Contribution of gene products encoded within the unique short segment of equine herpesvirus 1 to virulence in a murine model

The pathogenesis of three equine herpesvirus 1 (EHV-1) recombinants was assessed in a CBA mouse model. Sequences encoding the majority of glycoproteins I (gI) and E (gE) were deleted from the pathogenic EHV-1 strain RacL11 (L11ΔgIΔgE), and sequences comprising the 3859 bp deletion within the strain KyA U_S segment, which includes genes 73 (gI), 74 (gE), and 75 (putative 10 kDa protein 75), were re-inserted into attenuated KyA (KgI/gE/75). In addition, genes gE and 75 were inserted into KyA to generate the EHV-1 recombinant KgE/75. The insertion of the 3859 bp U_S segment was sufficient to confer virulence to KyA, as indicated by pronounced signs of clinical disease including substantial weight loss. A large plaque morphology was observed in cells infected with KgI/gE/75 compared with KyA, and a small plaque phenotype was observed in cells infected with L11ΔgIΔgE compared with RacL11. These data indicate that gI and/or gI and gE contribute to the ability of EHV-1 to spread directly from cell-to-cell. The deletion of both gI and gE from the pathogenic RacL11 strain did not reduce clinical signs of disease in infected mice, but did decrease mortality compared with RacL11. Furthermore, the insertion of genes 74 (gE) and 75 into the vaccine strain KyA did not alter the attenuated phenotype of this virus. Finally, KgI/gE/75 and RacL11 elicited the production of the proinflammatory chemokines MIP-1, MIP-1β, and MIP-2 in the lungs of infected mice, while KyA did not, suggesting that gI and/or gI and gE contribute to the up-regulation of these mediators of inflammation. These findings show that gI, and/or gI and gE restore a virulent phenotype to the EHV-1 KyA strain, and indicate that virulence factors, in addition to gI and gE, contribute to the pathogenesis of the RacL11 strain.     

A deletion in the gI and gE genes of equine herpesvirus type 4 reduces viral virulence in the natural host and affects virus transmission during cell-to-cell spread

In order to identify the role of the equine herpesvirus type 4 (EHV-4) glycoprotein I (gI) and E (gE) genes in determining viral virulence and their affect on the infection cycle, we constructed an EHV-4 recombinant strain containing a deletion in both gI and gE genes and its revertant. The recombinant was assayed in vitro in order to compare its growth kinetics with the parent and revertant viruses. Our results indicated that a deletion in the genes encoding gI and gE affected cell-to-cell spread of the virus in vitro. In order to assess the pathogenicity and vaccine efficacy of the recombinant in a natural host, colostrum-deprived foals were inoculated intranasally with the recombinant. Clinical signs obtained in foals upon the inoculation with the recombinant were milder than that for the revertant. This suggests that intact gI and/or gE genes are important factors in the expression of virulence in EHV-4 as in seen in the case of other herpesviruses. In addition, full protection against challenge infection was observed in foals, which had undergone a previous inoculation of the recombinant.     

Meningoencephalitis in Mice Infected with an Equine Herpesvirus 1 Strain KyA Recombinant Expressing Glycoprotein I and Glycoprotein E

One of the consequences of equine herpesvirus 1 (EHV-1) infection in the natural host is a neurological disease that can lead to paralysis. The pathology associated with EHV-1-induced neurological disease includes vasculitis of the small blood vessels within the central nervous system and subsequent damage to the surrounding neural tissue. In a previous study, an EHV-1 recombinant KyA virus (KgI/gE/75) was generated in which the sequences encoding glycoprotein I (gI) and glycoprotein E (gE) were repaired [Frampton et al. 2002 (Virus Research 90: 287-301)] using genes of the pathogenic EHV-1 strain 89c25. In contrast to the parental KyA virus that lacks gI and gE, the recombinant KgI/gE/75 was able to spread to the brains of CBA mice after intranasal infection. Infection resulted in a meningoencephalitis characterized by lymphocytic cuffing of small blood vessels within the brain, consistent with that observed in EHV-1-infected horses exhibiting neurological signs. KgI/gE/75 was able to elicit cytopathology in the lung prior to spread to the brain. However, like the attenuated KyA strain, KgI/gE/75 did not persist in the lung and was completely cleared from lung tissue by day 5 postinfection. We propose that gI and gE are neurovirulence factors for EHV-1, and that the CBA mouse model can be extended to study neurologic sequelae resulting after EHV-1 infection.     

Correlation between gI,gII, gIII,and gp 50 antibodies and virus excretion in vaccinated pigs infected with pseudorabies virus

Summary Eleven groups of 8 pigs were vaccinated with different vaccinal strains of pseudorabies virus deleted or not for non-essential glycoproteins (gI, gX, gp 63), then were challenged 8–9 weeks later with a virulent strain. Antibodies against the major viral glycoproteins (gII, gIII, gp 50) were titrated at the day of challenge. Excretion of the challenge strain, growth performance of the pigs, and gI antibody responses from pigs vaccinated with a gI minus strain, were monitored after the challenge. The results demonstrated a strong association between gII and gIII antibody titres before the challenge, with the amount of the challenge strain excreted and clinical protection after the challenge. Furthermore, gI antibody titres after the challenge were negatively correlated with the level of gII and gIII antibodies at the day of challenge, the virus excretion and the clinical protection after the challenge. Implications of these results for vaccination of pigs and gI antibody screening for the infection are discussed.     

A comparative study of pseudorabies virus (PRV) strains with defects in thymidine kinase and glycoprotein genes

In the course of two experiments, an examination was made of the virulence and neuroinvasiveness for pigs of two pseudorabies virus mutants (strain 6C2TK(-), with a defect in thymidine kinase (TK) function; and strain 6C2TK(-), gI(-)/gE(-), with defects in TK and glycoproteins I and E) and of the wild-type parent strain (86/27V). At various times after intranasal inoculation, pigs were killed and samples of tonsil, lung and different levels of the trigeminal and olfactory nervous pathways were examined by methods that included viral isolation, polymerase chain reaction assay and immunohistochemistry. Both mutant viruses were of reduced virulence, as indicated by no more than moderate clinical signs and lesions, and only sporadic isolation of virus; moreover, unlike the wild-type parent strain, the mutant viruses were not reactivated from the latent state by corticosteroid treatment. In addition, migration of the mutant strains to the central nervous system (olfactory and trigeminal nervous pathways) was reduced as compared with that of the wild-type strain. Thus, mutations in the genes encoding the TK enzyme and the gI/gE complex were associated with reduced virulence, reduced replication in peripheral target tissues, and reduced migration to the olfactory and trigeminal pathways.     

Protection of chickens against very virulent infectious bursal disease virus (IBDV) and Marek's disease virus (MDV) with a recombinant MDV expressing IBDV VP2.

To develop a herpes virus vaccine that can induce immunity for an extended period, a recombinant Marek's disease (MD) virus (MDV) CVI-988 strain expressing infectious bursal disease virus (IBDV) host-protective antigen VP2 at the US2 site (rMDV) was developed under the control of an SV40 early promoter. Chickens vaccinated with the rMDV showed no clinical signs and no mortality and 55% of the chickens were considered protected histopathologically after challenge with very virulent IBDV (vvIBDV), whereas all of the chickens vaccinated with the conventional IBDV vaccine showed no clinical signs and were protected. Chickens vaccinated with the CVI-988 or chickens in the challenge control showed severe clinical signs and high mortality (70-75%) and none of them were protected. Also, the rMDV conferred full protection to chickens against vvMDV just as the CVI-988 strain did, whereas 90% of the challenge control chickens died of MD. Antibody levels against IBDV and MDV following the vaccination increased continuously for at least 10 weeks. No histopathological lesions in the rMDV-vaccinated chickens and no contact transmission of the rMDV to their penmates were confirmed. These results demonstrate that an effective and safe recombinant herpesvirus-based IBD vaccine could be constructed by expressing the VP2 antigen at the US2 site of the CVI-988 vaccine strain.     

Construction of Oka varicella vaccine expressing human immunodeficiency virus env antigen

Oka varicella vaccine has been used to confer active immunity to varicella-zoster virus (VZV) in healthy and immunocompromised hosts. Based on its attenuated nature, Oka varicella vaccine expressing human immunodeficiency virus (HIV) env antigen was constructed by inserting the HIVenv gene into the viral genome and its immunogenicity was assessed in guinea pigs. The HIVenv gene encoding 296-463 amino acids was inserted between the sequences of the hepatitis B surface antigen and the thymidine kinase gene of the cloned plasmid and the recombinant virus was isolated by cotransfection of the chimeric plasmid with viral DNA. Insertion of the HIVenv gene into the viral genome was confirmed by PCR and sequencing of the viral genome of the recombinant virus. The recombinant virus expressed 30k HIVenv fusion protein in its infected cells. In guinea pigs, immunization with the recombinant virus induced an antibody response to both the HIV antigen and the V3 peptide of gp120 as well as VZV gE:gI. Cell-mediated immunity to the HIV antigen and gE:gI was assessed by the cutaneous reaction representing delayed type hypersensitivity. Immunized guinea pigs responded well to both the HIV antigen and gE:gI. Thus the recombinant Oka varicella vaccine expressing the HIVenv antigen induced both a humoral and cell-mediated immunity to the HIV antigen similar to VZV as Oka varicella vaccine induces humoral and cell-mediated immunity to VZV in the vaccinees. This recombinant Oka varicella vaccine expressing the HIVenv antigen may be evaluated for its immunogenicity as one of the AIDS vaccine candidates.     

Biosynthesis and interaction of glycoproteins E and I of canine herpesvirus.

In cells infected with canine herpesvirus (CHV), the mature form of glycoprotein E (gE) had a molecular weight of 94 kDa, and that of glycoprotein I (gI) had a broad range of molecular weights of 55-62 kDa. gE and gI formed a complex like gE and gI of other alphaherpesviruses. When cells were infected with the gI minus mutant of CHV (gI/Z), the mature form of the 94 kDa gE was not formed, but a 76 kDa gE polypeptide was found. Similarly, no mature gI was formed in cells infected with the gE minus mutant of CHV (gE/Z), but a 40 kDa gI polypeptide was formed. When cells were coinfected with gE/Z and gI/Z, the molecular masses of gE and gI were increased from 76 to 94 kDa and from 40 to 55-62 kDa, respectively. We constructed vaccinia virus recombinants which expressed CHV gE or CHV gI. Only when cells were coinfected with both the vaccinia recombinant which expressed gE and the vaccinia recombinant which expressed gI, gE and gI were processed into their mature forms. Our results suggest that the presence of both gE and gI is necessary for efficient processing of the precursors of gE and gI to their mature forms.     

Pathogenicity and vaccine efficacy of a thymidine kinase-deficient mutant of feline herpesvirus type 1 in cats

Summary We constructed a recombinant feline herpesvirus type 1 (FHV-1) which was deleted in a defined region (450 bp) within the thymidine kinase (TK) gene (C7301dlTK) [Yokoyama et al. (1995) J Vet Med Sci 57: 709–714]. In this report, we carried out two experiments to assess the pathogenicity and vaccine efficacy of the recombinant C7301dlTK in cats. The first experiment showed that, following multiple inoculation of the recombinant C7301dlTK by intraocular, intranasal and oral routes, the virus was sufficiently attenuated in cats, although a high titer of the virus was recovered from target organs (eye, nose, and mouth). In the second experiment, two intramuscular vaccinations with the recombinant C7301dlTK protected cats to a significant degree against subsequent challenge with the parent FHV-1 strain C7301 at 4 weeks after the last vaccination. These results demonstrate that the recombinant C7301dlTK is effective as a live attenuated vaccine with a clear genetic marker.     

A glycoprotein I- and glycoprotein E-deficient mutant of infectious laryngotracheitis virus exhibits impaired cell-to-cell spread in cultured cells

Summary. In alphaherpesviruses, glycoprotein I (gI) and glycoprotein E (gE) form a heterodimer that functions in cell-to-cell spread of the virus. Generally, alphaherpesvirus mutants that lack these glycoproteins are replication competent in cell culture but show a reduced capacity for cell-to-cell spread and hence smaller plaque sizes. Infectious laryngotracheitis virus (ILTV), or Gallid herpesvirus 1, is an alphaherpesvirus that causes respiratory disease in chickens. The roles of gI and gE in ILTV have not been investigated previously. In this study, a glycoprotein I and glycoprotein E deletion mutant of ILTV (gI/gE-ve ILTV) was generated by replacing the region of the ILTV genome coding for the adjacent gI and gE genes with the gene for enhanced green fluorescent protein (eGFP). This gI/E-ve ILTV was readily propagated in cell culture in the presence of wildtype ILTV (wt ILTV). However, in the absence of wt ILTV the propagation of gI/gE-ve ILTV was severely impaired. Infection of permissive cell cultures with gI/gE-ve ILTV failed to produce plaques but single infected cells could be identified by fluorescence microscopy. This suggests that gI/gE has a more significant role in the cell-to-cell spread of ILTV in vitro than in many other alphaherpesviruses.     

In vitro recombination of feline herpesvirus type 1

Summary.  We investigated the possibility of in vitro recombination of three different trains of feline herpesvirus type 1 (FHV-1), a virulent strain (C7805), a vaccine strain (F2), and a candidate vaccine strain with a deletion in the thymidine kinase gene (C7301dlTK). The results showed that recombination within different genotypes of FHV-1 strains could occur at a relatively high frequency ranging from 10.1% to 21.5% in vitro. These results indicate the generation of multiple mutant viruses by in vitro recombination experiments. The possibility for generating a virulent virus or novel type of FHV-1 by recombination in vivo is discussed. Accepted September 12, 1997 Received July 17, 1997     

Herpes simplex virus IgG Fc receptors induced using recombinant adenovirus vectors expressing glycoproteins E and I

Evidence has been presented that herpes simplex virus (HSV) immunoglobulin (IgG) Fc receptors are composed of a complex of two glycoproteins, gE and gI. In previous studies, cells infected with HSV-1 mutants lacking either gE or gI bound lower levels of soluble IgG than cells infected with wild-type viruses suggesting that both gE and gI were required for IgG binding. We have reevaluated the Fc receptor activity of these mutants using a more sensitive assay involving IgG-coated erythrocytes and have found that cells infected with a gE- mutant HSV-1 did not bind IgG-coated erythrocytes whereas cells infected with a gI- mutant retained some Fc binding activity. To further study HSV-induced Fc receptors recombinant adenovirus vectors expressing gE or gI were constructed. Cells expressing gE alone bound both soluble IgG and IgG-coated red cells, although the binding was consistently lower than that observed with HSV-infected cells or cells expressing both gE and gI. Cells expressing only gI were unable to bind either soluble IgG or IgG-coated erythrocytes. These results support the conclusion that both gE and gI are required for full Fc receptor activity, although gE alone can bind IgG to a lesser extent.     

Evaluation of a recombinant vaccinia virus containing pseudorabies (PR) virus glycoprotein genes gp50, gII,and gIII as a PR vaccine for pigs

Summary Pigs vaccinated twice intramuscularly with a highly attenuated strain of vaccinia virus (NYVAC) containing gene inserts for pseudorabies virus (PRV) glycoproteins gp50, gII, and gIII produced neutralizing antibodies for PRV and were less clinically affected than were nonvaccinated pigs following oronasal exposure to virulent PRV. Also, following oronasal exposure to virulent PRV the duration of virulent virus shedding by pigs that had been vaccinated intramuscularly with the recombinant virus was statistically less (p<0.05) than that of nonvaccinated pigs and like that of pigs vaccinated twice intramuscularly with inactivated PR vaccine. Intramuscular vaccination with the recombinant virus was compatible with the most commonly used differential diagnostic tests, namely those based on PRV glycoproteins gX and gI. Serum antibodies for these glycoproteins were absent from the sera of all pigs before and after vaccination with recombinant virus; whereas, they were present in the sera of all of the same pigs after they were exposed to virulent PRV. In contrast to the effectiveness of the recombinant virus administered intramuscularly, neither serum antibody nor clinical protection against PRV was detected when aliquots of the same recombinant virus preparation were administered either orally or intranasally. The latter finding suggests that recombinant virus replicates poorly, if at all, at these sites. If so, the dissemination of recombinant virus from vaccinated pigs to nonvaccinated pigs or other animals in contact seems unlikely.     

Induction of cellular immunity to varicella-zoster virus glycoproteins tested with pernasal coadministration of Escherichia coli enterotoxin in mice

A mutant of Escherichia coli enterotoxin promotes the induction of cellular immunity to a live varicella vaccine (the Oka strain) as a mucosal adjuvant in mice. An investigation was carried out to determine which of the purified glycoproteins of the virus among three induced cellular immunity with a single nasal administration. Spleen cells from mice immunized nasally with the vaccine and toxin produced interleukin-2 (IL-2) at the same level on restimulation in vitro with glycoprotein H: glycoprotein L (gH:gL), gB, and gE:gI, but not IL-4. The spleen cells from mice immunized with gH:gL, gB, or gE:gI and toxin produced IL-2 on restimulation with gH:gL, gB, or gE:gI, respectively, and the vaccine, but not IL-4. Immunization with gH:gL and the toxin showed increased thymidine uptake and production of IL-2 and interferon-gamma (IFN-gamma) of the spleen cells, but not IL-4, depending on the dose of gH:gL used for immunization and restimulation in vitro. Purified gE:gI and gB have been reported to be the strongest stimulators of cellular immunity to varicella upon subcutaneous injection and are useful as a subunit vaccine. All the glycoproteins tested are excellent stimulators of cellular immunity to the virus and itself on nasal co-immunization with the toxin.     

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus

Summary.  We constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301ddlTK- gag was confirmed to express the FIV Gag precursor protein in the cells. Accepted November 12, 1997 Received August 26, 1997     

The requirement of varicella zoster virus glycoprotein E (gE) for viral replication and effects of glycoprotein I on gE in melanoma cells

The glycoprotein E (gE) of varicella zoster virus (VZV), encoded by ORF68, is the most abundant viral glycoprotein. In the current experiments, we demonstrated that ORF68 deletion was incompatible with recovery of infectious virus from VZV cosmids. Replacing ORF68 at a nonnative AvrII site in the genome restored infectivity. Further, we found that VZV gE could be expressed under the control of the Tet-On promoter in stably transfected melanoma cell lines (Met-gE cells) without evidence of toxicity. In these Met-gE cells, gE colocalized with gamma-adaptin, a trans Golgi network marker, in perinuclear sites, but did not reach plasma membranes. In order to investigate how infection altered gE localization, we made a recombinant virus, vOka-MSPgE, with ORF68 from the VZV MSP strain. VZV MSP encodes a mutant gE protein (D150N) that lacks the mAb epitope, 3B3 (Santos et al., Virology 275, 306-317, 2000), whereas Met-gE protein binds mAb 3B3. Within 48 h after Met-gE cells were infected with vOka-MSPgE, the steady-state distribution of Met-gE protein extended beyond the perinuclear areas to other cytoplasmic sites and to plasma membranes. A second recombinant, vOka-MSPgE without gI (vOka-MSPgEdeltagI), was constructed to investigate Met-gE protein distribution in the absence of gI. The redistribution of Met-gE protein which was observed by 48 h after vOka-MSPgE infection did not occur until 5 days (140 h) within vOka-MSPgEdeltagI infected cells. After vOka-MSPgE infection of Met-gE cells, most Met-gE protein was in the final 94K mature form by 72 h. However, progression to predominance of mature gE was delayed in Met-gE cells infected with vOka-MSPgEdeltagI. These observations confirm our hypothesis that VZV gE is essential, based upon the demonstration of restored infectivity after replacing ORF68 in a nonnative site in the genome, and provide further evidence of the role of gI in facilitating the maturation and intracellular distribution of this critical VZV glycoprotein.     

Varicella-Zoster Virus Glycoproteins E and I Expressed in Insect Cells Form a Heterodimer That Requires the N-Terminal Domain of Glycoprotein I

Varicella-zoster virus (VZV) glycoproteins E and I (gE and gI), which are major components of the virion envelope, form a noncovalently linked complex. To understand their properties and functions, we expressed and purified soluble forms of gE and gI in the baculovirus system. Extracellular domains of gE and gI were cloned into baculoviruses, using either native or insect-derived signal peptides. Each recombinant virus yielded soluble protein in culture medium although a higher level of secretion was achieved with insect-derived signal peptides in recombinant gE baculoviruses. A soluble gE–gI complex was formed by co-infecting insect cells with recombinant gE and gI baculoviruses and detected by immunoprecipitation followed by Western blotting analyses. By gel filtration and cross-linking studies, we showed that the VZV gE–gI complex expressed in insect cells is a heterodimer. Interestingly, two recombinant gI proteins in which signal peptides were replaced with insect-derived signal peptides did not associate with gE. Amino-terminal sequencing and site-specific mutational studies showed that the replacement of only the signal peptides did not prevent complex formation but alterations in the processed amino-terminus of gI abrogated its ability to complex with gE. These findings indicate that the mature amino-terminus of gI is required for gE–gI complex formation by the external domains of VZV gE and gI.     

Assessment of vaccination of pigeons against paramyxovirus type 1 infection with inactivated aqueous-suspension or oil-emulsion vaccines

The immune response and resistance to PMV1 challenge of pigeons vaccinated with inactivated oil-emulsion (OE) or aqueous-suspension (AS) vaccines were compared. ASa was based on NDV LaSota strain, ASb on NDV Terumo strain, OEa on NDV Poletti strain and OEb on NDV Ulster 2C strain. Groups were each vaccinated subcutaneously with one of ASa (0.2ml), ASb (0.2ml), OEa (0.5ml), OEa (0.2ml) and OEb (0.5ml). An unvacci-nated group was kept as control. The immune response measured by HI tests was the highest with the single 0.2ml dose of ASa vaccine. The single 0.2ml dose of OEa vaccine produced a poor HI response. Challenge with 'pigeon' PMV1 (intramuscular + intranasal/ocular) one month after vaccination resulted in the vast majority of unvaccinated pigeons excreting virus in the laryngeal secretions and the faeces and all . birds died. In comparison, virus shedding and morbidity-mortality were significantly reduced in the five challenged vaccinated groups. However, morbidity-mortality was higher in the OE groups than in the AS groups. No ASa vaccinated pigeon developed clinical signs and only one ASb vaccinated pigeon presented nervous signs. In contrast, morbidity-mortality reached 30 and 35% in the OE vaccinated groups. Only the inactivated aqueous-suspension vaccines, especially that prepared from NDV LaSota strain, gave, after one dose injection (0.2ml), high resistance to a severe challenge with 'pigeon' PMV1.     

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.