A novel, cell-specific attenuation of a herpes simplex virus type 1 infection in vivo.

We have observed a cell-specific attenuation of herpes simplex virus type 1 strain 17syn+ in vivo that was dependent upon the cell type used to grow the virus. Direct corneal infection of rabbits with 17syn+ propagated in Vero cells caused 60% (6 of 10) to develop severe central nervous system (CNS) disease as evidenced by seizures and/or paralysis; all neurologically impaired rabbits died. In contrast, infection of rabbits with 17syn+ propagated in BHK-21 cells induced seizures and was fatal in 10% (1 of 10). The cell-specific attenuation of a 17syn+ occurred after one growth cycle in BHK-21 cells. To determine whether the decreased virulence of the BHK-21 cell-grown virus correlated with a less severe CNS inflammatory reaction, CNS tissues from rabbits infected with 17syn+ grown in Vero and BHK-21 cells were compared. Histopathological analyses revealed no differences in the location or severity of inflammatory lesions from rabbits infected with virus grown in either cell type. Virus-induced corneal disease was less dependent upon the cell type used to propagate the virus as there were no significant differences in the type or severity of observed corneal lesions. Possible explanations based on differences between Vero and BHK-21 cells are discussed.

PCR assessment of HSV-1 corneal infection in animals treated with rose bengal and lissamine green B.

PURPOSE: In vivo, the ophthalmic dye rose bengal displays profound antiviral effects against herpes simplex virus (HSV)-1, thus limiting its utility in diagnosis of epithelial keratitis when used before viral culture is performed. In contrast, lissamine green B does not possess significant antiviral activity in vivo. To determine whether polymerase chain reaction (PCR) could successfully detect HSV-1 DNA in ocular samples that have been exposed to ophthalmic dyes, animal models were used to observe the presence of infectious HSV-1 and viral DNA in eyes treated with rose bengal or lissamine green B. METHODS: Animals were bilaterally infected with HSV-1 strain H129, and at daily intervals up to 16 days post infection (dpi) rose bengal or lissamine green B was instilled in the left eyes. The right eyes were not treated with dyes. Swabs of the dye-treated and untreated eyes were assayed by PCR for viral infectivity by culture and the presence of DNA specific for a fragment of the HSV-1 DNA polymerase gene. RESULTS: A statistically equivalent number of samples from lissamine green B-treated and untreated eyes were positive by both viral culture and PCR. In contrast, rose bengal significantly decreased the infectious virus present in ocular secretions. A total of 44% and 78% of the rose bengal-treated and untreated eye samples, respectively, were positive by culture from 1 through 16 dpi. PCR was more sensitive than culture for detection of HSV-1 in rose bengal-treated eyes, in that 74% of rose bengal-treated samples were positive by PCR compared with 44% that were positive by culture during the 16-day period studied. It was also noted that both rose bengal and lissamine green B treatments slightly prolonged the period during which viral DNA was detectable in ocular secretions by PCR, possibly because the singlet oxygen produced by these photoreactive dyes compromised ocular cellular, humoral, and nonspecific immune factors allowing viral DNA to persist for slightly longer periods. CONCLUSIONS: PCR can successfully detect HSV-1 DNA in ocular samples that are culture negative and contain rose bengal or lissamine green B. Visualization of ocular epithelial defects with lissamine green B does not interfere with detection of infectious virus or HSV-1 DNA.

Comparative analyses of the latency-associated transcript promoters from herpes simplex virus type 1 strains H129, +GC and KOS-63.

We have analyzed the activity of a specific portion of the latency-associated transcript (LAT) promoter of three strains of herpes simplex virus type 1 (HSV-1) in neuronal and non-neuronal cell types. Restriction fragments containing the LAT promoter sequences and the 5'-end of the LATs were isolated from HSV-1 strains H129, +GC and KOS-63, sequenced and cloned into a chloramphenicol transferase (CAT) plasmid vector. These vectors were separately assayed for CAT production in human (SknSH) and mouse (C-1300) neuroblastoma cell lines and a human continuous cell line (HeLa). Strain KOS-63 contained a C to T base substitution within the LAT promoter binding factor element upstream of the cAMP response element binding sequence. In replicate experiments, in which the construct DNA was used for transfection, the CAT constructs from strains H129 and +GC functioned equally well in all three cell lines. In contrast, the strain KOS-63 CAT construct functioned significantly better in HeLa cells than in neuroblastoma cell lines and better than the identical CAT constructs from strains H129 and +GC. In addition, the construct from strain KOS-63 functioned less well in the human neuroblastoma cell line than in HeLa or C-1300 neuroblastoma cells. When LAT expression was examined directly in vivo by in situ hybridization, strain KOS-63 produced slightly less LAT RNA than strain H129 within trigeminal ganglionic neurons of latently infected rabbits. However, utilizing competitive gel-shift assays, DNA fragments containing the LAT promoter binding element from all three strains bound equivalent amounts of HeLa cell nuclear proteins. Together, these results suggest that the activity expressed by the strain KOS-63 LAT promoter in vivo and in vitro may relate to positive or negative effects of DNA binding proteins on LAT transcription, and that these effects are cell-type dependent.

An improved rapid method for purification of herpes simplex virus DNA using cesium trifluoroacetate.

A method for purification of herpes simplex virus DNA from cell culture is described which yields highly purified viral DNA within 8 h. The method involves the freezing and thawing of virus-infected cells followed by isopycnic centrifugation of the lysate supernatant in cesium trifluoroacetate. It was found that this method recovered DNA from most of the cell-associated virus particles in such sufficient purity that the DNA was digestible with restriction enzymes and could be used to transfect cells without the need for additional purification steps. Purification of viral DNA from cells that were not subjected to freezing and thawing was less efficient due to the amount of viral DNA that remained cell-associated.

Cloning and restriction endonuclease mapping of herpes simplex virus type-1 strains H129 and +GC.

EcoRI fragments of herpes simplex virus I (HSV-1) strains H129 and +GC were cloned and the EcoRI and BglII restriction enzyme sites were mapped. Comparison of these enzyme sites with the sequence of HSV-1 strain 17syn+ demonstrated that all EcoRI sites were identical. For H129, the BglII sites were also found to match strain 17syn+ BglII sites. With one exception, the BglII sites in strain +GC also aligned with the strain 17syn+ sequence. The one exception was a missing BglII site from strain +GC located between bases 25,149 and 25,154 in the EcoRI D fragment within the viral deoxyribonuclease gene (UL12). The BglII site represents the first difference to be mapped within HSV-1 strains H129 and +GC which have unique pathobiological properties in animal models of acute and reactivated infections.

Sequence and expression analysis of potential nonstructural proteins of 4.9, 4.8, 12.7, and 9.5 kDa encoded between the spike and membrane protein genes of the bovine coronavirus.

The nucleotide sequence between the spike and membrane protein genes in the bovine coronavirus (BCV) genome was determined by sequencing cDNA clones of the genome, and open reading frames potentially encoding proteins of 4.9, 4.8, 12.7, and 9.5 kDa, in that order, were identified. The 4.9- and 4.8-kDa proteins appear to be vestiges of an 11-kDa protein for which a single nucleotide deletion event in the central part of the gene gave rise to a stop codon. The consensus CYAAAC sequence precedes the 4.9-, 12.7-, and 9.5-kDa ORFs and predicts that transcription will start from each of these sites. Northern analyses using sequence-specific probes and oligo(dT)-selected RNA demonstrated that the predicted transcripts are made, and that these correspond to mRNAs 4, 5, and 5-1. BCV mRNA 4 appears to be a counterpart to mouse hepatitis virus (MHV) mRNA 4 which, in the MHV JHM strain, encodes the putative 15.2-kDa nonstructural protein. BCV mRNAs 5 and 5-1 appear to be used for the synthesis of the 12.7- and 9.5-kDa proteins, respectively, which demonstrates a pattern of expression strikingly different from that utilized by MHV. MHV makes its homologs of the 12.7- and 9.5-kDa proteins from the single mRNA 5. In vitro translation analyses demonstrated that the BCV 9.5-kDa protein, unlike its MHV counterpart, is poorly made from downstream initiation of translation. Thus, from a comparison between BCV and MHV we find evolutionary evidence for the importance of the CYAAAC sequence in regulating coronavirus transcription.

Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site.

The sequence of the spike (also called peplomer or E2) protein gene of the Mebus strain of bovine coronavirus (BCV) was obtained from cDNA clones of genomic RNA. The gene sequence predicts a 150,825 mol wt apoprotein of 1363 amino acids having an N-terminal hydrophobic signal sequence of 17 amino acids, 19 potential N-linked glycosylation sites, a hydrophobic anchor sequence of approximately 17 amino acids near the C terminus, and a hydrophilic cysteine-rich C terminus of 35 amino acids. An internal Lys-Arg-Arg-Ser-Arg-Arg sequence predicts a protease cleavage site between amino acids 768 and 769 that would separate the S apoprotein into S1 and S2 segments of 85690 and 65153 mol wt, respectively. Amino terminal amino acid sequencing of the virion-derived gp 100 spike subunit confirmed the location of the predicted cleavage site, and established that gp 120 and gp 100 are the glycosylated virion forms of the S1 and S2 subunits, respectively. Sequence comparisons between BCV and the antigenically related mouse hepatitis coronavirus revealed more sequence divergence in the putative knob region of the spike protein (S1) than in the stem region (S2).

Structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein.

The sequence of the hemagglutinin-esterase (HE) gene for the Mebus strain of bovine coronavirus was obtained from cDNA clones, and its deduced product is a 47,700-kilodalton apoprotein of 424 amino acids. Expression of the HE protein in vitro in the presence of microsomes revealed N-terminal signal peptide cleavage and C-terminal anchorage but not disulfide-linked dimerization. Dimerization was observed only after expression in vivo, during which HE was also transported to the cell surface.

Synthesis and processing of the bovine enteric coronavirus haemagglutinin protein.

The haemagglutinin molecule on the bovine enteric coronavirus has been identified as a glycoprotein of 140K composed of disulphide-linked subunits of 65K. In this study, we have shown the subunits to be identical by demonstrating an unambiguous amino-terminal amino acid sequence. The unglycosylated subunit was found to have an Mr of 42.5K and to undergo rapid disulphide linkage and glycosylation. Glycosylation was found to be of the asparagine-linked type and some of the oligosaccharides underwent processing to complex forms. Studies with inhibitors of glycosylation suggested that a processing of the haemagglutinin oligosaccharide takes place on the virion whilst it is in the Golgi apparatus. Each haemagglutinin subunit on the mature virion was estimated to possess six or seven carbohydrate chains of either the high-mannose or hybrid type, and three or four chains of the complex type.