Transduction of virulence in herpes simplex virus type 1 from a pathogenic to an apathogenic strain by a cloned viral DMA fragment

Herpes simplex virus (HSV) type 1 strain HFEM, the genome of which contains a deletion of about 4 kilobasepairs (kbp) between 0.7 and 0.8 viral map units, is apathogenic in the tree shrew when the animals are inoculated intravenously, intraperitoneally and/or subcutaneously. Similar results were obtained using Balb/c mice. Studies of the state of viral latency in animals infected with HSV-1 strain HFEM revealed that this strain was unable to colonize the ganglia of tree shrews. Infectious virus could be recovered only from the spleens of latently infected tree shrews. Thus, this system offers new opportunities for investigating the gene functions responsible for the virulence of HSV-1. Marker rescue experiments were performed by the contransfection technique using native DNA of HSV-1 strain HFEM and Bam H I DNA fragment B derived from the pathogenic HSV-1 strain F and cloned in a bacterial vector. A number of different intratypic recombinants were established in which the deleted region of HSV-1 strain HFEM had been repaired. The pathogenicity of these recombinants was examined in vivo. One of the recombinants (HSV-R-HFehx-C19) caused generalized and lethal herpes virus infection in juvenile and adult tupaias, indicating that the virulence of the pathogenic HSV-1 strain F can be transduced by the cloned Bam H I DNA fragment B to the apathogenic HSV-1 strain HFEM.     

Herpes simplex virus type 1 (HSV-1) UL56 gene is involved in viral intraperitoneal pathogenicity to immunocompetent mice

Summary A comparison of the pathogenicity in mice of the recombinant herpes simplex virus type 1 (HSV-1) strain HSV-1-M- LacZ, in which the U_L56 gene has been deleted, was made with its parental strain F, following infection in different mouse strains. The polymerase chain reaction (PCR) technique was used to study the migration of virus DNA in the mouse model. Tissues from adult mice infected intraperitoneally (IP) with one of three HSV-1 strains (F, HFEM or HSV-1-LacZ) were examined for the presence of viral DNA. DNA of the pathogenic strain F was detected in the adrenal glands, spinal cord, brain, liver and pancreas. DNA of HSV-1-M-LacZ was detected in the same tissues. However, DNA of the apathogenic strain HFEM was detected transiently (on days 2 and 3 p.i., but not days 1, 5 or 7), only in the adrenal glands and no viral DNA was detected in any of the other tissues. HSV-1 pathogenic strains injected intraperitoneally into newborn mice (7 days old) killed most of the mice. In the surviving mice viral DNA of the three virus strains was found in peritoneal exudate cells (PEC), adrenal glands, spinal cord, liver and spleen. It was found that HSV-1-M-LacZ, which lacks the U_L56 gene, resembled in pathogenicity to the newborn mice the pathogenic HSV-1 strains F and KOS. The PCR technique was used to trace viral DNA in tissues of the mice which survived HSV-1 infection at 7 weeks of age. Only HSV-1 (KOS) DNA was detected in the pancreas. The brains of these mice did not contain viral DNA. It is suggested that HSV-1 DNA may reside in surviving HSV-1- infected newborn mice in a latent state in nonneural tissues.     

Determination of the coding capacity of the BamHI DNA fragment B of apathogenic Herpes simplex virus type 1 strain HFEM by DNA nucleotide sequence analysis.

Herpes simplex virus type 1 (HSV-1) strain HFEM acquired an apathogenic phenotype due to a deletion within the DNA sequences of the BamHI DNA fragment B of the viral genome. In order to investigate the coding strategy of this particular region of the genome of HSV-1 strain HFEM the DNA nucleotide sequence of the BamHI DNA fragment B was determined. This analysis revealed that the BamHI DNA fragment B of HSV-1 strain HFEM comprises 6593 bp, corresponding to the nucleotide positions (np) 113322 to 117088 and np 120643 to 123465 of the genome of HSV-1 strain 17. According to these data the deletion of the genome of HSV-1 strain HFEM occurred between the np 117089 and 120642. The promoter region of the UL56 gene of HSV-1 strain HFEM is a part of the deleted DNA sequences. Therefore, this gene of HSV-1 strain HFEM is affected and cannot be expressed. The first 35 amino acid (AA) residues of the deduced amino acid sequence of the UL56 open reading frame (ORF) were found to be identical to the amino acid sequence of the UL56 genes of HSV-1 strains 17 and F. However, due to a deletion at np 3494 of the BamHI DNA fragment B of HSV-1 strain HFEM the amino acid composition of the predicted UL56 gene of HSV-1 strain HFEM is different from HSV-1 strain 17 between amino acid positions 36 and 233. In addition the deduced amino acid sequence of the IRL (inverted repeat of the long segment) copy of the IE110 gene of HSV-1 strain HFEM was found to be about 342 amino acids shorter than the amino acid sequence of IE110 gene of HSV-1 strain 17 (775 AA). This was based on a point mutation which was detected within the DNA sequences of Exon 3 of this copy of IE110 gene of HSV-1 strain HFEM.     

Effect of herpes simplex virus type-1 UL41 gene on the stability of mRNA from the cellular genes: β-actin,fibronectin, glucose transporter-1, and docking protein,and on virus intraperitoneal pathogenicity to newborn mice

Infection with HSV-1 is accompanied by the shut-off of cellular gene expression. The virion-associated function is encoded by the viral gene U_L41. An HSV-1 mutant, vhs-1, which has a genomic deletion in the U_L41 gene, is incapable of inducing the shut-off of cellular gene expression. The effect of HSV-1 infection on the shut-off of the cellular genes (or mRNA degradation) was studied specifically with the cellular genes for -actin, fibronectin, glucose transporter-1, and the docking protein. The level of these specific mRNAs was measured in cells infected with several HSV-1 strains and was compared to that of vhs-1- and mock-infected cells. It was possible to demonstrate a marked reduction in the level of the specific mRNA from these cellular genes in cells infected with several HSV-1 strains but not with the vhs-1 mutant. The pathogenicity of the HSV-1 vhs-1 mutant to newborn mice was studied. It was found that the mutant is less pathogenic to newborn mice than its parental strain HSV-1 KOS.     

Replacement of the deletion in the genome (0.762-0.789 mu) of avirulent HSV-1 HFEM using cloned MluI DNA fragment (0.7615-0.796 mu) of virulent HSV-1 F leads to generation of virulent intratypic recombinant

The HFEM strain of HSV-1 is apathogenic for the tree shrew by the intraperitoneal (i.p.) route because of a deletion in the genome coordinates 0.762-0.789. Insertion of the MluI DNA fragment (coordinates 0.7615-0.789) cloned from HSV-1 strain F, which is pathogenic for the tree shrew, restored the i.p. pathogenicity to strain HFEM. The recombinant designated R-M1-C1 was highly pathogenic for the tree shrew, but slightly virulent for inbred mouse strain A. It thus appears that the viral DNA sequence involved in the i.p. pathogenicity of HSV-1 is located within the genome coordinates 0.761-0.796. This sequence is recognized differently by the cellular elements involved in HSV-1 infection in the tree shrew and the mouse.     

Determination of the nucleotide sequence flanking the deletion (0.762 to 0.789 map units) in the genome of an intraperitoneally avirulent HSV-1 strain HFEM

Herpes simplex virus type 1 (HSV-1) strain HFEM which harbours a deletion of 4.1 kbp in its genome (0.762 to 0.789 map units, HpaI DNA fragment P of HSV-1), is apathogenic for mice and tree shrews by the intraperitoneal application route. The exact position of this deletion was determined by DNA sequence analysis. This analysis was performed using the recombinant plasmid pU18HSHF-XmI-B which harbours the flanking genome regions (0.752 to 0.762 and 0.789 to 0.7895 map units) of the deletion in the genome of HSV-1 HFEM, and the recombinant plasmids pU18HSF-XmI-B, pU18HSF-AS, and pHSF-BB-BsH-D, harbouring particular regions of the genome of the virulent HSV-1 strain F at the coordinates 0.752 to 0.761, 0.786 to 0.790, and 0.762 to 0.771, respectively. The comparison of the DNA sequence of this region with the DNA sequences of the corresponding genome regions of the pathogenic HSV-1 strain F and HSV-1 strain 17 showed that the 5' end of the deletion in the genome of HSV-1 HFEM starts at the nucleotide position 3774 of the BamHI DNA fragment B from HSV-1/17. This position is 71 bp upstream of the UL/RL junction of the HSV-1 genome. The 3' terminus of the deletion ends at the nucleotide position 7226 of the BamHI DNA fragment B from HSV-1/17. The position is within the incomplete ninth repetitive box (ACTCC-CACGCACCCCC) and is located 36 bp upstream of the 3' end of the IE 110 mRNA.     

A sequence in HpaI-P fragment of herpes simplex virus-1 DNA determines intraperitoneal virulence in mice

The virulence of herpes simplex virus-1 (HSV-1) strains by the intraperitoneal (ip) route of injection in mice depends on the presence of an intact sequence in the HpaI DNA fragment P within coordinates 0.762 to 0.787. Deletion of the HpaI-P region (e.g., strain HFEM) abrogates the ability of the virus to infect mice by the ip route without affecting pathogenicity by the intracerebral (ic) route. A recombinant virus (M1C1) derived from DNA of the HSV-1 HFEM strain and the MLUIDNA fragment (coordinates 0.761 to 0.796) spanning the HpaI-P sequence of the pathogenic strain F regained pathogenicity for mice by the ip route.     

Correlation of pathogenicity and gene constellation of an influenza a virus (fowl plague) I. Exchange of a single gene

Recombinants of fowl plague virus (FPV) with other influenza A prototype strains of human and animal origin in which only a single gene (RNA segment) is not derived from FPV were tested for their pathogenicity in chickens. Most of these recombinants had a lowered pathogenicity, while some were completely apathogenic. The apathogenic recombinants induced high titers of antibodies against the surface components of FPV in the infected chickens which were protected against a challenge infection with wild type FPV. Loss of pathogenicity depended on the gene which was exchanged as well as on the influenza A strain of which the foreign gene was derived, but no specific gene constellation could be detected for apathogenic recombinants. There is no clear correlation between growth rate and pathogenicity, indicating that other factors such as organ tropism might also play an important role in pathogenicity of influenza viruses.     

Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice.

Pathogenicity of Rickettsia tsutsugamushi for laboratory mice is known to be influenced by at least three factors: (i) route of inoculation, (ii) antigenic strain, and (iii) natural resistance of the host. By using Karp, Gilliam, and Kato strains of R. tsutsugamushi, we examined the effect of these three pathogenicity factors on the kinetics of infection and the development of immunity in BALB/cDub and C3H/HeDub mice. The appearance of rickettsemia in the pathogenic infections generally preceded infections of reduced pathogenicity by 1 to 2 days in both magnitude and time of onset. Mice infected by the subcutaneous route with normally pathogenic rickettsiae, i.e., Gilliam-infected C3H/HeDub mice and Karp-infected BALB/cDub mice, consistently maintained a detectable rickettsemia over a 1-year period. Rickettsiae were recovered from the spleens of 95% (19 of 20) of these mice 52 weeks postinfection. In contrast, mice with infections of reduced pathogenicity, i.e., BALB/cDub mice infected by intraperitoneal and subcutaneous inoculation with Gilliam, did not have detectable rickettsemia from week 20 through week 52 postinfection except for a single mouse on week 44 postinfection. Rickettsiae were detected in the spleens of only 40% (8 of 20) of these mice after 1 year. In both Gilliam-infected mouse strains, protection against heterologous challenge with Karp or Kato rickettsial strains was incomplete up to 7 days postimmunization. Infections of reduced pathogenicity did not result from an enhanced systemic immune response by the host. The onset of the humoral response was not different for the pathogenic and reduced-pathogenicity infections. Pathogenicity differences seemed to result from the more rapid growth of the rickettsiae in the pathogenic infections.     

Effects of avian viruses on cultured chicken bone-marrow-derived macrophages

Cultured chicken bone-marrow-derived macrophages have been assayed for their susceptibility to infection with various avian viruses. Three criteria of infection were employed: (1) Virus-induced alterations in cell morphology ; (2) presence of intracellular viral antigens detectable by immunofluorescence; (3) kinetics of virus release by infected macrophages. Macrophages proved to be resistant to Marek's disease virus (MDV), herpesvirus of turkeys (HVT-FC126), infectious bronchitis virus (IBV) and reticuloendotheliosis virus (REV). MDV included the pathogenic HPRS-16 strain prepared from feather follicles, and the apathogenic HPRS-24 strain adapted to growth in chick embryo fibroblast cultures. IBV included both embryo-propagated and tissue culture-adapted variants of the apathogenic Beaudette strain and a pathogenic Massachusetts-type strain. REV comprised the strains REV-C, CSV and oncogenic virus of the REV-F strain. Adenovirus, infectious laryngotracheitis (ILT) virus, reovirus, infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) replicated in macrophages causing different but characteristic cytopathic effects, or alterations in cell morphology associated with macrophage activation. The most prominent effect of IBDV and lentogenic NDV infection were morphological signs of macrophage activation, i.e. enlargement or 'transformation' of cells which tended to survive in infected cultures and were usually free of detectable amounts of immunofluorescent viral antigens. Macrophage cultures were less susceptible to infection with adenovirus (OTE strain), pathogenic ILT virus and lentogenic NDV (B1 strain) than permissive chicken kidney cell (CKC) cultures. In contrast, macrophage cultures were significantly more susceptible to infection with reovirus than CKC cultures, indicating that bone-marrow-derived macrophages might be the major target cells of this virus species. Virus restriction by cultured bone-marrow-derived macrophages was expressed to various degrees among the different avian virus species and among different strains of the same virus species, however, it was not generally correlated with the pathogenicity of these viruses in chickens.     

Analysis of the transcription pattern of HSV-1 UL52 and UL53 genes

The UL52 and UL53 genes of herpes simplex virus type-1 are both located in theBamHI-L DNA fragment, with an overlap of 14 amino acids. An RNase protection experiment was designed to determine the 5′ termini of both the UL52 and UL53 mRNAs. The 5′ end of the UL52 mRNA was found to be located 100 bp upstream of its ATG initiation codon. Surprisingly, the 5′ terminus of the UL53 gene was found to be downstream of its putative initiation codon. Therefore, it was suggested that the translation of the UL53 open reading frame (ORF) starts at an internal initiation codon that is located 55 codons downstream of the putative one. A hybrid selection experiment was performed in which the UL53-specific mRNA was selected from BSC-1 cells infected with HSV-1 KOS and translated in vitro. The translation product of the UL53 message was found to be 32 kD (shorter than the original 37.5 kD ORF). The size of the protein obtained corresponds with the expected translation product starting at the downstream initiation codon. Analysis of the sequence upstream of this initiation codon reveals the presence of a promotor sequence. Therefore, we suggest that the UL53 protein is 54 amino acids shorter than was previously suggested and is located at coordinates 112,341–113,193.     

Herpes Simplex Virus Type 1 (HSV-1) Strain HSZP Host Shutoff Gene: Nucleotide Sequence and Comparison with HSV-1 Strains Differing in Early Shutoff of Host Protein Synthesis

The UL41 gene of the HSZP strain of herpes simplex virus type 1 (HSV-1) defective with respect to the early shutoff of host protein synthesis was sequenced and compared with the corresponding HSV-1 strain KOS and 17 gene sequences. In comparison with strain 17, nine mutations (base changes) were HSZP specific, five KOS specific and four were common for both strains. Nine mutations caused codon changes. Three of these mapped to the nonconserved regions and the others to the conserved regions of the functional map of UL4l gene. One KOS specific mutation mapped to the region responsible for the binding of the virion host shutoff (vhs) protein to the alpha-transinducing factor (VP16). The possible relationship between mutations and host shutoff function is discussed. The nucleotide sequence data of the UL41 gene of HSZP and KOS have been submitted to the Genbank nucleotide database and have been assigned the accesion numbers Z72337 and Z72338. This revised version was published online in July 2006 with corrections to the Cover Date.     

Routine test for in vitro differentiation of pathogenic and apathogenic Listeria monocytogenes strains.

The exosubstance of Rhodococcus equi in a prepurified form strongly enhanced the hemolytic effect of certain strains of Listeria monocytogenes. The strains which produced positive synergic hemolysis with this exosubstance were also pathogenic for guinea pigs and white mice. The other strains, which remained nonhemolytic in the presence of the R. equi. exosubstance, were apathogenic for those animals. A routine test was devised for the in vitro determination of the pathogenicity of L. monocytogenes strains.     

Characterisation of IE and UL5 gene products of equine herpesvirus 1 using DNA inoculation of mice

Summary.  The equine herpesvirus 1 (EHV-1) strain HVS25A regulatory genes IE and UL5, encoding homologues of herpes simplex virus 1 (HSV-1) ICP4 and ICP27 respectively, were cloned into a eukaryotic expression vector and the DNA injected intramuscularly into mice. Antibodies produced in this way detected the IE or UL5 gene products as diffuse material in nuclei of RK13 cells transfected with the individual genes but as discrete punctate or large aggregates in RK13 cells infected with EHV-1. Western blotting on EHV-1 infected RK13 cells showed multiple IE products of 120–200 kDa and a UL5 product of 52 kDa. Inoculation with plasmids expressing EHV-1 IE or UL5 provided limited protection against EHV-1 challenge in mice as determined by increased virus clearance from lungs on day 2 post-challenge and a reduction in severity of lung histopathology. However, this protection was relatively weak compared with that provided by inoculation of DNA encoding EHV-1 glycoprotein D (gD), possibly reflecting the importance of neutralising antibody in this model. Accepted May 19, 2000 Received March 20, 2000     

Herpes simplex virus type 1 and 2 in the adrenal glands: Replication and histopathology

Summary The adrenal glands were shown to be the most severely infected organs in the early phase of HSV-1 infections (up to 10 days p. i.) after i. p. infections in mice. Virus could be isolated from the adrenal glands as early as one hour after infection with pathogenic and apathogenic strains. Infection of the adrenal glands is a result of viremia. The content of HSV-1 (5 strains) was much higher in the adrenals than in spleen and liver. It peaked at 3–4 days p. i. compared to 1–2 days in spleen and liver. Only strain 17 syn⁺ produced low tissue titres in the adrenal glands.Morphologic alterations by HSV-1 infections commenced with distinct foci 2 days after infection in the zona fasciculata, detected immunohistochemically by HSV-specific peroxidase-staining. Necrotic cells could be observed. The foci became confluent until day 4 and remained in this status up to day 7 p. i. During infection immunocompetent cells (macrophages, granulocytes, many T-helper — but only few T-cytotoxic/suppressor lymphocytes) could be observed. On day 10 p. i. the viral antigen had been completely eliminated.In contrast, intraperitoneal infections with 5 strains of HSV-2 resulted in infection of the adrenal glands only to a low degree. The titer of virus was low (exception: strain HG 52). This correlates well with the type of disease produced by either HSV-1 or 2. By comparing the replication of different strains of HSV-1 and 2, three types of tropism after i. p. infection of mice can be distinguished.With 7 FiguresIn partial fulfillment of his M. D.'s degree.     

Neurovirulence of herpes simplex virus type 1 depends on age in mice and thymidine kinase expression

The susceptibility of mice of different ages (from four to 28 days) to infection with herpes simplex virus type 1 (HSV-1) mutants inoculated onto scarified corneas was studied. The TK+ isolate from wild type virus was pathogenic in mice of all age groups. An HSV-1 mutant (designated TK1/4) with a less active thymidine kinase (TK) gene expressing 25 per cent of the TK activity of the TK+ isolate was pathogenic for mice up to 10 days of age. In older mice, virus pathogenicity was dependent on the inoculum dose: increasing the TK1/4 virus dose tenfold raised the level of TK activity and thus the virulence of the virus. A TK- mutant with no TK activity was pathogenic for four to eight day old mice that have TK activity in the brain, but not in older mice. Thus, resistance to HSV-1 that is age-dependent in mice can be determined by the extent to which the virus strain is liable to express its TK gene and by the amount of TK activity present in the brain.