Comparative studies on activation of latent herpes simplex virus in rabbits

Latent infection was established in 113 rabbits by inoculation of herpes simplex virus type 1 (HSV-1) strain Kupka into the right scarified cornea. In animals succumbed from 88-387 days post-infection (p.i.), HSV-1 was detected in the cultured right cornea (RC) fragments of 2 out 45 rabbits (4.4%), while the cultured right trigeminal ganglion (RTG) fragments yielded virus in 60 out 70 animals (85.7%). Spontaneous virus shedding occurred nearly in the half (35) of 74 animals followed for a mean of 211 days; providing that swabbing of both eyes was made 1-2 times weekly, the positive isolation rate was 1.3% (73 out total 5648 samples). The following treatments were used for provocation: cyclophosphamide (CPA) alone, mechanical trauma to cornea in association with local administration of either xylene or adrenalin, and a combination of both CPA administration and corneal irritation. In the course of these treatments continued for 4-8 days, virus was reisolated in 9 out 59 rabbits, the average positive isolation rate from RC being 6.9% (29 out 420 samples). HSV-specific antigens were searched for in about 56 000 semiserial sections prepared from noncultured as well as cultured samples of RC, both Gasserian ganglia and brainstem coming from 87 rabbits with established latency, of which 42 underwent provocation. By direct fluorescent antibody (FA) staining single neurons positive for structural HSV antigens were found in 14% of noncultured RTG samples from untreated rabbits as compared to 37% of such samples from animals subjected to provocation. In the course of above treatments, explanted RC fragments were positive in 17 out of 42 animals (40.4%) indicating a 9-fold increase in comparison to non-stimulated rabbits. The results confirm the absence of any virus, either infectious or covert, at the inoculation site in 95.6% of rabbits autopsied later than 12 weeks p.i. They also imply that a reversed transport of HSV occurred upon either spontaneous of artificial virus activations.     

Evidence for control of herpes simplex virus mutagenesis by the viral DNA polymerase

We have measured the effect of mutations associated with the DNA polymerase of herpes simplex virus type 1 on the production of spontaneous mutants resistant to bromodeoxyuridine. Bromodeoxyuridine-resistant mutants contain mutations in the viral thymidine kinase gene. The conditional lethal mutation in strain C7, which prevents growth at 39° and is located in the DNA polymerase gene, reduced slightly the yields of bromodeoxyuridine-resistant progeny compared to the parent virus. In contrast, a temperature-resistant derivative of C7, ts⁺C, produced levels of resistant progeny 11-fold greater than normal. Evidence is presented that the ts⁺CO derivative is not a true revertant of C7, but instead contains a second site mutation which suppresses the C7 temperature-sensitive mutation. This suppressor mutation appears closely linked to the original C7 mutation. Possible mechanisms for the elevated production of bromodeoxyuridine-resistant progeny by ts⁺C are discussed.     

Enhanced in vitro reactivation of latent herpes simplex virus from neural and peripheral tissues with hexamethylenebisacetamide

Summary We evaluated the effect of the demethylating agent hexamethylenebisacetamide on reactivation of latent herpes simplex virus type 2 (HSV-2) from guinea pig neural and extraneural tissues. Four explant cultures from the dorsal root ganglia of 42 latently infected guinea pigs and vaginal and cervical explant cultures from 33 animals were divided so that half received 5 mM of hexamethylenebisacetamide supplemented media and half media alone. HSV-2 was recovered earlier and from a greater percentage of treated cultures than controls. For example, seven days after explant, HSV-2 was recovered from 35 of 84 (42%) treated dorsal root ganglia cultures compared to seven of 84 control cultures (p<0.0001). Likewise, HSV-2 was recovered seven days after explant from 11 of 66 (17%) treated external genital skin cultures and 2 of 66 control cultures (p<0.009), Hexamethylenebisacetamide had no effect on productive HSV-2 infection in guinea pig dorsal root ganglia cultures. This study provides evidence for a role of demethylation in the reactivation of latent HSV from neural as well as peripheral tissues and suggests that latent virus exists at these sites in a similar state. Hexamethylenebisacetamide should be useful in studies of herpes virus latency because it decreases the time necessary to recover virus from latently infected tissues and enhances the recovery of virus.     

Detection of viral antigens in cerebrospinal fluid of patients with herpes simplex virus encephalitis

Thirty-two cerebrospinal fluid (CSF) samples from eighteen patients with confirmed herpes simplex encephalitis (HSE) were assayed by an indirect enzyme-linked immunosorbent assay (ELISA) for the presence of viral antigens. The results are expressed as an antigen ratio distinguishing between herpes simplex virus (HSV) antigens containing samples and negative samples. Judged by this criterion a positive result was obtained in 33% of the patients. Overall, 25% of the CSF samples from HSE patients were positive. In one out of 33 control patients with other neurological disorders a positive antigen ratio was found. Two or more CSF samples were available from eleven patients. In six of these, the second or later samples showed a decreased antigen ratio when compared to the first CSF sample. An increase of the anti-HSV antibody titer was seen in the CSF of five of these six patients. Five out of six patients with a decreasing antigen ratio had an unfavorable outcome of their encephalitis, while a favorable outcome was seen in four of the five patients with an increasing or steady antigen ratio. A decrease of the antigen ratio in the course of HSE can be explained by the presence of immune complexes in CSF and may indicate a poor prognosis.     

Restriction of latent herpes virus infection in rabbits immunized with subviral herpes simplex virus vaccine

The ability of an experimental herpes simplex virus type 1 (HSV) vaccine to influence the establishment of latent infection was examined. The virion-free vaccine was prepared from HSV 1-infected LEP cells by extraction with Nonidet P 40. Albino rabbits were immunized with three doses of the subviral vaccine (strain KOS) and challenged with 10(5) PFU of HSV 1 (Kupka strain) into the right scarified cornea. Non-immune controls and rabbits immunized with formalin-inactivated virion vaccine (strain KOS) were infected in a similar way. At intervals from 53 to 164 days p. i., the animals were killed and fragments of both trigeminal ganglia were kept separately in culture for 10 days. In 18 out of 22 immunized animals (81.8%), latency was established in the homolateral Gasserian ganglion. The proportion of HSV-yielding fragments, was considerably higher in ganglia from non-immune animals (42.6%) as compared to those from ganglia of rabbits immunized with the subviral vaccine (5.4%) or with the whole virion vaccine (14.6%). The immunity resulting from previous vaccination restricted about 5 times the number of ganglion cells, which become virus carriers.     

Typing of herpes simplex virus strains by analyzing the viral DNA using restriction endonucleases

Analysis of the products of viral DNAs cleavage by restrictive endonucleases showed the strains of herpes simplex virus Us and L2 used for herpes vaccine manufacture to belong to type 1 and the VN strain to type 2 of herpes simplex virus. This method is found to be optimal for typing of herpes simplex virus strains.     

Detection of antibody to viral proteins following primary infection with herpes simplex virus

Sera from patients with primary genital infection with herpes simplex virus (HSV) were tested by immunoblotting (IB) and radioimmunoprecipitation (RIP) analysis to determine the protein targets of antibody elicited by infection. The two tests detected antibody to different antigens: IB primarily detected reactivity with p40, a phosphorylated capsid protein, and RIP detected antibody to glycoprotein B, a viral envelope component. Furthermore, RIP detected antibody at an earlier stage of infection then IB. A nondenaturing version of IB was developed and used to investigate the role that the solubilisation of antigen plays in the sensitivity of each test for antibodies with different specificities.     

Detection of viral DNA polymerase activity in salmon tumour tissue induced by herpes virus, Oncorhynchus masou virus.

DNA polymerase activities were surveyed in tumour tissue and normal tissue of cherry salmon (Oncorhynchus masou). High activity of DNA polymerase alpha was detected in the tumour tissue but not in the normal tissue. This indicates that the tumour cells replicate prosperously. Viral DNA polymerase activity was detected only in the tumour tissue, indicating that Oncorhynchus masou virus (OMV) DNA should replicate there. DNA polymerase beta activity was of same level in both tissues. This is the first evidence that herpesvirus DNA polymerase was detected in tumour tissue in association with herpesvirus.     

Activation of herpes simplex virus 1 gamma 2 genes by viral DNA replication

The expression of gamma 2 or true gamma genes resident in the herpes simplex virus 1 genome requires functional products of genes expressed earlier in infection and viral DNA synthesis. To determine whether the requirement for viral DNA synthesis for the expression of gamma 2 genes reflects a trans-acting function of a product of one or a few genes made or activated during viral DNA synthesis or whether it reflects a cis effect of DNA synthesis at specific sites, 143 thymidine kinase (TK) minus cells were sequentially infected with a virus carrying a 700-bp deletion of the TK gene and 6 hr later with wild-type or recombinant viruses carrying an alpha-TK or a gamma 2-TK gene in either the presence or absence of viral DNA synthesis. These experiments indicated that the gamma 2-TK gene contained in an unreplicated viral genome was not expressed by trans-acting factors specified before and during DNA synthesis by the first infecting virus, and, therefore, the induction of gamma 2 genes appears to be mediated by a cis-acting function associated with viral DNA synthesis.     

Typing of clinical herpes simplex virus isolates with mouse monoclonal antibodies to herpes simplex virus types 1 and 2: comparison with type-specific rabbit antisera and restriction endonuclease analysis of viral DNA

A total of 122 clinical isolates of herpes simplex virus (HSV) from 107 patients were typed by using an indirect immunoperoxidase technique with commercially available type-specific rabbit antisera, recently developed mouse monoclonal antibodies to HSV types 1 and 2, and restriction endonuclease analysis of viral DNA. With the commercially available type-specific rabbit antisera, 34% of clinical HSV isolates were of indeterminate type; 63% of them were typed as HSV type 1 and 37% as HSV type 2 by using monoclonal antibody and restriction enzyme typing systems. Typing by immunofluorescence assay with the monoclonal antibodies gave identical results to those obtained by restriction enzyme analysis. Simultaneous infection with both HSV types was demonstrated by monoclonal antibody typing in five isolates from three patients. These findings were subsequently confirmed by plaque purification and restriction endonuclease analysis of viral DNA. Monoclonal antibodies were as sensitive as restriction enzyme analysis for the typing of clinical HSV isolates. Because of their simplicity, they are more amenable to use in clinical laboratories than is restriction endonuclease analysis.     

Detection of herpes simplex virus DNA sequences in human blood and bone marrow cells

Herpes simplex virus type 1 (HSV1) establishes latent infections in neural tissues of humans and experimental animals. Utilizing a sensitive polymerase chain reaction (PCR) assay we detected HSV DNA sequences in blood ceils of healthy prospective bone marrow transplant (BMT) donors and patients. In three healthy individuals studied, HSV DNA sequences were found in all blood cell types and also in bone marrow cells as well as in stem cell progenitor colonies isolated from in vitro cultures. Studies of BMT donor-recipient pairs suggested that HSV reactivation may occur in hematopoietic cells after transplantation, as the PCR signal intensity increased over time simultaneous with an increased antibody liter to HSV. In a mouse model for HSV infection, HSV DNA sequences were found in blood and bone marrow cells at the latent stage of infection, after intravenous (IV) inoculation, but not after ocular inoculation. These studies suggest that bone marrow cells may be an additional site of HSV latency capable of reactivation after BMT. These studies have broad implications for understanding pathogenesis of HSV disease and are of particular significance in situations where allo-geneic bone marrow cells are given therapeutically. © 1994 Wiley-Liss, Inc.     

Studies on herpes simplex virus in tissue culture

Conclusions and summary The Z strain of herpes simplex virus as well as three freshly isolated strains of the same virus caused a complete degeneration of the fibroblastic outgrowth in roller tube cultures of human embryonic lung. Infectivity titrations made in such tissue cultures regularly gave higher values than the lethality titers obtained by yolk sac inoculation of embryonated eggs. Furthermore, the virus amounts found in the fluid phase of roller cultures were comparable to those obtained in the allantoic fluid of eggs. Three strains of herpes simplex virus have so far been isolated directly in tissue culture from herpetic vesicles. However, more work is needed, involving isolation experiments from pathological specimens simultaneously in tissue cultures and other hosts before the relative sensitivity of the tissue culture method for primary isolation purposes can be evaluated.     

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk ⁺)mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tkgene expression during lytic HSV infections. This finding suggests that cell-associated viral tkgene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk ⁺ cell line to those present in tk ^– revenant and tk ⁺ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.     

Capsid assembly and DNA packaging in herpes simplex virus

The genome of HSV-1 contains 80–85 open reading frames. Genetic and biochemical evidence suggests that at least 39 of these genes encode proteins that are components of the HSV-1 virion. The architecture of the HSV-1 virion consists of a trilaminar lipid envelope, an amorphous layer known as the tegument, a capsid shell, and a DNA-containing core. The capsid is an icosahedral shell whose major morphological features are 162 capsomers. It is composed of a major capsid protein called VP5 and three less abundant proteins, VP19C, VP23 and VP26. VP5 is the structural subunit of all 162 capsomers while VP19C and VP23 are located in the space between the capsomers. In addition to the structural proteins, capsid assembly involves participation of the HSV-1-encoded protease and the scaffolding protein, preVP22a. DNA packaging involves participation of DNA, empty capsids, and at least seven additional HSV-1-encoded proteins. Considerable advances have been made in understanding the structure of the capsid shell, largely as the result of applying cryoelectron microscopy techniques. Use of recombinant baculoviruses has allowed for a detailed analysis of the proteins required for capsid assembly. More recently, an in vitro system has been developed which has aided in defining the assembly pathway by identifying intermediates in the assembly of intact capsids. The in vitro system has identified a fragile roundish procapsid which matures into the polyhedral capsid in a transition similar to that undergone by bacteriophage proheads. This review is a summary of our present knowledge with respect to the structure and assembly of the HSV-1 capsid and what is known about the seven genes involved in DNA packaging. © 1997 John Wiley & Sons Ltd.     

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.     

Expression of an immediate early polypeptide and activation of a viral origin of DNA replication in cells containing a fragment of herpes simplex virus DNA

A thymidine kinase cotransformation procedure has been used to introduce the sequences encoding the herpes simplex virus type 1 (HSV-1) immediate early protein, Vmw175, into permissive cells either in the presence or the absence of the adjacent origin of viral DNA replication. Cells transformed by either origin-plus or origin-minus DNA were capable of expressing functional Vmw175 as indicated by their ability to complement the growth at the nonpermissive temperature of an HSV-1 mutant, ts K, containing a temperature-sensitive lesion in the Vmw175 gene. A proportion of the virus yield from cells transformed with the origin-plus, but not the origin-minus, plasmid exhibited a ts+ phenotype. The generation of ts+ virus correlated with an amplification of input plasmid DNA sequences which occurred following superinfection, suggesting that recombination between the ts mutant and the amplified viral DNA sequences had taken place. Encapsidation of the amplified DNA sequences was also detected, suggesting that in addition to a functional origin of replication and Vmw175 gene the transformed cells also retain the viral DNA packaging signals.     

Detection of herpes simplex virus DNA from genital lesions by in situ hybridization.

Lesion specimens from 118 episodes of recurrent genital herpes were used to compare herpes simplex virus (HSV) isolation with a direct specimen test for in situ DNA hybridization utilizing a biotinylated probe. The frequency of detection of HSV was similar with both tests; HSV was isolated from 81% of vesicular lesions, 76% of pustules, and 67% of ulcers, while HSV DNA was detected in 77, 76, and 55% of lesions in these stages, respectively. Utilizing both methods, HSV was identified in 91, 94, and 79%, respectively. The sensitivity and specificity of the DNA probe in comparison to standard viral isolation in tissue culture were 92 and 63%, respectively. Seven DNA-positive, viral isolation-negative specimens were obtained from patients who had positive culture confirmation at some time subsequent or prior to enrollment, suggesting that these were true positive results. The sensitivity of the DNA probe was dependent on cellular content of the specimen, and 36 (28%) of the 127 submitted specimens had fewer than 20 nonsuperficial cells. The DNA probe was rapid and convenient; its major disadvantage was the lack of type-specific information. The performance of the probe in lower-prevalence populations and in asymptomatic shedding of HSV remains to be evaluated.     

Recovery of latent herpes simplex virus from human trigeminal nerve roots

Summary Herpes simplex virus type 1 (HSV-1) has been isolated from the trigeminal nerve roots from five of eighteen human cadavers. All virus isolates were identified by type specific immunofluorescent staining. This finding suggests latent HSV is not confined to the autonomic and sensory ganglia of humans.With 1 Figure