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.

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.

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.

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-5gEÄ), thymidine kinase (BoHV-5TKÄ) and both proteins (BoHV-5gEÄTKÄ). 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-5gEÄ 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-5TKÄ (N = 8) or BoHV-5gEÄTKÄ (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-5TKÄ and BoHV-5gEÄTKÄ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

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.

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 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.

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.

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.

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.

In the absence of glycoprotein I (gI), gE determines bovine herpesvirus type 5 neuroinvasiveness and neurovirulence.

Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus that causes fatal encephalitis in calves. Envelope glycoproteins E (gE) and gI of alphaherpesviruses are important for the pathogenesis in vivo. Previously the authors determined that BHV-5 gE is important for BHV-5 neurovirulence. To determine the role of gI in BHV-5 neurovirulence, the authors have constructed gI-deleted and gI-revertant BHV-5 and analyzed their neuropathogenic properties in a rabbit seizure model. Following intranasal infection, 40% of the rabbits infected with the gI-deleted virus showed severe neurological signs. gI-deleted BHV-5 invaded all the central nervous system (CNS) structures invaded by the gI-revertant BHV-5; however, the number of neurons infected by the gI-deleted virus was similar or slightly reduced (two to four fold). Thus, the gI-deleted virus retained significant neurovirulence and/or neuroinvasive properties when compared with the gE-deleted BHV-5. Pulse-chase analysis revealed that the gE of gI-deleted virus was processed to a larger and a diffused 94- to 100-kDa protein (instead of 94 kDa). The 94- to 100-kDa protein was processed in the Golgi with delayed kinetics but it was endoglycosidase H (EndoH) resistant. In cells infected with gI-deleted virus, there was a reduction in cell-surface gE expression compared to wild-type, which correlated to reduced amount of gE processed in the Golgi. The authors believe that in the absence of gI, BHV-5 gE is sufficient for BHV-5 neurovirulence.

A glycine-rich bovine herpesvirus 5 (BHV-5) gE-specific epitope within the ectodomain is important for BHV-5 neurovirulence.

The bovine herpesvirus 5 (BHV-5) gE ectodomain contains a glycine-rich epitope coding region (gE5 epitope), residues 204 to 218, that is significantly different from the corresponding gE region of BHV-1. Deletion of the gE epitope significantly reduced the neurovirulence of BHV-5 in rabbits. Pulse-chase analyses revealed that the epitope-deleted and wild-type gE were synthesized as N-glycosylated endoglycosidase H-sensitive precursors with approximate molecular masses of 85 kDa and 86 kDa, respectively. Like the wild-type gE, epitope-deleted gE complexed with gI and was readily transported from the endoplasmic reticulum. Concomitantly, the epitope-deleted and wild-type gE acquired posttranslational modifications in the Golgi leading to an increased apparent molecular mass of 93-kDa (epitope-deleted gE) and 94-kDa (wild-type gE). The kinetics of mutant and wild-type gE processing were similar, and both mature proteins were resistant to endoglycosidase H but sensitive to glycopeptidase F. The gE epitope-deleted BHV-5 formed wild-type-sized plaques in MDBK cells, and the epitope-deleted gE was expressed on the cell surface. However, rabbits infected intranasally with gE epitope-deleted BHV-5 did not develop seizures, and only 20% of the infected rabbits showed mild neurological signs. The epitope-deleted virus replicated efficiently in the olfactory epithelium. However, within the brains of these rabbits there was a 10- to 20-fold reduction in infected neurons compared with the number of infected neurons within the brains of rabbits infected with the gE5 epitope-reverted and wild-type BHV-5. In comparison, 70 to 80% of the rabbits exhibited severe neurological signs when infected with the gE5 epitope-reverted and wild-type BHV-5. These results indicated that anterograde transport of the gE epitope-deleted virus from the olfactory receptor neurons to the olfactory bulb is defective and that, within the central nervous system, the gE5 epitope-coding region was required for expression of the full virulence potential of BHV-5.

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis.

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.

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.

Neurovirulence of glycoprotein C(gC)-deleted bovine herpesvirus type-5 (BHV-5) and BHV-5 expressing BHV-1 gC in a rabbit seizure model.

Herpesvirus glycoprotein C (gC) is one of the major virus attachment proteins. Bovine herpesvirus type 1 (BHV-1) causes respiratory and genital diseases in cattle, whereas BHV-5 causes acute meningoencephalitis in calves. The gC gene sequence of these two viruses are substantially different. To determine the contribution of the BHV-5 glycoprotein gC (gC5) to the neuropathogenesis of BHV-5, we have constructed two BHV-5 recombinants: gC-deleted BHV-5 (BHV-5gCDelta) and BHV-5 expressing BHV1 gC (BHV-5gC1). Neurovirulence properties of these viruses were analyzed using a rabbit seizure model that distinguishes BHV-1 and -5 based on their differential neuropathogeneses. Intranasal inoculations of BHV-5gCDelta and BHV-5gC1 viruses produced neurological signs in 30% and 40% of the infected rabbits, respectively. Immuno-histochemistry results showed that the number of infected neurons was 2 - 4-fold less with the gC-deleted BHV-5 than with the wild-type BHV-5. The gC-deleted BHV-5 did not invade the hippocampus but invaded additional sites not invaded by wild-type BHV-5. Similarly, the BHV-5gC1 virus failed to invade the hippocampus, but it did not invade the additional sites. Virus isolation results suggest that these recombinants replicate less efficiently in the brain than the wild-type and gC-revertant viruses. However, compared to the gC-deleted BHV-5, the gC-exchanged BHV-5gC1 replicated better within the CNS. These results indicate that gC regulates BHV-5 neurotropism in some areas of the olfactory pathway. Additionally, gC is important for BHV-5 neurovirulence in the olfactory pathway but it is not essential.

Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neurovirulence in the olfactory pathway of the rabbit.

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEDelta), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295-310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEDelta and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.

Spread of bovine herpesvirus type 5 (BHV-5) in the rabbit brain after intranasal inoculation.

Following intranasal inoculation of wild-type BHV-5 in rabbits, we studied the sequential transneuronal passage of the virus in the CNS by immunocytochemistry, histopathology, and virus isolation. At 4 and 6 days postinfection (d.p.i.), rabbits had no or mild neurological signs, and virus was isolated only from the olfactory bulbs. At 8 and 9 d.p.i., infected rabbits had severe neurological signs, and virus could be isolated from multiple regions of the brain segments. In these rabbits, high titers of virus were consistently present in the anterior and posterior cortices, including frontal, piriform/entorhinal, temporal, parietal, and occipital cortices, the hippocampus and the amygdala. Virus was isolated occasionally from the midbrain/diencephalon and pons/medulla. Virus was not isolated from the cerebellum and trigeminal ganglion of rabbits examined from 2-12 d.p.i. Immunocytochemistry revealed virus-specific antigens at 4 d.p.i. within the glomerular layer, external plexiform layer, and mitral cell layer of the main olfactory bulb. At 6 d.p.i., virus-specific antigens were also present within the inner granular layer of the main olfactory bulb. At 8 and 9 d.p.i., widespread BHV-5-specific staining occurred in the areas of the brain connected to the main olfactory bulb, including the frontal/cingulate cortex, anterior olfactory nucleus, lateral olfactory tubercle, piriform/entorhinal cortex, hippocampus, amygdala, dorsal raphe, and locus coeruleus. In the trigeminal ganglion, specific staining was detected within a few neurons at 2,4, 6, 8 d.p.i. However, further spread of the virus along the trigeminal pathway was not evident. These data indicate that BHV-5 replicates and spreads preferentially in the olfactory pathway following intranasal instillation and that this viral spread correlated with the severity of neurological symptoms and histopathological lesions.

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.