiment, young calves (100-120 days-old) were vaccinated (n=15) or kept as controls (n=5) and subsequently challenged with a BoHV-1 isolate. Control calves developed moderate to severe rhinitis and respiratory distress; two were euthanized in extremis at days 5 and 9 pi, respectively. In contrast, vaccinated animals were protected from challenge and only a few developed mild and transient nasal signs. The duration and titers of virus shedding after challenge were reduced (p<0.05) in vaccinated animals comparing to controls. In both experiments, vaccinated animals developed antibodies to gE only after challenge. These results demonstrate homologous and heterologous protection and are promising towards the use of the recombinant BoHV-5gEΔTKΔ in vaccine formulations to control BoHV-5 and BoHV-1 infections.

The bovine herpesvirus type 1 envelope protein Us9 acidic domain is crucial for anterograde axonal transport.

In this study, we examined the functional role of bovine herpesvirus type 1 (BHV-1) Us9 acidic domain residues 83-90 in the anterograde axonal transport of the virus in calves (natural host), rabbits, and in cultured neurons. A mutant virus strain lacking Us9 residues 83-90 (BHV-1 Us9 Δ83-90) and the rescued virus (BHV-1 Us9 R83-90) replicated efficiently in the nasal and ocular epithelium during primary infection and established latency in the trigeminal ganglia (TG). However, upon reactivation from latency, only the BHV-1 Us9 R83-90 virus was detected in nasal and ocular swabs of animals. In compartmentalized, rabbit primary dorsal root ganglia (DRG) neuron cultures, the Us9-deleted BHV-1, BHV-1 Us9 Δ83-90 and BHV-1 Us9 R83-90 viruses were transported efficiently in the retrograde direction. However, only the BHV-1 Us9 R83-90 virus was transported in an anterograde direction. These studies suggested that the Us9 acidic domain residues located between 83 and 90 were required for axonal anterograde transport.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both.

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric beta-galactosidase gene. Subsequently, using the BoHV-5 gE virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE) and TK + gE (BoHV-5 gE/TK) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE, BoHV-5 TK and BoHV-5 gE/TK) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK) gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits.

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEDelta), thymidine kinase (BoHV-5TKDelta) and both proteins (BoHV-5gEDeltaTKDelta). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEDelta developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKDelta (N = 8) or BoHV-5gEDeltaTKDelta (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKDelta and BoHV-5gEDeltaTKDelta are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

Glycoprotein E (gE) specified by bovine herpesvirus type 5 (BHV-5) enables trans-neuronal virus spread and neurovirulence without being a structural component of enveloped virions.

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alpha-herpesvirus that causes fatal encephalitis in calves. We previously demonstrated that deletion of a glycine-rich epitope in the gE ectodomain dramatically reduced BHV-5 neurovirulence. To investigate the role of gE cytoplasmic tail sequences in the neuropathogenesis of BHV-5 in rabbits, we constructed a BHV-5gE recombinant virus with a short residual cytoplasmic domain lacking the YXXL motifs and the acidic (BHV-5gEAm480). In vitro, BHV-5gEAm480 produced on the average smaller plaques, compared with wild-type BHV-5, but it produced on the average substantially larger plaques than the gE ORF-deleted BHV-5. The truncated gE was not phosphorylated, and was not endocytosed from the cell surface. Importantly, the truncated gE was not incorporated into enveloped infectious virions, but its glycosylation and interaction with gI were not affected. In a rabbit model of infection, the BHV-5gEAm480 remained highly virulent, while the gE-null virus was avirulent. The gEAm480 mutant virus invaded most of the central nervous system (CNS) structures that are invaded by the wild-type BHV-5. The number of neurons infected by BHV-5gEAm480 was very similar to the number infected by BHV-5 wild-type and gEAm480-rescued viruses. Collectively, the results suggest that gE functions in transsynaptic transmission of BHV-5 and neurovirulence without being a structural component of the virion particle.

Envelope protein Us9 is required for the anterograde transport of bovine herpesvirus type 1 from trigeminal ganglia to nose and eye upon reactivation.

In this study, the authors examined the role of bovine herpesvirus type 1 (BHV-1) Us9 in the anterograde transport of the virus from trigeminal ganglia (TG) to nose and eye upon reactivation from latency. During primary infection, both BHV-1 Us9-deleted and BHV-1 Us9-rescued viruses replicated efficiently in the nasal and ocular epithelium. However, upon reactivation from latency, only the BHV-1 Us9-rescued virus could be isolated in the nasal and ocular shedding. By real-time polymerase chain reaction, comparable DNA copy numbers were detected in the TGs during latency and reactivation for both the viruses. Therefore, Us9 is essential for reactivation of the virus in the TG and anterograde axonal transport from TG to nose and eye.

The Us9 gene of bovine herpesvirus 1 (BHV-1) effectively complements a Us9-null strain of BHV-5 for anterograde transport, neurovirulence, and neuroinvasiveness in a rabbit model.

The alphaherpesvirus envelope protein Us9 is a type II viral membrane protein that is required for anterograde spread of bovine herpesvirus 5 (BHV-5) infection from the olfactory receptor neurons to the brain. In a rabbit seizure model, Us9-deleted BHV-5 failed to invade the central nervous system (CNS) following intranasal infection. However, when injected directly into the olfactory bulb, retrograde-spread infection from the olfactory bulb (OB) to the piriform cortex and other areas connected to the OB was not affected. In contrast to BHV-5, wild-type BHV-1 failed to invade the CNS following intranasal infection. In this study, we show that mature BHV-1 Us9 is a 30- to 32-kDa protein, whereas mature BHV-5 Us9 is an 18- to 20-kDa protein. In vitro, BHV-1 Us9 is expressed at 3 h postinfection (hpi), whereas BHV-5 Us9 is expressed at 6 hpi. Despite these differences, BHV-1 Us9 not only complemented for BHV-5 Us9 and rescued the anterograde-spread defect of the BHV-5 Us9-deleted virus but conferred increased neurovirulence and neuroinvasiveness in our rabbit seizure model. Rabbits infected with BHV-5 expressing BHV-1 Us9 showed severe neurological signs at 5 days postinfection, which was 1 to 2 days earlier than BHV-5 wild-type or Us9-reverted BHV-5 virus. The data underscore the importance of both Us9 genes for virion anterograde transport and neuroinvasiveness. However, Us9 is not the determinant of the differential neuropathogenesis of BHV-1 and BHV-5.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.