A physicochemical model for rationalizing SARS-CoV-2 concentration in sewage. Case study: The city of Thessaloniki in Greece.

Detection of SARS-CoV-2 in sewage has been employed by several researchers as an alternative early warning indicator of virus spreading in communities, covering both symptomatic and asymptomatic cases. A factor that can seriously mislead the quantitative measurement of viral copies in sewage is the adsorption of virus fragments onto the highly porous solids suspended in wastewater, making them inaccessible. This depends not only on the available amount of suspended solids, but also on the amount of other dissolved chemicals which may influence the capacity of adsorption. On this account, the present work develops a mathematical framework, at various degrees of spatial complexity, of a physicochemical model that rationalizes the quantitative measurements of total virus fragments in sewage as regards the adsorption of virus onto suspended solids and the effect of dissolved chemicals on it. The city of Thessaloniki in Greece is employed as a convenient case study to determine the values of model variables. The present data indicate the ratio of the specific absorption (UV254/DOC) over the dissolved oxygen (DO) as the parameter with the highest correlation with viral copies. This implies a strong effect on viral inaccessibility in sewage caused (i) by the presence of humic-like substances and (ii) by virus decay due to oxidation and metabolic activity of bacteria. The present results suggest days where many fold corrections in the measurement of viral copies should be applied. As a result, although the detected RNA load in June 2020 is similar to that in April 2020, virus shedding in the city is about 5 times lower in June than in April, in line with the very low SARS-CoV-2 incidence and hospital admissions for COVID-19 in Thessaloniki in June.

Major histocompatibility complex class II (DRB1*, DQA1*, and DQB1*) and DRB1*04 subtypes' associations of Hashimoto's thyroiditis in a Greek population.

Hashimoto's thyroiditis (HT) is an autoimmune disease resulting from complex interactions between genetic and environmental factors. The disease is associated with certain human leukocyte antigen (HLA) class II alleles in various populations. We aimed to determine in this study, for the first time in a Greek population, the association of HLA-DRB1*, -DQA1*, and -DQB1* alleles with HT. HLA-DRB1*, -DQA1*, and -DQB1* alleles' and -DRB1*04 subtypes' distribution was evaluated in 125 patients with HT and in 500 healthy control individuals by using a DNA-based sequence-specific primer method. Chi(_)squared tests and Bonferroni correction method were applied in the statistical analysis of the data. Significantly higher frequency of DRB1*04 (24.8% vs 7.7%, P < 0.0001) was observed in HT patients, while HLA-DRB1*07 was significantly decreased (2.8% vs 7.9%, P < 0.05). HLA-DRB1*04 subtyping showed a significant increase of DRB1*0405 (21% vs 7.8%, P < 0.0001) in HT patients. Also significant high frequencies of DQB1*0201 (14.8% vs 8.2%, P < 0.001), DQB1*0302 (18.8% vs 7.0%, P < 0.0001), and DQA1*0301 (25.6% vs 7.8%, P < 0.0001) were recorded in the patient group. Conducting the first research of this kind in a Greek population, our study tries to provide an evaluation of the prevalence of HT relating to HLA-DRB1*0405, and we report a relative risk of 2.7 for HT in a Greek population.

Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins.

We report the construction of two HSV-1 recombinants encoding chimeric forms of the E2 glycoprotein of HCV-1a composed of the ectodomain of E2 (aa384-611 or 384-711) fused to different parts of the transmembrane and cytoplasmic domain of the HSV-1 gC glycoprotein (gC). The parental HSV-1, known as KgBpK(-)gC(-), is deleted for gC and the main heparan sulphate (HS) binding domain of gB, and it exhibits impaired binding (ca. 80%) to HS compared to the wild type virus KOS [Laquerre, S., Argnani, R., Anderson, D.B., Zucchini, S., Manservigi, R., Glorioso, J.C., 1998. Heparan sulphate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J. Virol. 72, 6119-6130]. We show that gC:E2 proteins are efficiently expressed and transported to the cell surface. We also demonstrate that HSV-1 can incorporate both gC:E2 chimeric proteins into particles and show that incorporation of both chimeric molecules in the viral envelope partially restored binding (ca. 20%) of the HSV-1 recombinants to heparan sulphate. Finally, we showed that the gC:E2ScaI chimeric glycoprotein was able to bind a recombinant form of hCD81 and virion-expressed gC:E2ScaI permitted the binding of the HSV-1 recombinant virus to the hCD81 molecule.

No correlation of five gene polymorphisms with periodontal conditions in a Greek population.

BACKGROUND: Various studies have examined possible correlations between a number of cytokine gene polymorphisms and periodontal disease in populations of different origins. The present study sought the correlation between four single-nucleotide polymorphisms (IL1A+3954, IL1B+4845, TNFA-308, COL1A1 Sp1), a variable number of tandem repeats polymorphism (IL1RN intron 2) and periodontal conditions in subjects of Greek origin. METHODS: One hundred and ninety-two healthy subjects, stratified as non-periodontitis and periodontitis (chronic and aggressive) cases, participated in the present study. Genotyping was performed by polymerase chain reaction-based techniques using the primers and conditions described in the literature. The frequencies of genotypes between study groups were compared using Genepop v3.3 genetic software and Instat statistical package. RESULTS: No differences were observed among the groups concerning the distributions of genotypes under investigation. CONCLUSIONS: Carriage rates of the polymorphisms under investigation in systemically healthy subjects of Greek origin are well within the range reported for Caucasians but these polymorphisms cannot discriminate between non-periodontitis and periodontitis (chronic or aggressive) cases.

Identification and characterization of gene products of ORF U90/89 of human herpesvirus 6.

Human herpesvirus 6 (HHV-6) isolates can be classified into two variants, A and B. Comparison of genomic sequences of these variants has highlighted sequence variability in the region spanning U86 to U100. This region includes the immediate early A (IE-A) locus that was defined as positional homologue of the major IE locus of Human cytomegalovirus (HCMV) with little recognizable sequence homologies. A 3.5 kb transcript, one of the four spliced transcripts identified in the IE-A locus, is derived from the U90/89 ORF encoding the IE1 protein. We expressed six Escherichia coli fragments spanning the HHV-6A U90/89 ORF as IE1 fusion proteins. The bacterially expressed fusion protein was used to raise monospecific polyclonal antiserum for detection and identification of the IE1 protein product(s). Using this antiserum we detected 165, 190, and > 190 K proteins in HHV-6A- and HHV-6B-infected cells and the 165 K protein in cells transfected with an IE1 cDNA construct. The IE1 proteins exhibited perinuclear and cytoplasmic localization in infected cells. There was a correlation between the expression level of IE1 and the degree of permissiveness for virus growth in various cell lines. In transient expression experiments a 140 bp fragment from the upstream IE-A region was shown to possess promoter activity. The C-terminal region of IE1 delineated by amino acids (aa) 588 to 636 showed a DNA binding activity in Southwestern blot analysis.

A single amino acid substitution in the cytoplasmic tail of the glycoprotein B of herpes simplex virus 1 affects both syncytium formation and binding to intracellular heparan sulfate.

Herpes simplex virus 1 (HSV-1) (S) is a spontaneous syncytial mutant derived from the prototype HSV-1(F) after extensive plaque purification, and produces large syncytial plaques on Vero cells. Marker transfer experiments and DNA sequence analysis mapped the syncytial phenotype to a T-C base substitution at codon 787 of the cytoplasmic domain of mature gB, that results in Leu to Pro substitution and consequently belongs to the syn 3 locus. Both the cytoplasmic and the extracellular domains of gB are active in the fusion event since the addition of anti-gB monoclonal antibodies that recognize the extracellular domain of gB prevent HSV-1(S) induced cell fusion. Similarly, gD also participates in cell fusion since addition of anti-gD monoclonal antibodies also prevent HSV-1(S) induced cell fusion. Furthermore the glycoproteins B and D formed complexes in cells infected with mutant or wild type viruses. The amount of gB bound to total heparan sulfate is lower in the mutant than in the wild type strain. This difference becomes particularly profound when gB is associated with a portion of heparan sulfate intercalated to the membranes. The discrepancy in the binding of the mutant and wild type gB to heparan sulfate may be related to the mechanism of cell fusion induced by HSV-1(S).

Antiviral properties of isoborneol, a potent inhibitor of herpes simplex virus type 1.

Isoborneol, a monoterpene and a component of several plant essential oils, showed dual viricidal activity against herpes simplex virus 1 (HSV-1). First, it inactivated HSV-1 by almost 4 log10 values within 30 min of exposure, and second, isoborneol at a concentration of 0.06% completely inhibited viral replication, without affecting viral adsorption. Isoborneol did not exhibit significant cytotoxicity at concentrations ranging between 0.016% and 0.08% when tested against human and monkey cell lines. Isoborneol specifically inhibited glycosylation of viral polypeptides based on the following data: (1) the mature fully glycosylated forms of two viral glycoproteins gB and gD were not detected when the virus was replicated in the presence of isoborneol, (2) no major changes were observed in the glycosylation pattern of cellular polypeptides between untreated and isoborneol treated Vero cells, (3) isoborneol did not affect the glycosylation of gB produced from a copy of the gB gene resident in the cellular genome, and (4) other monoterpenes such as 1,8-cineole and borneol, a stereoisomer of isoborneol, did not inhibit HSV-1 glycosylation.

Insecticidal activity of strains of Bacillus thuringiensis on larvae and adults of Bactrocera oleae Gmelin (Dipt. Tephritidae).

The olive fly, Bactrocera oleae, is the key pest on olives in the Mediterranean area. The pest can destroy, in some cases, up to 70% of the olive production. Its control relies mainly on chemical treatments, sometimes applied by aircraft over vast areas, with their subsequent ecological and toxicological side effects. Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects, including the orders of Lepidoptera, Diptera, and Coleoptera and other invertebrates. The aim of this study was to search for isolates toxic to B. oleae. Several hundred B. thuringiensis isolates were obtained from olive groves and olive presses in different areas of Greece, Sardinia (Italy), and Spain and from cooperating scientists throughout the world. Some isolates were found toxic only to adults or larvae and some to both stages of the olive fly. In addition, the most toxic isolates were assayed on Opius concolor Szepl. (Hym. Braconidae), the most important parasitoid of the olive fruit fly. Only 3 isolates out of 14 gave significant mortality against this parasitoid. Several of the most toxic crystalliferous isolates may contain novel toxins since they gave no PCR products when probed with primers specified for 39 known toxin genes.

Axonal transport of herpes simplex virus-1 in an in vitro model based on the isolated sciatic nerve of the frog Rana ridibunda.

An in vitro model for the study of the axonal transport of herpes simplex virus-1 (HSV-1) in the nerve fibres of the sciatic nerve of the frog Rana ridibunda, has been developed. The nerve was placed along a three-chambered bath consisting of three isolated chambers arranged in series: the stimulating, perfusion and recording chambers. The HSV-1 inoculum was placed in the stimulating chamber, where the proximal part of the isolated sciatic nerve was immersed. HSV-1 was detected after 24-36 h in the recording chamber, where the distal part of the nerve was immersed in Dulbecco's Modified Eagle Medium (DMEM), indicating an axonal transport speed of 46-60 mm/day. The evoked maximum compound action potentials generated in the stimulating chamber was monitored continuously in the recording chamber as an indication of the viability of the nerve during axonal transport. The in vitro method presented here is a useful tool for the pharmacological study of various parameters, e.g. drugs diluted in the perfusion chamber, ionising radiation and temperature, which may affect the axonal transport or other properties of HSV-1.

Application of a transformed cell line constitutively expressing HSV-1 polypeptides for the detection of HSV antibodies in human sera by an enzyme immunoassay.

We have previously reported the construction of a cell line BA4, constitutively producing the glycoproteins gD, gG, and alpha 4, the major regulatory protein of HSV-1. These cells have been selected in stepwise increasing concentrations of methotrexate and shown to produce much higher amounts of gD than non-selected cells. Extracts of the selected cells were used in an enzyme linked immunosorbent assay to detect HSV antibodies in human sera obtained from Greek blood donors. We report here that (i) the assay developed is able to distinguish HSV antibody positive from negative human sera and (ii) that its application in an epidemiological survey showed that the incidence of HSV infection in the general population in Greece is 90.4%.

Transformed cells producing the glycoprotein D of HSV-1 are resistant to infection with clinical strains of HSV.

The generality of the resistance exhibited by gD producing cells to HSV-1 infection was tested. We tested three different cell lines producing various amounts of gD for resistance against three HSV-1 strains. The strains used were the prototype laboratory F strain and two recently isolated low passage local clinical strains, VG and VD. The results indicate that: (i) the resistance of the cell lines is directly related to the amount of gD they produce, (ii) the cell lines showed greater resistance against the two local clinical HSV-1 strains than against the laboratory strain, and (iii) the resistance is not mediated at the level of virus adsorption to the cell membranes.

Regulation of glycoprotein D synthesis of herpes simplex virus 1 by alpha 4 protein, the major regulatory protein of the virus, in stably transformed cell lines: effect of the relative gene copy numbers.

Earlier studies concerning gamma 1 gene regulation by the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1), in stably transformed cell lines, reported conflicting results, i.e., alpha 4 protein positively regulated the gamma 1 gB gene in alpha 4/gB cells, while it negatively regulated the gamma 1 gD gene in alpha 4/BJ cells. Both cell lines were derived from a common parental cell line alpha 4/c 113 that contains 1 copy of the alpha 4 gene, and the only apparent difference between them was the relative copy number of the gB and gD sequences (1 and 30-50, respectively) resident in the cell genome. We investigated this disparity by constructing a cell line (BA 4) that contains one copy each of the alpha 4 and gamma 1 gD sequences, by fusion of alpha 4/c 113 and BJt cells, containing and expressing respectively 1 copy of the alpha 4 and gD genes. BA 4 cells constitutively expressed both the alpha 4, gD genes inherited from the parental cell lines (alpha 4/c 113 and BJt). In BA 4 cells that alpha 4 protein positively regulates the gD gene as evidenced from (i) higher levels of gD expression than the parental BJt cells lacking the alpha 4 gene, and (ii) significant decrease in gD expression under conditions that render the alpha 4 protein produced in BA 4 cells non-functional. In addition the gamma 2gG gene contained within the DNA fragment encoding the gD gene, is also expressed in BA 4 cells. On the basis of these data, we propose that gamma gene regulation by the alpha 4 protein is affected by the relative copy number of these genes, resident in the cell genome.

Mapping of the functional domains of the alpha 4 protein of herpes simplex virus 1.

Truncated alpha 4 genes were introduced into BHK tk- cells along with the neomycin phosphotransferase gene, that confers resistance to the eukaryotic antibiotic G418, driven by the HSV-1 beta tk promoter (beta tk- neor). Stably transformed cell lines were obtained and studied for the ability of the resident truncated alpha 4 genes to regulate the expression of the beta tk- neor, and for the ability of the truncated alpha 4 polypeptides to localize to the nuclei of transformed cells. The results indicated that the domain(s) for beta gene induction and for nuclear localization of the alpha 4 protein are located within the N-terminal 288 amino acids of the protein.

A subset of type-specific epitopes map in the amino terminus of herpes simplex virus type 1 glycoprotein B.

We constructed a recombinant herpes simplex virus (HSV) containing the transcribed coding and non-coding sequences of HSV-1 strain F glycoprotein B (gB) gene, a gamma 1 gene, fused to the promoter-regulatory sequences of the HSV-1 alpha 4 gene and inserted into the thymidine kinase gene of RH1G44, an HSV-1 x HSV-2 recombinant that contains an HSV-2 gB gene at the natural locus. Phenotypic analyses of the insertion mutant, R3145, showed that the alpha gB gene was transcribed in the presence of cycloheximide but underwent partial conversion to the HSV-2 form. Nucleotide sequencing of the gene indicated that the 5' crossover occurred between nucleotides 107 and 117 upstream from the translation initiation site and that the 3' crossover occurred between the sequences specifying amino acids 402 and 412 of the HSV-1 gB. The chimeric protein consisted of an N-terminal 405 to 415 amino acids encoded by the HSV-2 gene and a C-terminal 462 to 472 amino acids encoded by the HSV-1 gene. Comparison of the reactivity of the parental and recombinant gB with type-specific monoclonal antibodies indicated that the chimeric gB lost reactivity with four HSV-1-specific antibodies but gained reactivity with three HSV-2-specific antibodies.

Herpes simplex virus glycoprotein D is sufficient to induce spontaneous pH-independent fusion in a cell line that constitutively expresses the glycoprotein.

Spontaneous small polykaryocytes were detected in a cell line designated BJ-o that harbors the BamHI J fragment of herpes simplex virus 1 DNA and expresses constitutively glycoprotein D (gD). The fusion activity of BJ-o cells correlated with gD production and was drastically reduced following exposure of the cells to monoclonal antibody HD1 to gD. Studies on the characteristics and requirements of cell fusion dependent on gD led to the conclusion that the characteristics and requirements for gD-mediated fusion activity of BJ-o cells are similar to those previously reported for cell fusion induced by the virus in that (i) polykaryocytosis was not augmented by exposure to medium of low pH with or without prior exposure to trypsin, (ii) the number of polykaryocytes was reduced following removal of terminal sialic acid residues by neuraminidase, and (iii) the number of polykaryocytes was augmented by masking of high-mannose N-linked oligosaccharides with concanavalin A or with its reduced form, succinyl concanavalin A. This effect was reversed by competition with mannose.

The glycoprotein C gene of herpes simplex virus 1 resident in clonal L cell lines manifests two regulatory domains conferring a dominant B and a subordinate gamma 2 regulation.

Earlier studies have shown that late, gamma 2, genes of herpes simplex virus 1 stably incorporated into the environment of the cell are regulated as beta genes. For example, the induction of the intact gene specifying glycoprotein C (gC) resident in a clonal L cell line superinfected with a gC- virus was enhanced in the presence of inhibitory concentrations of phosphonoacetate (PAA), a viral DNA synthesis inhibitor. Moreover, the gene was induced by superinfection at the nonpermissive temperature with tsHA1, a temperature-sensitive (ts) DNA- virus mutant. In the viral genome, the gC gene is not expressed by the tsHA1 mutant at the nonpermissive temperature or by the mutant or wild-type virus at the permissive temperature in the presence of inhibitory concentrations of PAA. We report that expression of a truncated gC gene introduced in the same fashion and resident in a clonal L cell line was at least partially sensitive to PAA and was not induced by tsHA1 at the nonpermissive temperature. The gene was transcribed from a prokaryotic transcription initiation site in the plasmid. Induction of gene expression by superinfecting virus did not result in appreciable amplification of the gene. We conclude that gC, a gamma 2 gene, contains two regulatory domains. The gene domain upstream from nucleotide +22 confers beta-like regulation, whereas the gene domain downstream from +22 confers gamma 2 regulation. In the gene resident in cells in culture the upstream regulatory domain is dominant, whereas the converse is true for the gene resident in the viral genome.

Regulation of glycoprotein D synthesis: does alpha 4, the major regulatory protein of herpes simplex virus 1, regulate late genes both positively and negatively?

Earlier studies have described the alpha 4/c113 baby hamster kidney cell line which constitutively expresses the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1). Introduction of the HSV-1 glycoprotein B (gB) gene, regulated as a gamma 1 gene, into these cells yielded a cell line which constitutively expressed both the alpha 4 and gamma 1 gB genes. The expression of the gB gene was dependent on the presence of functional alpha 4 protein. In this article we report that we introduced into the alpha 4/c113 and into the parental BHK cells, the HSV-1 BamHI J fragment, which encodes the domains of four genes, including those of glycoproteins D, G, and I (gD, gG, and gI), and most of the coding sequences of the glycoprotein E (gE) gene. In contrast to the earlier studies, we obtained significant constitutive expression of gD (also a gamma 1 gene) in a cell line (BJ) derived from parental BHK cells, but not in a cell line (alpha 4/BJ) which expresses functional alpha 4 protein. RNA homologous to the gD gene was present in significant amounts in the BJ cell line; smaller amounts of this RNA were detected in the alpha 4/BJ cell line. RNA homologous to gE, presumed to be polyadenylated from signals in the vector sequences, was present in the BJ cells but not in the alpha 4/BJ cells. The expression of the HSV-1 gD and gE genes was readily induced in the alpha 4/BJ cells by superinfection with HSV-2. The BJ cell line was, in contrast, resistant to expression of HSV-1 and HSV-2 genes. The BamHI J DNA fragment copy number was approximately 1 per BJ cell genome equivalent and 30 to 50 per alpha 4/BJ cell genome equivalent. We conclude that (i) the genes specifying gD and gB belong to different viral regulatory gene subsets, (ii) the gD gene is subject to both positive and negative regulation, (iii) both gD and gE mRNAs are subject to translational controls although they may be different, and (iv) the absence of expression of gD in the alpha 4/BJ cells reflects the expression of the alpha 4 protein in these cells.

Entry of herpes simplex virus 1 in BJ cells that constitutively express viral glycoprotein D is by endocytosis and results in degradation of the virus.

The BJ cell line which constitutively expresses herpes simplex virus 1 glycoprotein D is resistant to infection with herpes simplex viruses. Analysis of clonal lines indicated that resistance to superinfecting virus correlates with the expression of glycoprotein D. Resistance was not due to a failure of attachment to cells, since the superinfecting virus absorbed to the BJ cells. Electron microscopic studies showed that the virions are juxtaposed to coated pits and are then taken up into endocytic vesicles. The virus particles contained in the vesicles were in various stages of degradation. Viral DNA that reached the nucleus was present in fewer copies per BJ cell than that in the parental BHKtk- cells infected at the same multiplicity. Moreover, unlike the viral DNA in BHKtk- cells which was amplified, that in BJ cells decreased in copy number. The results suggest that the glycoprotein D expressed in the BJ cell line interfered with fusion of the virion envelope with the plasma membrane but not with the adsorption of the virus to cells and that the viral proteins that mediate adsorption to and fusion of membranes appear to be distinct.

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.

Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line.

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody.