Parental Wild-type Virus Research Articles

Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys.

The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3' noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5' boundary of the 3' noncoding region and whose 3' boundary did not extend beyond the last 113 nt of the 3' end were viable. With the exception of mutant 3'd 303-183, which contained a deletion of nt 303 to 183 from the 3' terminus, deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK2 cells than parental wild-type virus. Significantly, mutant 3'd 303-183 grew to a high titer and was least restricted in growth. Mutant 3'd 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3'd 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization. The immunogenicity of these 3' DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK2 cells. One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

Identification and characterization of pseudorabies virus dUTPase.

Sequence analysis within the long segment of the pseudorabies virus (PrV) genome identified an open reading frame of 804 bp whose deduced protein product of 268 amino acids exhibited homology to dUTPases of other herpesviruses. The gene was designated UL50 because of its colinearity with the homologous gene of herpes simplex virus type 1. An antiserum raised against a bacterially expressed fragment of PrV UL50 specifically detected a 33-kDa protein in lysates of infected cells, which is in agreement with the predicted molecular mass of the PrV UL50 protein. A UL50-negative PrV mutant (PrV UL50-) was constructed by the insertion of a beta-galactosidase expression cassette into the UL50 coding sequence. A corresponding rescuant (PrV UL50resc) was also isolated. The interruption of the UL50 gene led to the disappearance of the 33-kDa protein, whereas restoration of UL50 gene expression restored detection of the 33-kDa protein. Enzyme activity assays confirmed that UL50 of PrV codes for a dUTPase which copurifies with nuclei of infected cells. PrV UL50- replicated with an only slightly reduced efficiency in epithelial cells in culture compared with that of its parental wild-type virus strain. Our results thus demonstrate that UL50 of PrV encodes a protein of 33 kDa with dUTPase activity which copurifies with nuclei of infected cells and is dispensable for replication in cultured epithelial cells.

Differences in the Role of Glycoprotein C of HSV-1 and HSV-2 in Viral Binding May Contribute to Serotype Differences in Cell Tropism

Heparan sulfate serves as a receptor for several herpesviruses. For herpes simplex virus 1 (HSV-1), pseudorabies virus, and bovine herpesvirus 1, glycoprotein C homologues have been shown to mediate the binding to cell-surface heparan sulfate. It has been assumed that glycoprotein C of HSV-2 (gC-2) plays a similar role in HSV-2 entry, but this has not been established experimentally. We first determined, using heparin-affinity chromatography, that gC-2 is a heparin-binding glycoprotein. To examine the role of gC-2 in HSV-2 infection, we constructed a gC-2 deletion mutant, HSV-2(G)gC−. In contrast to results obtained for the other α herpesviruses, we found that the HSV-2(G)gC−virus showed no loss in specific binding activity (particles bound/cell) or specific infectivity (PFU/particle) compared to the parental wild-type virus. Moreover, while gC-1 mutants show a marked lag in the rate of viral penetration, the gC-2-deletion virus did not. We did find that gC-2, like gC-1, protects virus from complement-mediated neutralization. These results suggest that, in contrast to HSV-1, gC-2 does not play the key role in viral binding. The major role of gC-2 may be to protect virus from complement-mediated neutralization. We speculate that serotype differences in the contribution of gC to viral binding may contribute to serotype differences in cell tropism.

The persistent variant of influenza C virus carries one characteristic point mutation in RNA segment 1

Influenza C/Ann Arbor/1/50-pi (C/AA-pi) virus causes persistent infections in MDCK and Wi38 cells, but sets limited, wild-type like infections in other cells. Concluding that persistence itself is dependent on the host environment, we determined the nucleotide sequence of the C/AA-pi analogous gene to the basic polymerase 2 (PB2) of influenza A virus, which is known to be a determinant for the host range. C/AA-pi and the parental wild-type virus (C/AA-wt) have 16 nucleotides in common that are different to a previously published PB2 sequence (C/JJ/50). These variations, which are probably due to divergent passage histories, are scattered along the sequence and are partially found in another published isolate (C/Berlin/1/85). One single mutation, however, is unique to the persistent variant. Nucleotide 28 mutated from T to C which leads to a change of amino acid 3 from Leu to Phe. This substitution is stably associated with the persistent phenotype throughout multiple passages in different cell lines and eggs and cannot be found in any other influenza C viruses.

Characterization of a Highly Attenuated Japanese Encephalitis Virus Generated from Molecularly Cloned cDNA

Six recombinant Japanese encephalitis virus (JEV) isolates were recovered from infectious RNAs transcribed by T7 RNA polymerase from molecularly cloned cDNA templates. Three of the recombinant viruses had characteristics similar to the wild-type parent virus, JaOArS982. The other 3 recombinant viruses exhibited an attenuated phenotype in mice. An avirulent recombinant virus, IC47, was characterized and compared with the wild-type parent virus and a virulent recombinant virus, IC37. IC47 produced smaller plaques than parent or IC37 viruses and exhibited no viremia or neuroinvasion in young adult mice inoculated subcutaneously and no mortality when inoculated intracerebrally. IC47 was also immunogenic and protective in the murine model. The probable basis for attenuation, revealed by nucleotide sequence analysis, was a single amino acid substitution at position 138 (Glu to Lys) in the E protein.

A Cold-Passaged, Attenuated Strain of Human Respiratory Syncytial Virus Contains Mutations in the F and L Genes

In previous studies, a mutant (cp-RSV) of the RSV A2 strain derived from 52 serial cold passages in bovine embryonic tissue culture was highly attenuated in seropositive adults and children but caused upper respiratory tract disease in seronegative infants. We investigated the genetic basis for this attenuation phenotype by comparing the complete genomic RNA sequence of this virus with the published sequence of strain A2 as well as with that of its unattenuated wild-type parent (HEK-7) virus. RNA was extracted from virions grown in tissue culture, reverse transcribed, amplified by the polymerase chain reaction (PCR), cloned, and sequenced. Changes from the published A2 wild-type sequence were confirmed on independently derived cDNA clones and by direct sequencing of PCR fragments. The HEK-7 parent virus was then analyzed at these positions by direct sequencing of PCR fragments. Fifteen nucleotide differences between the published A2 wild-type virus and cp-RSV were found. None appeared to involve cis-acting RNA sequences. Of the 15 nucleotide differences, only 1 occurred outside a translational open reading frame (ORF), and 2 which did occur within ORFs were silent at the amino acid level. The remaining 12 nucleotide differences encoded amino acid changes. All 3 of the mutations which were silent at the amino acid level, and 8 of the 12 which resulted in amino acid differences, were also present in the HEK-7 parent virus and therefore were not changes acquired during the cold passages. Thus, the remaining 4 nucleotide differences and the attendant 4 amino acid changes are associated with the attenuation phenotype of the cp-RSV. Two of the changes occur in the F gene and two in the L gene. These results confirm the previously described RSV genomic sequence, provide the first sequence of a live attenuated RSV vaccine strain, provide the first sequence of an RSV strain which has been evaluated in chimpanzees and humans, and indicate that attenuation in humans of a pneumovirus can be associated with a relatively small number of nucleotide and amino acid changes.

Two open reading frames (ORF1 and ORF2) within the 2.0-kilobase latency-associated transcript of herpes simplex virus type 1 are not essential for reactivation from latency.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are dispensable for establishment and maintenance of latent infection. However, the LATs have been implicated in reactivation of the virus from its latent state. Since the reported LAT deletion and/or insertion variants that are reactivation impaired contain deletions in the putative LAT promoter, it is not known which LAT sequences are involved in reactivation. To examine the role of the 2.0-kb LAT in the process of reactivation and the functional importance of the putative open reading frames (ORF1 and ORF2) contained within the 2.0-kb LAT, we have constructed an HSV-1 variant that contains a precise deletion and insertion within the LAT-specific DNA sequences using site-directed mutagenesis. The HSV-1 variant FS1001K contains an 1,186-bp deletion starting precisely from the 5' end of the 2.0-kb LAT and, for identification, a XbaI restriction endonuclease site insertion. The FS1001K genome contains no other deletions and/or insertions as analyzed by a variety of restriction endonucleases. The deletion in FS1001K removes the entire 556-bp intron within the 2.0-kb LAT, the first 229 nucleotides of ORF1, and the first 159 nucleotides of ORF2 without having an affect on the RL2 (ICP0) gene. Explant cocultivation reactivation assays indicated that this deletion had a minimal effect on reactivation of the variant FS1001K compared with the parental wild-type virus using a mouse eye model. As expected, Northern (RNA) blot analyses have shown that the variant virus (FS1001K) does not produce the 2.0-kb LAT or the 1.45- to 1.5-kb LAT either in vitro or in vivo; however, FS1001K produces an intact RL2 transcript in tissue culture. These data suggest that the 2.0-kb LAT putative ORF1 and ORF2 (or the first 1,186 bp of the 2.0-kb LAT) are dispensable for explant reactivation of latent HSV-1.

Genes in the HindIII J fragment of the murine cytomegalovirus genome are dispensable for growth in cultured cells: insertion mutagenesis with a lacZ/gpt cassette

The organization and function of the genes encoded within the HindIII J region of the murine cytomegalovirus genome were analyzed by transcript mapping and cDNA isolation, nucleotide sequence analysis and identification of open reading frames (ORFs), and construction of recombinant viruses carrying insertions disrupting five of the seven ORFs. This region was found to encode five beta transcripts and one gamma transcript in addition to two beta transcripts previously mapped to the sgg1 locus. Seven open reading frames were identified, and one was recognized as a homolog of a human cytomegalovirus US22 gene family. The five largest ORFs contained within the HindIII J fragment (sgg1, HJ4, HJ5, HJ6, and HJ7) were each disrupted by the insertion of a lacZ/gpt genetic marker cassette. The growth kinetics of all recombinant viruses were investigated and found to be the same as wild-type parental virus, indicating that these five ORFs were dispensable for growth in cell culture.

Studies of the pathogenesis of wild-type virus and six temperature-sensitive mutants of mouse cytomegalovirus.

A comparison of six temperature-sensitive (ts) mutants with the parental wild-type (wt) virus showed that, when 1-week-old BALB/c mice were inoculated intraperitoneally with 300 pfu of mouse passaged virus, the viruses could be broadly categorized into two groups. Two viruses (wt and tsm6) were lethal at this dose (10 and 2 LD50 respectively); animals died within 4-6 days of inoculation and the virus became generalized infecting heart, lung, liver, spleen, kidney, and salivary glands to high titre (> 4.3 log10 pfu/g). In contrast, for viruses (tsm1, tsm3, and tsm4) not lethal at this dose (300 pfu = 0.1 to 0.25 LD50), viral replication was poor (< 3.4 log10 pfu/g) except in the salivary glands (5.6 to 7.5 log10 pfu/g). Mutant tsm5 failed to replicate in any tissue while mutant tsm2, lethal at this dose (300 pfu = 1 LD50), produced levels of virus similar to those found with tsm1, tsm3, and tsm4. Comparison of all viruses at sub-lethal doses (0.1 to 0.25 LD50) did show minor differences in their replication in heart, and in levels of virus and duration of infection in kidney and salivary glands. More marked differences were evident between the viruses in their ability to be reactivated from the latent state during immunosuppression. Wild-type virus was most easily reactivated in that 67% of animals exhibited virus in salivary glands, heart, lung, spleen, and kidney. Mutants tsm1, tsm2, tsm3 and tsm6 could be reactivated but from fewer animals (33%, 33%, 18%, and 38% respectively) and fewer tissues. Mutants tsm4 and tsm5 could not be reactivated. Differences in the ability of the viruses to replicate in the lungs and to cause pneumonitis in intranasally-inoculated immunosuppressed mice were also seen. Although immunosuppression was necessary for the induction of severe pneumonitis, differences in severity of pneumonitis resulted from differences in the ability of the mutants to replicate in the lung in vivo. These different mutants should prove useful for examining the viral and host factors involved in CMV-induced pneumonitis, and for examining mechanisms involved in latency and reactivation.

Mapping of herpes simplex virus 1 genes with mutations which overcome host restrictions to infection.

Earlier studies have shown that the thymidine kinase-negative baby hamster kidney (BHKTK-) cell lines expressing constitutively the herpes simplex virus 1 (HSV-1) glycoprotein D (gD), designated BJ, restrict infection by HSV-1 at the level of virus entry. U10, a HSV-1 mutant not restricted by the BJ cells, carried the substitution of proline for Leu25 in the gD gene, suggesting that gD encodes a specialized domain which precludes virus entry into cells expressing gD. Analyses of a new series of 36 unrestricted viral mutants showed the following. (i) Only two mutants contained mutations at a site which did not overlap with the previously reported mutation. A representative of a previously mapped mutant and one of the two new mutants were examined in detail. Thus, in the gD of mutant U30 Ala185 was replaced by threonine, whereas in gD of U21, Ala185 and Leu25 were replaced with threonine and proline, respectively. U30 and U21 multiplied better than the wild-type parent virus in the parental BHKTK- cells. (ii) Transfer of the gD gene from U21 or U30 to wild-type parent virus or to the gD- virus FgD beta yielded recombinants which, while capable of infecting BJ cells, were considerably less efficient than the parent unrestricted mutants, suggesting that the latter contained additional mutations which were responsible in part for the unrestricted phenotype. Conversely, marker rescue of mutant viruses with wild-type gD reduced but did not abrogate entirely the unrestricted phenotype. (iii) Mutations in gD which conferred the unrestricted phenotype were not random. (iv) gD plays a role in the restriction, inasmuch as preincubation of cells expressing gD with antibodies to gD abolished restriction. (v) In mutant R5000, the gD substitution Ser140 to Asn was capable of overcoming a restriction of a BHKTK- clonal line which does not express gD but conferred very low ability to replicate on BJ cells. We conclude that (a) uncloned stocks of BHKTK- cells exhibit a low level restriction to infection with wild-type virus, (b) clonal lines of BHKTK- cells which vary with respect to the stringency of restriction express either allelic genes differing in the properties of their products or products of different genes, and (c) both the restricted and unrestricted phenotypes reflect the interactions of gD with these cellular products. The implications of these conclusions with respect to the restriction imposed on BHK cells by the expression of gD are discussed.

State News

One year after the emergence of the 2009 influenza (H1N1) pandemic, most cases have been relatively mild; however, the possibility of enhanced viral growth ability by reassortment with seasonal viruses cannot be overlooked. Here, we show that reassortant viruses containing a seasonal H1 HA and swine-origin NA and M genes have enhanced virus growth over their wild-type parental viruses. The emergence of such viruses in nature could, therefore, represent a threat.

Immunization with replication-defective mutants of herpes simplex virus type 1: sites of immune intervention in pathogenesis of challenge virus infection.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) were used as a new means to immunize mice against HSV-1-mediated ocular infection and disease. The effects of the induced immune responses on pathogenesis of acute and latent infection by challenge virus were investigated after corneal inoculation of immunized mice with virulent HSV-1. A single subcutaneous injection of replication-defective mutant virus protected mice against development of encephalitis and keratitis. Replication of the challenge virus at the initial site of infection was lower in mice immunized with attenuated, wild-type parental virus (KOS1.1) or replication-defective mutant virus than in mice immunized with uninfected cell extract or UV-inactivated wild-type virus. Significantly, latent infection in the trigeminal ganglia was reduced in mice given one immunization with replication-defective mutant virus and was completely prevented by two immunizations. Acute replication in the trigeminal ganglia was also prevented in mice immunized twice with wild-type or mutant virus. The level of protection against infection and disease generated by immunization with replication-defective mutant viruses was comparable to that of infectious wild-type virus in all cases. In addition, T-cell proliferative and neutralizing antibody responses following immunization and corneal challenge were of similar strength in mice immunized with replication-defective mutant viruses or with wild-type virus. Thus, protein expression by forms of HSV-1 capable of only partially completing the replication cycle can induce an immune response in mice that efficiently decreases primary replication of virulent challenge virus, interferes with acute and latent infection of the nervous system, and inhibits the development of both keratitis and systemic neurologic disease.

Isolation and preliminary characterisation of twenty-five temperature-sensitive mutants of mouse cytomegalovirus.

To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive (ts) mutants. Six of these (tsm9, tsm13, tsm20, tsm22, tsm28 and tsm30) failed to replicate in mice and were avirulent. Five mutants (tsm14, tsm18, tsm19, tsm25 and tsm27) were of similar virulence to the parental wild-type (wt) virus, five (tsm7, tsm15, tsm24, tsm26 and tsm31) were 12-100 fold less virulent, five (tsm8, tsm12, tsm16, tsm23 and tsm29) were 150-1500 fold less virulent and four (tsm10, tsm11, tsm17 and tsm21) were between 2,000 and 85,000 fold less virulent than wt. One mutant (tsm28) did not plaque or replicate at 39 degrees C while 5 other mutants (tsm7, tsm9, tsm23, tsm24 and tsm27) also failed to plaque at 39 degrees C but only failed to replicate or replicated poorly at 40 degrees C. A further two mutants (tsm10 and tsm13) were able to plaque and replicate at 39 degrees C but not 40 degrees C. Six other mutants (tsm14, tsm15, tsm16, tsm21, tsm22 and tsm30) failed to form plaques at 40 degrees C and were severely restricted in their replication at 40 degrees C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.

The US 9, 10, 11, and 12 Genes of Herpes Simplex Virus Type 1 Are of No Importance for Its Neurovirulence and Latency in Mice

We have studied the pathogenic and latency/reactivation potential of a herpes simplex virus type 1 (HSV-1) variant (N38) which has a deletion of four genes, US9, US10, US11, and US12, in the short unique region of the HSV-1 genome. N38 was as pathogenic as the parental wild-type virus following intracerebral infection of mice and replicated with wild-type kinetics in the brain. After intraperitoneal infection, some restricted replication of N38 was observed in the adrenal gland and the deletion virus was about 20-fold less virulent than the parental virus. There was no significant difference between two viruses in the frequency of reactivation or in reactivation time. The results indicate that US9, US10, US11, and US12 gene products are dispensable for its neurovirulence and latency in mice.

Phenotypic and Functional Characterization of Mouse Attenuated and Virulent Variants of Foot-and-Mouth Disease Virus Type O 1 Campos

A series of genetically related variants arising from a parental wild-type isolate of O 1 Campos and its tissue culture adapted variant were differentiated by various cell culture markers (temperature sensitivity, plaque size, viral yield) and lethality in mice. These isolates were additionally characterized functionally and biochemically by examining poly(C) length, RNA synthesis, protein synthesis, and cell receptor binding. In primary bovine kidney cells, the virulent isolates had greater levels of protein synthesis, whereas in baby hamster kidney cells, the attenuated variant outproduced the wild-type parent. The tissue culture adapted variant had substantially greater ability to attach to cells than the parental wild type. The parental wild-type and the tissue culture-adapted variant were similarly neutralized by various sera against whole virus, but the parental wild type was less effectively neutralized by sera prepared from either full or truncated (variable region) bacterially expressed VP1 polypeptides. The capsid region of the genomes of both these variants was sequenced and a nucleotide substitution resulting in a change in amino acid 56 in VP3 was found. The nucleotide sequence change for the remaining two variants was that of the parental wild-type virus.

Latent Infection Can Be Established with Drastically Restricted Transcription and Replication of the HSV-1 Genome

Viral functions essential for the establishment of latent infection in murine sensory neurons in vivo were investigated by employing a herpes simplex virus type 1 (HSV-1) variant (KD6/B11) deleted for expression of the ICP4 gene and therefore unable to replicate. Since the viral DNA persisted in these cells, the latency-associated transcripts were expressed for prolonged periods of time, and the variant was biologically retrievable by superinfection with an ICP4-competent agent, we concluded that a latent infection had been established. In situ hybridization experiments designed to investigate gene expression during the acute phase of infection with the variant revealed a highly restricted pattern compared to that of the wild-type parent virus HSV-1 KOS(M). While latency-associated transcripts were detected in a large number of infected neurons, expression of other virus genes was limited to a subset of immediate-early and early genes (ICP0, ICP8, ICP27, and HSV-1 DNA polymerase genes). Expression was further limited to a small proportion of the infected neurons (approximately 1% of neurons expressing latency-associated transcripts). No hybridization was detected with probes specific for the viral TK gene and late genes VP5 and gC. Quantitative assays of vital DNA during the acute phase of infection indicated that the input viral DNA did not replicate. From these results we conclude that HSV-1 latent infection can be established in murine sensory neurons under conditions in which vital genetic expression and DNA replication are severely restricted.

Increased synthesis of polycistronic mRNA associated with increased polyadenylation by vesicular stornatitis virus

Electron microscopy suggested that the mRNA produced in vitro by tsG16(I), a temperature-sensitive mutant of vesicular stomatitis virus, contained an increased proportion of polycistronic mRNAs. Using hybrid selection, we found that the poly(A) + mRNA synthesized in vitro by tsG16(I) contained approximately two to three times more polycistronic mRNA than did poly(A) + mRNA synthesized in vitro by the parental wild-type ( wt) virus. The increase in polycistronic mRNA occurred at all intergenic junctions examined. In vitro, tsG16(I) has an increased polyadenylation phenotype and a temperature-sensitive transcriptase activity that appear to be due to different mutations. Partial revertants of tsG16(I), which have lost the aberrant polyadenylation phenotype but retain the in vitro thermosensitive transcriptase, produced wt amounts of polycistronic mRNA. This suggested that the increased production of polycistronic mRNA by tsG16(I) may be associated with the increased polyadenylation phenotype of this mutant. These data further support the hypothesis that an increase in size of poly(A) tracts is associated with increased production of polycistronic mRNA.

The myristylated virion proteins of herpes simplex virus type 1: investigation of their role in the virus life cycle.

Herpes simplex virus type 1 (HSV-1) gene UL11 encodes a myristylated virion protein. In this paper we have characterized the UL11 product further and investigated its role in the virus life cycle. Wild-type HSV-1 strain 17syn+ expresses three electrophoretically distinguishable UL11 polypeptide species. Analysis of single plaque isolates demonstrated that two virus populations exist within the 17syn+ stock: a major population encoding only the two higher Mr species, and a minor population encoding the lowest Mr species alone. DNA sequence analysis suggests that the latter polypeptide differs from the former ones at a single amino acid residue only. The UL11 polypeptides are synthesized as delayed early gene products and are phosphorylated in vitro. Following subcellular fractionation of infected cells, they are found predominantly associated with membranes. Within the virus particle, they appear to reside within the tegument. An insertion mutant containing the lacZ gene from Escherichia coli within the UL11 open reading frame is viable in tissue culture, although it gives smaller plaques and is impaired for growth compared to the wild-type parent or revertant viruses; it does not have a temperature-sensitive or host-range phenotype. Thus, although required for efficient replication, the myristylated HSV-1 virion protein, in contrast to those of many other viruses, is not essential for virus growth in tissue culture.

The clinical significance of antiviral drug resistance

When we refer to drug resistance in relation to antivirals we are usually referring to a decrease in sensitivity to the drug measured in a tissue culture system. The value is usually quoted as an IC50, the concentration required to produce a 50 % inhibition of infectivity, and it may be measured in focal systems such as plaque reduction assays, or in yield reduction systems. In the latter assays, virus yield can itself be assessed in different ways including the use of surrogate markers of virus replication such as cytotoxicity, production of virus antigens or enzymes, or by estimating the extent of genome replication. It is important to appreciate that the actual values for ICs0 will vary widely according to the systems used to measure them, and they are therefore only meaningful if compared with values obtained for parental wild-type viruses. This comparison is not always possible, particularly when looking at clinical isolates, and judgement about whether or not an isolate is resistant will then have to be based on available base-line data derived using the same system with viruses that have never been exposed to the drug.

The UL20 gene of herpes simplex virus 1 encodes a function necessary for viral egress

A recombinant virus from which the start codon and 53% of the UL20 open reading frame had been deleted was constructed and characterized. We report the following: (i) The UL20- mutant formed small plaques in 143 tk- cells but failed to form plaques in Vero cells. Virus yields were approximately 10- to 100-fold lower than those of wild-type virus in all cell lines tested. (ii) Electron microscopic examination of Vero cells infected with the UL20- mutant revealed that enveloped and unenveloped capsids accumulated in the cytoplasm, possibly in the space between the inner and outer lamellae of the nuclear membrane, and that virtually no virus was present in the extracellular space. (iii) Glycoproteins B, C, D, E, H, and I recovered from lysates of cells infected with the UL20- mutant could not be differentiated from those present in lysates of cells infected with the wild-type parent virus with respect to the electrophoretic mobility of mature and precursor forms. (iv) Repair of the deleted sequences restored the wild-type phenotype. (v) The gene product of the UL20 gene was shown to be associated with cellular membranes and to possess characteristics of integral membrane proteins. We conclude that the UL20 gene encodes an integral membrane protein with a hitherto unrecognized function in that it enables the transit of virions to the extracellular space. The function of the UL20 gene product is complemented by some cell lines but not by Vero cells. The vesicles which serve to transport virions may have an origin different from those associated with transport of normal cellular proteins.