Efficacy of generic oral directly acting agents in patients with hepatitis C virus infection.

Novel direct-acting antivirals (DAAs) are now the standard of care for the management of hepatitis C virus (HCV) infection. Branded DAAs are associated with high sustained virological response at 12 weeks post-completion of therapy (SVR12), but are costly. We aimed to assess the efficacy of generic oral DAAs in a real-life clinical scenario. Consecutive patients with known HCV infection who were treated with generic-oral DAA regimens (May 2015 to January 2017) were included. Demographic details, prior therapy and SVR12 were documented. Four hundred and ninety patients (mean age: 38.9 ± 12.7 years) were treated with generic DAAs in the study time period. Their clinical presentations included chronic hepatitis (CHC) in 339 (69.2%) of cases, compensated cirrhosis in 120 (24.48%) cases and decompensated cirrhosis in 31 (6.32%) cases. Genotype 3 was most common (n = 372, 75.9%) followed by genotype 1 (n = 97, 19.8%). Treatment naïve and treatment-experienced (defined as having previous treatment with peginterferon and ribavirin) were 432 (88.2%) and 58 (11.8%), respectively. Generic DAA treatment regimens included sofosbuvir in combination with ribavirin (n = 175), daclatasvir alone (n = 149), ribavirin and peginterferon (n = 80), ledipasvir alone (n = 43), daclatasvir and ribavirin (n = 37), and ledipasvir and ribavirin (n = 6). Overall SVR12 was 95.9% (470/490) for all treatment regimens. SVR12 for treatment naïve and experienced patients was 97.0% (419/432) and 87.9% (51/58), respectively, P = .005. High SVR12 was observed with various regimens, irrespective of genotype and underlying liver disease status. There were no differences in SVR12 with 12 or 24 weeks therapy. No major adverse event occurred requiring treatment stoppage. Generic oral DAAs are associated with high SVR rates in patients with HCV infection in a real-life clinical scenario.

Anti-envelope antibody responses in individuals at high risk of hepatitis C virus who resist infection.

Injection drug users uninfected by hepatitis C virus (HCV) despite likely repeated exposure through high-risk behaviour are well documented. Factors preventing infection in these individuals are incompletely understood. Here, we looked for anti-HCV-envelope antibody responses in a cohort of repeatedly exposed but uninfected subjects. Forty-two hepatitis C diagnostic antibody- and RNA-negative injection drug users at high risk of exposure were studied and findings compared to healthy controls and cases with chronic HCV infection. Purified IgGs from sera were tested by ELISA for binding to genotype 1a and 3a envelope glycoproteins E1E2 with further testing for IgG and IgM reactivity against soluble E2. Virus-neutralizing activity was assessed using an HCV pseudoparticle system. Uninfected subjects demonstrated significantly greater IgG and IgM reactivities to envelope glycoproteins than healthy controls with IgG from 6 individuals additionally showing significant neutralization. This study is the first to describe humoral immunological responses targeting the HCV envelope, important for viral neutralization, in exposed uninfected individuals. A subset of these cases also had evidence of viral neutralization via anti-envelope antibodies. In addition to confirming viral exposure, the presence of specific anti-envelope antibodies may be a factor that helps these individuals resist HCV infection.

Elevated interferon-stimulated gene transcription in peripheral blood mononuclear cells occurs in patients infected with genotype 1 but not genotype 3 hepatitis C virus.

Hepatitis C virus (HCV) can be classified into seven distinct genotypes that are associated with differing pathologies and respond differently to antiviral therapy. In the UK, genotype 1 and 3 are present in approximately equal proportions. Chronic infection with HCV genotype 3 is associated with increased liver steatosis and reduced peripheral total cholesterol levels, which potentially influences peripheral immune responses. To understand these differences, we investigated host gene transcription in peripheral blood mononuclear cells by microarray and quantitative PCR in patients with genotype 1 (n = 22) or genotype 3 infection (n = 22) and matched healthy controls (n = 15). Enrichment of genes involved in immune response and inflammatory pathways were present in patients infected with HCV genotype 1; however, no differences in genes involved in lipid or cholesterol metabolism were detected. This genotype-specific induction of genes is unrelated to IL28B genotype or previous treatment failure. Our data support the hypothesis that genotype 1 infection drives a skewed Type I interferon response and provides a foundation for future investigations into the host-pathogen interactions that underlie the genotype-specific clinical outcomes of chronic HCV infection.

Suspected tick-borne flavivirus meningoencephalomyelitis in dogs from the UK: six cases (2021).

OBJECTIVES: Tick-borne encephalitis virus and louping ill virus are neurotropic flaviviruses transmitted by ticks. Epidemiologically, tick-borne encephalitis is endemic in Europe whereas louping ill's predominant geographical distribution is the UK. Rarely, these flaviviruses affect dogs causing neurological signs. This case series aimed to describe the clinical, clinicopathological, and imaging findings, as well as the outcomes in six dogs with meningoencephalitis and/or meningomyelitis caused by a flavivirus in the UK in 2021. MATERIALS AND METHODS: Observational retrospective case-series study. Clinical data were retrieved from medical records of dogs with positive serological or immunohistochemical results from three different institutions from spring to winter 2021. RESULTS: Six dogs were included in the study. All dogs presented an initial phase of pyrexia and/or lethargy followed by progressive signs of spinal cord and/or intracranial disease. Magnetic resonance imaging showed bilateral and symmetrical lesions affecting the grey matter of the thalamus, pons, medulla oblongata, and thoracic or lumbar intumescences with none or mild parenchymal and meningeal contrast enhancement. Serology for tick-borne encephalitis virus was positive in five dogs with the presence of seroconversion in two dogs. The viral distinction between flaviviruses was not achieved. One dog with negative serology presented positive immunohistochemistry at post-mortem examination. Three dogs survived but presented neurological sequelae. Three dogs were euthanased due to the rapid progression of the clinical signs or static neurological signs. CLINICAL SIGNIFICANCE: These cases raise awareness of the presence of tick-borne encephalitis as an emergent disease or the increased prevalence of louping ill virus affecting dogs in the UK.

Vaccination saves lives: a real-time study of patients with chronic diseases and severe COVID-19 infection.

OBJECTIVES: This study aims to describe the demographic and clinical profile and ascertain the determinants of outcome among hospitalized coronavirus disease 2019 (COVID-19) adult patients enrolled in the National Clinical Registry for COVID-19 (NCRC). METHODS: NCRC is an on-going data collection platform operational in 42 hospitals across India. Data of hospitalized COVID-19 patients enrolled in NCRC between 1st September 2020 to 26th October 2021 were examined. RESULTS: Analysis of 29 509 hospitalized, adult COVID-19 patients [mean (SD) age: 51.1 (16.2) year; male: 18 752 (63.6%)] showed that 15 678 (53.1%) had at least one comorbidity. Among 25 715 (87.1%) symptomatic patients, fever was the commonest symptom (72.3%) followed by shortness of breath (48.9%) and dry cough (45.5%). In-hospital mortality was 14.5% (n = 3957). Adjusted odds of dying were significantly higher in age group ≥60 years, males, with diabetes, chronic kidney diseases, chronic liver disease, malignancy and tuberculosis, presenting with dyspnoea and neurological symptoms. WHO ordinal scale 4 or above at admission carried the highest odds of dying [5.6 (95% CI: 4.6-7.0)]. Patients receiving one [OR: 0.5 (95% CI: 0.4-0.7)] or two doses of anti-SARS CoV-2 vaccine [OR: 0.4 (95% CI: 0.3-0.7)] were protected from in-hospital mortality. CONCLUSIONS: WHO ordinal scale at admission is the most important independent predictor for in-hospital death in COVID-19 patients. Anti-SARS-CoV2 vaccination provides significant protection against mortality.

Therapeutic control of hepatitis C virus: the role of neutralizing monoclonal antibodies.

Liver failure associated with hepatitis C virus (HCV) accounts for a substantial portion of liver transplantation. Although current therapy helps some patients with chronic HCV infection, adverse side effects and a high relapse rate are major problems. These problems are compounded in liver transplant recipients as reinfection occurs shortly after transplantation. One approach to control reinfection is the combined use of specific antivirals together with HCV-specific antibodies. Indeed, a number of human and mouse monoclonal antibodies to conformational and linear epitopes on HCV envelope proteins are potential candidates, since they have high virus neutralization potency and are directed to epitopes conserved across diverse HCV genotypes. However, a greater understanding of the factors contributing to virus escape and the role of lipoproteins in masking virion surface domains involved in virus entry will be required to help define those protective determinants most likely to give broad protection. An approach to immune escape is potentially caused by viral infection of immune cells leading to the induction hypermutation of the immunoglobulin gene in B cells. These effects may contribute to HCV persistence and B cell lymphoproliferative diseases.

Transition-state affinity chromatography of trypsin-like proteinases with dipeptidyl argininal ligands.

Dipeptidyl argininal (arginine aldehyde) affinity resins of general formula R-(X-Y-argininal) (where R = resin matrix and X, Y = amino acids of varied structure) are synthesized in a solid-phase procedure in which the dipeptide (-X-Y-) is first attached to the resin, followed by the joining of the Y amino acid to argininal semicarbazone, and decomposition of the semicarbazone in a methanol/acetic acid/formaldehyde reagent. An R-(Gly-Gly-argininal) resin binds urokinase tightly, but does not bind thrombin. However, thrombin binds strongly to R-(Phe-Pro-argininal), whereas urokinase does not bind. Accordingly, the X-Y-argininal ligands selectively bind proteinases of identical primary binding site specificity to arginine, but different secondary site specificity in -X-Y-. The selectivity is due to an amplification of peptide binding specificity caused by the transition-state analog properties of the ligands. While the affinity constants between peptide aldehyde and proteinase approach those of antibody-antigen interactions, the elution with semicarbazide (aldehyde-trapping reagent) buffers easily remove tightly bound proteinases without proteinase inhibitors or denaturation. Conditions for the binding and elution of proteinases, methods of regeneration and other characteristics of the resins are described.

Regulation of the protein A-encoding gene in Staphylococcus aureus.

The synthesis of several exoproteins, including protein A (SpA) in Staphylococcus aureus is coordinately regulated by the agr locus. Different constructs of the SpA-encoding gene (spa) were introduced into Agr+ and Agr- derivatives of a spa- strain of S. aureus. Plasmid-located spa with deletions at the 3' end expressed a truncated SpA which was almost exclusively extracellular and which confirmed the role of C-terminal region X in cell-wall binding. In the Agr- host, the production of SpA was elevated severalfold. Transcriptional and translational fusions were constructed to study the agr- mediated regulation of spa gene expression. Translational fusions of a beta-lactamase (Bla)-encoding ApR reporter gene with the spa promoter and N-terminal coding sequences expressed elevated levels of Bla activity in the Agr- host. In contrast, a transcriptional fusion of the spa gene with a promoter of the positively regulated staphylococcal epidermolytic toxin A (ETA)-encoding gene synthesized higher levels of SpA in an Agr+ host, as compared to Agr-. Moreover, the synthesis of SpA in the Agr+ strain was switched on during the transition from the exponential to stationary phase in a similar fashion to ETA itself. These data strongly indicate that the regulation of both SpA and ETA occurs at the transcriptional level in S. aureus. The agr-regulated spa promoter was defined by deletion analysis and by transcript mapping.

The N-terminal 22 amino acids encoded by the gene specifying the major secreted protein of vaccinia virus, strain Lister, can function as a signal sequence to direct the export of a foreign protein.

Cells infected with vaccinia virus strain Lister secrete a polypeptide of approximate molecular weight 35,000 (35K) into the medium. Previous studies identified a cleavable, hydrophobic region of 17 amino acids in the 35K protein which could potentially function as a signal peptide to target the protein to the secretory pathway. Here we report the use of the expression-secretion signals derived from the 35K gene to direct export and secretion of a foreign protein. Vaccinia virus recombinants carrying the bacterial chloramphenicol acetyl transferase gene (cat) immediately downstream from the promoter and the N-terminal coding sequences of the 35K gene were constructed. Our studies show that the N-terminal 22 or 42 amino acids of the 35K protein direct efficient secretion of the CAT protein. However, due to a cryptic glycosylation site within CAT, glycosylated protein was secreted, which reduced enzymatic activity. Activity was restored in the presence of tunicamycin. Removal of the glycosylation site by site-directed mutagenesis abolished glycosylation with no effect on secretion, although CAT activity was again reduced, possibly due to an effect on the active site. The results presented here demonstrate the feasibility of using the promoter and the signal sequence of the 35K gene to generate recombinant viruses for overexpression and secretion of foreign proteins.

A rare case of primary otoscleroma of the middle ear.

OBJECTIVE: We report an extremely rare case of primary otoscleroma. METHOD: We present a case report and a review of the world literature concerning otoscleroma. RESULTS: An adult woman presented with chronic suppurative otitis media with tubotympanic disease and conductive hearing loss. On mastoid exploration, dark granulations were seen, which were identified as otoscleroma on histopathological examination. The patient responded well to streptomycin. CONCLUSION: To the best of our knowledge, this is the first report of primary otoscleroma in the world literature. This case indicates that Frisch's bacillus can also spread to the middle ear.

Oncogenic role of DDX3 in breast cancer biogenesis.

Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.

The product of the UL6 gene of herpes simplex virus type 1 is associated with virus capsids.

We report on the analysis of the UL6 and UL7 open reading frames of the herpes simplex virus type 1 (HSV-1) genome. The UL6 and UL7 transcripts were identified in HSV-1-infected cells by Northern blotting and shown to be coterminal at their 3' ends. Both transcripts were synthesized in the presence of phosphonoacetic acid, although in reduced amounts, indicating that UL6 and UL7 are expressed as delayed-early or gamma-1 genes. The 5' ends of the two transcripts were mapped by S1 nuclease and primer extension analysis. A polyclonal antiserum directed against an Escherichia coli-expressed 6 x His-UL6 fusion protein identified a protein of approximate M(r) 75,000 in cells infected with either HSV-1 or with a vaccinia virus recombinant expressing the HSV-1 UL6 protein. As with the transcript, the UL6 protein was synthesized at reduced levels in the absence of viral DNA replication. Western immunoblotting showed that the UL6 protein was present in purified virions but not in L-particles of HSV-1, and that it was located exclusively in the tegument/capsid fraction of virion. Further analysis of the UL6 protein revealed that this protein was associated with virus capsids.

Characterization of Bunyamwera virus defective interfering particles.

In an attempt to isolate conditional lethal amber nonsense mutants of Bunyamwera virus, five variants were found which produced small plaques on BHK and mouse L cells. Characterization of these variants by Northern blotting showed that they synthesized defective (subgenomic) RNAs derived from the L RNA segment. No subgenomic M or S segment RNAs were detected. The defective L RNAs were shown to be packaged into virus particles, and four of five preparations caused interference with the multiplication of standard virus. When defective-containing preparations were mixed with standard virus and grown in doubly infected cells a reduction in titre of standard virus of up to 400-fold was observed. Hence these preparations most probably contained defective interfering (DI) particles. Novel DI-specific polypeptides were synthesized in DI virus-infected cells. These novel proteins could be precipitated by antisera raised against either the N or C terminus, or both, of the L protein. Nucleotide sequence analysis of cloned cDNA to prominent DI RNAs in three different defective virus preparations revealed that the DI RNA in each case had suffered a single internal deletion of the L segment while retaining the 5'- and 3'-terminal sequences. The extent of the deletion ranged between 72% and 77% of the L RNA segment. Our results suggest that these DI particles may have arisen during the attempted isolation of Bunyamwera virus amber mutants on mouse L cells, since defective/subgenomic RNAs derived from the L and M segments were readily generated in mouse L cells but not in BHK cells, following infection with wild-type virus.

Hepatitis C virus core protein interacts with a human DEAD box protein DDX3.

Several studies have implicated hepatitis C virus (HCV) core in influencing the expression of host genes. To identify cellular factors with a possible role in HCV replication and pathogenesis, we looked for cellular proteins that interact with the viral core protein. A human liver cDNA library was screened in a yeast two-hybrid assay to identify cellular proteins that bind to core. Several positive clones were isolated, one of which encoded the C-terminal 253 amino acids of a putative RNA helicase, a DEAD box protein designated DDX3. Bacterially expressed glutathione-S-transferase-DDX3 fusion protein specifically pulled down in vitro translated and radiolabeled HCV core, confirming a direct interaction. Immunofluorescent staining of HeLa cells with a polyclonal antiserum showed that DDX3 is located predominantly in nuclear speckles and at low levels throughout the cytoplasm. In cells infected with a recombinant vaccinia virus expressing HCV structural proteins (core, E1, and E2), DDX3 and core colocalized in distinct spots in the perinuclear region of the cytoplasm. The regions of the proteins involved in binding were found by deletion analysis to be the N-terminal 59 amino acid residues of core and a C-terminal RS-like domain of DDX3. The human DDX3 is a putative RNA helicase and a member of a highly conserved DEAD box subclass that includes murine PL10, Xenopus An3, and yeast Ded1 proteins. Their role in RNA metabolism or gene expression is unknown. The significance of core-helicase interaction in HCV replication and pathogenesis is discussed.

The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the E1 glycoprotein and native complex formation.

Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, that interact to form both native and aggregated complexes in tissue culture cells. In native complexes, E1 and E2 are associated by noncovalent interactions and such complexes are considered to constitute the authentic interactions between the proteins. By contrast, the proteins are linked by covalent, disulfide bonds in aggregated complexes. From studies with a mutant in which cysteine residues in E1 have been substituted with other amino acids, we show that E1 continues to associate with E2, although the migratory patterns of the proteins on gels are consistent with the formation of aggregated complexes. Therefore, such complexes can be stabilized by noncovalent as well as covalent interactions. To further examine the requirements for native complex formation, segments of foreign glycoproteins were linked to regions of E2. Our data provide direct evidence for the requirement of C-terminal sequences in E2 that contain the transmembrane domain to permit oxidation of E1 and assembly of a native complex. By contrast, native complexes and oxidized E1 are not found in the presence of chimeric proteins containing the E2 ectodomain. These data suggest that interaction of E1 with the E2 transmembrane domain is critical for native complex formation.

Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus 8325-4.

The gene coding for protein A (spa) has been mapped close to nov on the genetic map of the chromosome of Staphylococcus aureus 8325-4. A rapid mapping procedure has been developed which first allowed the region of the chromosome carrying the spa gene to be identified by blot +hybridization of large DNA fragments which had been separated by pulsed-field gel electrophoresis. Restriction endonuclease SmaI fragment G was shown to carry the spa gene. An insertion mutation in spa was constructed by in vitro insertion of a fragment of DNA expressing resistance to kanamycin and neomycin. A spa::Kan(r)Neo(r) mutation was isolated in S. aureus 8325-4 by allele replacement. This provided a selectable marker which allowed the spa gene to be mapped by transformation analysis.

Covalent interactions are not required to permit or stabilize the non-covalent association of hepatitis C virus glycoproteins E1 and E2.

Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which are thought to locate to the envelope of virus particles. These proteins form two complexes in tissue culture systems, a high molecular mass aggregate that contains intermolecular covalent bonds and a native complex in which E1 and E2 associate by non-covalent interactions. The contribution of either complex to the structures of the proteins on virus particles is not known. Using dithiothreitol to reduce inter- and intramolecular disulphide bonds in situ, we have studied the nature of the interactions within the aggregate and the role of covalent bonds in the early stages of E1-E2 association. Results with two HCV type 1a strains, Glasgow and H77, showed that the aggregate contains not only covalent interactions but also non-covalent associations between E1 and E2. These non-covalent associations are complex since deletion mutant analysis failed to identify any single region which was required for non-covalent interaction. Complex formation by de novo synthesized proteins was not arrested under reducing conditions which prevented the production of inter- and intramolecular disulphide bonds. Moreover, a conformation-specific antibody continued to recognize the E2 protein in reduced complexes, indicating that covalent bonds do not stabilize certain structures of E2 that can interact with E1. These data suggest that disulphide bonds are not required either to allow association between the proteins or to stabilize E1-E2 complexes.

Different effects of opiate withdrawal on dopamine turnover, uptake, and release in the striatum and nucleus accumbens.

The purpose of these experiments was to further characterize changes in dopaminergic function that follow withdrawal from chronic opiate treatment. Withdrawal after treatment to a maximum dose of 120 mg/kg of morphine did not alter dopamine concentrations in the substantia nigra, ventral tegmental area, striatum, or nucleus accumbens; but did decrease concentrations of DOPAC and the ratio of DOPAC to dopamine in the lateral striatum and nucleus accumbens. Uptake of tritiated dopamine was diminished for withdrawn slices obtained from the striatum with no effect observed for tissue from the nucleus accumbens. Deficits of in vitro release of tritiated dopamine also occurred following withdrawal, with the nucleus accumbens being sensitive to dependence produced by a lower dose of morphine. In conclusion, opiate withdrawal produces a complex pattern of effects on dopaminergic function that is specific for the striatum and nucleus accumbens.

Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.

The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitro-constructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M. O'Reilly, J.C.S. de Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathogen. 1:125-138, 1986). A double Spa- Hly- mutant was constructed by transduction. The virulence of Spa- and Hly- mutants was tested by experimental infection of mice. When subcutaneous injections were given, Hly- mutants formed a flat, darkened lesion, whereas Hly+ strains caused a raised, cream lesion. Alpha-toxin was shown to be a major factor in forming subcutaneous lesions and in causing the death of mice injected intraperitoneally. Spa- mutants were slightly less virulent than their Spa+ counterparts, which suggests that protein A is also a virulence factor of S. aureus.

PREPs: herpes simplex virus type 1-specific particles produced by infected cells when viral DNA replication is blocked.

Herpes simplex virus (HSV)-infected cells produce not only infectious nucleocapsid-containing virions but also virion-related noninfectious light particles (L-particles) composed of the envelope and tegument components of the virus particle (J. F. Szilágyi and C. Cunningham, J. Gen. Virol. 62:661-668, 1991). We show that BHK and MeWO cells infected either with wild-type (WT) HSV type 1 (HSV-1) in the presence of viral DNA replication inhibitors (cytosine-beta-D-arabinofuranoside, phosphonoacetic acid, and acycloguanosine) or with a viral DNA replication-defective mutant of HSV-1 (ambUL8) synthesize a new type of virus-related particle that is morphologically similar to an L-particle but differs in its relative protein composition. These novel particles we term pre-viral DNA replication enveloped particles (PREPs). The numbers of PREPs released into the culture medium were of the same order as those of L-particles from control cultures. The particle/PFU ratios of different PREP stocks ranged from 6 x 10(5) to 3.8 x 10(8), compared with ratios of 3 x 10(3) to 1 x 10(4) for WT L-particle stocks. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analyses revealed that true late proteins, such as 273K (VP1-2), 82/81K (VP13/14), and gC (VP8), were greatly reduced or absent in PREPs and that gD (VP17) and 40K proteins were also underrepresented. In contrast, the amounts of proteins 175K (VP4; IE3), 92/91K (VP11/12), 38K (VP22), and gE (with BHK cells) were increased. The actual protein composition of PREPs showed some cell line-dependent differences, particularly in the amount of gE. PREPs were biologically competent and delivered functional Vmw65 (VP16; alpha TIF) to target cells, but the efficiency of complementation of the HSV-1 (strain 17) mutant in1814 was 10 to 30% of that of WT L-particles.