Experimental reactivation of bovine herpesvirus 1 (BHV-1) by means of corticosteroids in an intranasal rabbit model

Summary Intranasal inoculation of the rabbit was shown to be a viable alternative to eye inoculation as a model to study latency and reactivation of bovine herpesvirus 1 (BHV-1). In four different experiments, separate groups of rabbits were intranasally inoculated with BHV-1. In two experiments some rabbits were inoculated instead with a TK-defective (TK^–) mutant strain of BHV-1. The development of a specific antibody response was monitored by both virus neutralization and ELISA assays. Cell-mediated immunity was measured by means of a skin test. Many weeks after virus inoculation the rabbits were treated with corticosteroid. Antibody formation after treatment was markedly different in wild type and in TK^– virus inoculated groups. In the former, virus reactivation was suggested by a sudden rise in serum antibody levels with kinetics closely resembling those reported in infected calves following corticosteroid administration, whereas in the case of the TK^– group no significant increase in antibody activity was measured. Histopathological changes in trigeminal ganglia also indicated reactivation of virus in the wild-type virus infected animals. Further evidence for reactivation was obtained by virus isolation from nasal swabs after corticosteroid treatment.     

Changes in the bovine herpesvirus 1 genome during acute infection,after reactivation from latency,and after superinfection in the host animal

Summary Three subtypes, as defined byHindIII restriction endonuclease (RE) analysis patterns [17], of bovine herpesvirus 1 (BHV 1) were used to inoculate seronegative, BHV 1-free cattle. These included: infectious bovine rhinotracheitis virus (IBRV), subtype 1.1; infectious pustular vulvovaginitis virus (IPPV) isolate K22, subtype 1.2b; and IPVV isolate FI, subtype 1.2a. Nasal, vaginal, and buffy coat samples were taken for virus isolation from each animal. RE analysis was done on virus isolates collected during acute infection, after reactivation from latency, and after reactivation followed by superinfection with a subtype of BHV 1 that differed from the primary inoculation virus. Changes occurred in the BHV1 genome after only 1 passage in the host animal, and varied from tissue to tissue within the same animal. Viruses reactivated from latency also displayed genome variability. Only animals that received IPVV as the primary inoculation virus were successfully superinfected. After superinfection, cattle shed both superinfecting and reactivated viruses, and genome variability was observed. These data suggest that the application of RE analysis in diagnostic and epidemiologic studies of BHV 1 is limited to analysis between types and subtypes, and is not applicable for the examination of isolates from within a BHV 1 subtype.     

Two different strains of an alphaherpesvirus can establish latency in the same tissue of the host animal: evidence from bovine herpesvirus 1

Summary We inoculated plaque-purified bovine herpesvirus type 1 (BHV 1), strain K22, subtype BHV 1.2b, intravenously into susceptible cattle. Five months later, we reactivated latent virus with dexamethasone and then super-infected the same cattle intranasally and intravaginally with a different plaquepurified BHV1, strain Cooper, subtype BHV 1.1. After a second dexamethasone treatment four months later, reactivated viruses were isolated and examined with restriction endonucleases. We showed that both virus subtypes were reactivated, proving that 2 different strains of an alphaherpesvirus can establish latency in the same tissue in the host animal.     

A thymidine kinase deficient HSV-2 strain causes acute keratitis and establishes trigeminal ganglionic latency,but poorly reactivates in vivo

The incidence of herpetic keratitis following in-tranasal or direct ocular infection with thymidine kinase-negative (TK^?) strains of herpes simplex virus (HSV)-2 has not been well studied, and the role of the TK gene in the establishment of latency and virus reactivation is controversial. To determine whether a TK^? strain of HSV-2 could establish trigeminal ganglionic latency and be reactivated in vivo to produce recurrent keratitis or nervous system infection, an animal model of acute and recurrent infection was utilized. Rabbits were infected by the intranasal or ocular routes, and latency was reactivated by immuno-suppression. Virus shedding in nasal and ocular secretions was monitored, and the eyes were examined for the presence of corneal epithelial lesions during acute and reactivated infections. Central nervous system (CNS) and trigeminal ganglionic tissues were assayed by histologic, virologic, and in situ hybridization techniques. All rabbits intranasally infected shed virus in both ocular and nasal secretions, whereas only 30% of rabbits infected in the eyes shed virus in nasal secretions. Virus was recovered from co-cultivation cultures, but not from cell-free ho-mogenates, of trigeminal ganglionic and CNS tissues from animals inoculated by both routes. The incidence of keratitis was much greater after direct ocular inoculation, although both routes of inoculation produced CNS and ganglionic inflammatory lesions. Keratitis healed in 92% of the animals infected by the ocular route by 26 days post infection. Of rabbits initially infected in the eyes and then subjected to drug-induced reactivation, only 30% shed virus, which was limited to a 24 hour period; there was no reappearance of epithelial keratitis, no animal became blind, and none died. In contrast, latently infected control rabbits uniformly reactivated. These studies show that this TK^? HSV-2 strain (i) replicates in the eye, (ii) is neuroinvasive but non-neurovirulent following intranasal and direct ocular infection; (iii) sheds in the eye more frequently and for longer periods after ocular than after intranasal inoculation; (iv) induces epithelial keratitis that usually heals spontaneously; (v) establishes latency in trigeminal ganglionic neurons, but no other ganglionic cells; and, (vi) reactivates in a small proportion of animals, but does not produce recurrent ocular lesions following drug-induced immunosuppres-sion. Thus, the TK gene appears directly involved in HSV latency and reactivation in vivo. © 1994 Wiley-Liss, Inc.     

Latency of a thymidine kinase-negative pseudorabies vaccine virus detected by the polymerase chain reaction

Summary Latent viral DNA was detected by the polymerase chain reaction in trigeminal ganglia of all of 10 pigs that were necropsied 81 or more days after they had been infected intranasally with a thymidine kinase-negative (TK^–) vaccine strain of pseudorabies virus (PRV). Failure to reactivate virus from any of the same pigs by earlier treatment with dexamethasone suggested that even though latency can be established with TK^– PRV, subsequent reactivation may be a relatively rare event.     

Pathogenesis of genital herpes simplex virus infection in mice

This study was undertaken to establish the role of virulence of various herpes simplex virus (HSV) strains in the course of infection when applying the virus to the non-injured mucous membranes of mice.Wild-type HSV-type 1 (HSV-1) strains with marked differences in their neurovirulence following intracerebral inoculation showed minor differences in virulence after vaginal inoculation, but essentially their neurovirulence in cerebral infection corresponded to their virulence on the mucous membranes.In comparison with the wild-types, however, there were pronounced differences among syn- and TK^–-mutants of HSV-1 and HSV-2 in the degree of virulence at different sites in the course of virus infection. Whereas syn-mutants proved avirulent on the mucous membranes but not in neural tissues, TK^–-mutants were avirulent both on mucous membranes and in neural tissues.Ts-mutants of HSV-2 were not found to establish themselves when administered to the non-injured mucous membranes, nor did they induce neutralizing antibodies, but a later challenge with the wild-type virus at the same site lead only to an attenuated course of infection.Supported by the Deutsche Forschungsgemeinschaft Schn 174/6-2     

Influence of double infections on the induction of thymidine kinase by UV-irradiated herpes simplex virus types 1 and 2 and pseudorabies virus

Summary Simultaneous infection of primary rabbit kidney cells with HSV type 1 TK⁺ and a TK^– strain results in a mutual influence of both viruses on the induction of thymidine kinase (TK). TK⁺ virus has an enhancing and TK^– virus a depressing effect on TK induction by a superinfecting TK⁺ virus. The enzyme induction depends on the ratio of multiplicities of both viruses. The mutual influence on TK induction depends further on the time of addition of the superinfecting virus: the effect of the second virus can still be observed when given 6 hours after primary infection. Identical phenomena can be observed using combinations with HSV type 2 or Pseudorabies viruses.The ability of HSV to induce TK is progressively inactivated with increasing the time of UV-irradiation. The depressing effect of a TK^– strain and the stimulating effect of a TK⁺ strain on superinfecting TK⁺ strains is UV-sensitive: after 6 minutes of UV-irradiation neither inhibition nor stimulation of TK induction by a superinfecting TK⁺ strain can be observed. Infection by long-term (20 minutes) UV-irradiated TK⁺ strains results in a depression of TK induction by a superinfecting TK⁺ virus. Long-term irradiation of the TK^– virus does not show this effect.Cytosine-arabinoside has no effect on the mutual influence of TK induction by TK⁺ and TK^– strains; the phenomenon of mutual depression therefore has to be considered an early process.With 5 Figures     

Detection of latent thymidine kinase-deficient herpes simplex virus in trigeminal ganglia of mice using the polymerase chain reaction

Summary Latency of thymidine kinase-negative mutants of herpes simplex virus (TK^– HSV) could not be detected by reactivating the virus from the ganglia of infected mice. Because Southern blot hybridization was not sensitive enough to detect viral DNA, positive results obtained by dot blot hybridization were ascertained by the highly specific and sensitive polymerase chain reaction (PCR), which detected both latent TK^– HSV type 1 and 2 DNA from the trigeminal ganglia of infected mice.     

Herpes simplex virus thymidine kinase expression in infection of the trigeminal ganglion.

Infection of the trigeminal ganglion was investigated using standard thymidine kinase-positive (TK⁺) herpes simplex virus (HSV) and two TK^? mutants. After corneal inoculation of mice with TK^? HSV, the incidence of acute and latent trigeminal ganglion infection was markedly decreased compared to TK⁺ virus. When cell-free virus was titered from mice 1 hr to 5 days post-corneal inoculation, ocular replication of TK^? HSV was found to be similar to TK⁺ HSV, but whereas TK⁺ HSV replicated well and was found in substantial amounts in trigeminal ganglia (2 × 10³ PFU/mg ganglion tissue), TK^? HSV did not replicate in the ganglia. Mean TK^? trigeminal ganglion virus titers were 10,000-fold less than TK⁺ titers. When a TK⁺ revertant of TK^? mutant virus was tested, however, trigeminal ganglion virus replication was similar to that with the parental TK⁺ virus. The results obtained were interpreted as being consistent with impaired axonal transport of TK^? HSV from cornea to trigeminal ganglion neurons or more likely, with impaired replication of TK^? HSV in ganglionic neurons.     

Stimulation of thymidine kinase activity in baculovirus infected cells is not due to a virus-coded enzyme

Summary A polyhedrin-positive recombinant autographa californica nuclear polyhedrosis virus (AcNPV) carrying a herpes simplex virus thymidine kinase gene under the control of theSyn XIV promoter, a fusion of synthetic and linker-modified polyhedrin promoters, has been constructed. When this recombinant baculovirus was used to infect a variant ofSpodoptera frugiperda cells deficient in thymidine kinase (TK^–), a high level of TK activity was detected. These results, in conjunction with the demonstration that AcNPV could replicate in TK^– S. frugiperda cells and no TK activity was found throughout infection, imply that the wild type virus-stimulated TK activity observed inS. frugiperda (TK⁺) cells is not contributed by a virus-coded enzyme.     

Herpes simplex virus thymidine kinase expression in trigeminal ganglion infection: correlation of enzyme activity with ganglion virus titer and evidence of in vivo complementation

Experimental trigeminal ganglion and corneal infection in mice was studied with three thymidine kinase-positive (TK⁺) strains of herpes simplex virus type 1 (HSV-1) and eight TK^? HSV-1 mutants. Viruses were extensively tested in cell culture to determine whether any were temperature sensitive (ts) for virus replication or for TK activity. TK^? mutants were no more ts than were TK⁺ viruses. By arabinosylthymine testing and measurement of thymidine phosphorylation, it was apparent that some TK^? mutants were, in fact, intermediate for TK activity (TK^±). After corneal inoculation of individual viruses it was observed with one exception that TK^?, TK^±, and TK⁺ viruses replicated in ocular tissues (3 days postinoculation, 2.2 × 10³–1.1 × 10⁵ PFU/eye). However, TK^? mutants were rarely isolated from trigeminal ganglia (<2.5–<5.6 × 10^?1 PFU/mg), whereas TK⁺ and to a lesser degree TK^± viruses were frequently (1.2 × 10³–1.2 × 10⁴ PFU/mg and 3.2 × 10⁰–2.1 × 10³ PFU/mg). In vivo complementation studies were performed by corneal inoculation of TK^??TK^? and TK^??TK^? virus mixtures. TK⁺ HSV complemented TK^? virus since significant amounts of TK^? HSV were isolated from trigeminal ganglia (complementation indices were >60–>1600). In addition, after inoculation of certain TK^?–TK^? pairs, complementation in ganglia was observed (complementation indices were >2.4–>120). These studies support the hypothesis that HSV-1 TK expression is necessary for sensory ganglion (neuron) infection in three ways: HSV-1 TK mutants that replicated in ocular tissues and were not ts mutants did not replicate in vivo in trigeminal ganglia; (ii) there was a correlation between level of viral TK activity and trigeminal ganglion virus titer; and (iii) when complemented by TK⁺ or TK^? HSV, TK^? virus replicated in trigeminal ganglia.     

Evaluation of Newly Developed Immunoperoxidase Monolayer Assays for Detection of Antibodies against Bovine Herpesvirus 4

This study describes the evaluation of immunoperoxidase monolayer assays (IPMAs) for detection of antibodies against bovine herpesvirus 4 (BHV4) DN-599 or BHV4 LVR 140 in sera of cattle. We compared the quality of these IPMAs with the quality of a BHV4 indirect enzyme-linked immunosorbent assay (ELISA). In addition, a preliminary serological survey of BHV4 antibodies was carried out to estimate the seroprevalence of BHV4 in Dutch cattle at different ages. The specificities of both BHV4 IPMAs were 1.00. The geometrical mean titers (detection limit) of the BHV4 IPMAs were twice as high as that of the BHV4 indirect ELISA. In experimentally infected cattle, BHV4 antibodies were detectable by IPMAs 16 to 18 days postinfection, which was almost 2 weeks earlier than in the indirect ELISA. The reproducibility of the BHV4 DN-599 IPMA (_κD value, 0.92) and of the BHV4 LVR 140 IPMA (_κD value, 0.87) were good. For field sera the overall agreement between the BHV4 indirect ELISA and the two BHV4 IPMAs, DN-599 and LVR 140, was 95 and 96%, respectively. The serological-survey study showed that the estimated seroprevalence of BHV4 in Dutch cattle was 16 to 18% and that the percentage of BHV4-positive animals varied by age category (between 6 and 43%). In summary, the two BHV4 IPMA formats have several advantages that make IPMA a useful alternative to the BHV4 indirect ELISA for detecting BHV4 antibodies in cattle.     

Analysis of the TK enzyme complex induced by HSV types 1 and 2 by means of isoelectric focusing and polyacrylamide gel electrophoresis

Summary Recently we have described that the Herpes simplex virus(HSV)-induced thymidine kinase (TK) induces AMP- and ADP-dThd-5-phosphotransferase activities. We now demonstrate the heterogeneity of the described activities in isoelectric focusing experiments and polyacrylamide gel electrophoresis. A TK^–-mutant of HSV type 1 fails to induce these activities. The activities of the type 1 enzyme complex was neutralized by an anti-HSV-serum. The TK-enzyme complex expressed in LTK^–-cells transformed to a TK⁺-phenotype by sheared HSV-1 DNA was compared with the wild type TK complex in isoelectric focusing experiments. Additionally we demonstrate that the HSV type 1 enzyme complex has thymidylate kinase activity, while the type 2 TK complex did not exhibit thymidylate kinase activity.Feedback regulation mechanisms by dTMP, dTDP and dTTP were investigated using partially purified enzyme preparations of HSV types 1 and 2 infected TK^–-cells.With 7 FiguresIn part presented at the meeting of the Section Virologie of the Deutsche Gesellschaft für Hygiene und Mikrobiologie, Oktober 1981, Göttingen.     

Interferon production and virus replication in lymphoblastoid cells infected with different viruses

Summary A semicontinuous infection system was used to test viral replication and interferon induction in lymphoblastoid cells: measles virus, Newcastle disease virus (NDV), Sendai virus, human parainfluenza virus (type II and III), Semliki forest virus (SFV) and Vesicular stomatitis virus (VSV). With the exception of Sendai virus, all viruses replicated in the Namalva cell line. Only measles virus was able to induce high levels of interferon. Three other cell lines, NC37, Raji (TK⁺-variant) and Raji (TK^–-variant) were tested using measles virus as inducer. The interferon yields from these cells were inferior to those obtained from Namalva cells.     

Thymidine kinase-negative bovine herpesvirus type 1 mutant is stable and highly attenuated in calves

Summary Calves were vaccinated intranasally (IN) or intravenously (IV) with a thymidine kinase-negative (tk^–) BHV-1 mutant. Vaccinated calves developed neutralizing antibodies but did not show clinical signs of infectious bovine rhinotracheitis (IBR). Vaccination also prevented clinical signs of IBR disease following IN challenge exposure of the calves to parental Los Angeles (LA) and USDA Cooper strains of tk⁺ BHV-1. Nasal swabs were collected for 10 days after the vaccination and the challenge exposures to monitor BHV-1 multiplication. At both 91 and 121 days post vaccination (PV), calves were also stressed for 5 days with dexamethasone (DEX) to induce reactivation of BHV-1 and nasal swabs were obtained. tk^– BHV-1 multiplied in the nasal mucosa of IN vaccinated calves and was also recovered after DEX treatment. Likewise, tk^– BHV-1 was isolated from the buffy coat fraction of IV vaccinated calves, but not from nasal swabs. tk^– BHV-1 vaccination reduced the multiplication of tk⁺ BHV-1 in the nasal mucosa, but did not completely prevent development of a persistent infection by the challenge virus. The phenotypes of viruses isolated from the nasal swabs and buffy coats were analyzed by enzyme assays of extracts from virus-infected cells and by plaque autoradiography. These assays showed that tk^– BHV-1 can persist for at least 3 months in vaccinated calves and may also be transmitted from vaccinated to control calves without revertingin vivo to tk⁺. The results demonstrate that the tk^– BHV mutant is attenuated and efficacious as a vaccine.With 1 Figure     

Correlation of virus replication,cytokine (TNF-α and IL-1) producing cells,neuronal necrosis and inflammation after intranasal infection of mice with herpes simplex virus strains of different virulence

Summary The number of TNF- and IL-1 producing cells was investigated during the acute replication phase of herpes simplex virus (HSV) in trigeminal ganglia after intranasal infection with strains of different virulence. The highly virulent strain WAL replicated strongly and induced many cytokine producing cells early in the ganglia. The low virulent strain HFEM replicated less, only few cytokine producing cells were detected late. The thymidine-kinase negative (TK^–) virus 1301 did not replicate but produced some lymphocytic inflammation. The higher the virulence of strains of HSV-1 or -2 was, the stronger was the extent of histopathological lesions; moreover, a dissociation in time between replication and cellular reaction (granulocytic and lymphocytic) could be observed after infection with strains HFEM and TK^– virus 1301. CD4 and CD8 positive cells could be detected mainly at the rim of necrotic areas, TNF- and IL-1 producing cells, however, were scattered throughout the ganglia.     

Complementation of genetic disease: A velocity sedimentation procedure for the enrichment of heterokaryons

Methodology is described to enrich for heterokaryons after mammalian cell fusion. A heterogeneous cell mixture can be separated on a Sta-Put apparatus into fractions of uniform size cells by sedimentation through a 1% bovine serum albumin-5% Ficoll gradient. Unfused RAG and LM/TK^– cells, differing by 10% in diameter, have been sorted by size; following fusion, larger and faster sedimenting cells were shown to be hybrids. This methodology can be utilized in genetic complementation studies of human genetic diseases where selection procedures for proliferating hybrids do not exist. When fibroblasts from individuals with Tay-Sachs disease [deficient in hexosaminidase A (HEX A^–)] and Sandhoff-Jatzkewitz disease (HEX A^– and HEX B^–) are fused, HEX A is generated, demonstrating complementation of two different mutations. After Sta-Put fractionation, the HEX A complementation product was associated with the faster sedimenting multinuclear cells and not with the mononuclear parental cells. This methodology will facilitate detection of genetic differences in fibroblasts from related inherited disorders.     

Minimal infective dose of rotavirus

Summary We studied the minimal infective dose of the gastroenteritis virus, rotavirus. Increasingly lower doses [10⁴, 10³, 10¹, 1, 10^–2 plaque forming units (PFU)] of the OSU strain of porcine rotavirus were administered to highly susceptible (colostrum deprived, cesarean derived) newborn miniature swine piglets.In vitro studies showed that virus infectivity was inactivated in piglet gastric juice, both by low pH and by pH- and concentration-dependent factor(s). These factors remain unidentified, but to prevent intragastric viral inactivation, sodium bicarbonate was administered prior to oral virus inoculation of piglets with virulent (non-tissue culture passaged) virus. The lowest dose of virus to induce clinical illness or to demonstrate viral replication by recovery of significantly more infectious virus than was administered, or both, was 1 PFU. These results should help establish standards for virus contamination of water and recommendations for evaluating disinfection procedures for rotaviruses.     

Construction and immune response characterization of a recombinant pseudorabies virus co-expressing capsid precursor protein (P1) and a multiepitope peptide of foot-and-mouth disease virus in swine

Foot-and-mouth disease (FMD) is the most contagious and devastating disease of livestock. Our previous studies demonstrated that TK⁻/gG⁻/P1⁺, a recombinant expressing the FMDV capsid precursor protein (P1) based on attenuated pseudorabies virus (PRV) TK⁻/gG⁻, could be used as a recombinant vaccine to protect pigs against both pseudorabies and FMD. However, because the P1 expression cassette is inserted into the gG locus of the genome of PRV TK⁻/gG⁻, this bivalent vaccine cannot be used in conjunction with the PRV gE-ELISA, an extensively used discriminatory serological test in eradication programs for pseudorabies, which limits the clinical use of this bivalent vaccine. To circumvent this shortcoming, in this study, an expression cassette containing synthetic multiepitope gene “FHG” consisting of six potential B cell epitopes and two potential T cell epitopes of FMDV, under the control of CMV promoter, was further inserted into the gE/gI locus of genome of TK⁻/gG⁻/P1⁺, resulting in a new recombinant FHG/P1/PRV. The immunogenicity of FHG/P1/PRV was evaluated and compared with TK⁻/gG⁻/P1⁺ in piglets. Our results clearly showed that FHG/P1/PRV performed better than or comparable with TK^-/gG^-/P1⁺, as demonstrated by comparable PRV-specific neutralizing antibodies, enhanced FMDV-specific neutralizing antibodies, and cellular immune responses. More importantly, no gE- and gG-specific antibodies could be detected in pigs immunized with FHG/P1/PRV. These data indicate that FHG/P1/PRV is a promising bivalent vaccine candidate with more extensive potential application than TK⁻/gG⁻/P1⁺ against both pseudorabies and FMD.     

Protection of rabbits against HTLV-II infection with a synthetic peptide corresponding to HTLV-II neutralization region

Summary Rabbit immune sera raised against synthetic peptides of the HTLV-II envelope gp46 region were examined for HTLV-II neutralization ability by HTLV-vesicular stomatitis virus (VSV) pseudotype assay and syncytium inhibition assay. HTLV-II neutralization activity was detected in the sera against HTLV-II Env gp46, 80–103 but not in those to HTLV-II Env gp46, 171–196. Three rabbits immunized with the synthetic peptide of HTLV-II Env gp46, 80–103 and three non-immunized rabbits were challenged with intravenous inoculation of an HTLV-II-producing human cell line (MOT, 1×10⁷ cells). The non-immunized rabbits showed seroconversion for HTLV-II after 2 weeks and maintained persistent infection but the immunized rabbits were protected from HTLV-II infection. Nested or repeated polymerase chain reaction revealed the presence of HTLV-II provirus sequences in the non-immunized rabbits but not in the immunized rabbits. These results suggest that peptide vaccination with a synthetic peptide corresponding to the HTLV-II neutralization region is useful for preventing HTLV-II infection.