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Kinetics of Tupaia herpesvirus strain 2 (THV-2) DNA synthesis was measured by labeling with radioactive thymidine and determining the amount of newly synthesized DNA. Viral DNA synthesis reached a maximum between 24 to 36 hr postinfection, preceded by a transient stimulation of host cell DNA synthesis. Blot hybridizations of labeled recombinant plasmids harboring inserts of both genomic termini of THV-2 and of individual viral terminal DNA fragments to DNA of virus-infected cells and combined restriction enzyme analysis of labeled intracellular viral DNA revealed the presence of linear concatemeric and/or circular viral DNA molecules.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.

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.

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.

Virions of fish lymphocystis disease virus (FLDV), a member of the iridovirus family, were isolated directly from lymphocystis disease lesions of individual flatfishes and purified by sucrose and subsequent cesium chloride gradient centrifugation to homogeneity as judged by electron microscopy. The isolated FLDV DNAs appear to be heterogeneous in size. Contour length measurements of 43 DNA molecules gave an average length of 49 +/- 23 microns, corresponding to 93 +/- 44 X 10(6) D. Molecular weight estimations of FLDV DNA by restriction enzyme analysis resulted in only 64.8 X 10(6) D indicating an excess length of the DNA of about 50%. FLDV DNA was sensitive to lambda 5'-exonuclease and to E. coli 3'-exonuclease III without preference of any one terminal DNA restriction fragment. Denaturation and reannealing experiments of FLDV DNA resulted in the formation of circular DNA molecules of 34.25 microns contour length (= 65.22 X 10(6) D). This result suggests that FLDV DNA contains directly repeated sequences at both ends and that it is terminally redundant. FLDV DNA is methylated in cytosine. FLDV DNA did not hybridize with frog virus DNA indicating that the two iridoviruses are not closely related to each other. Restriction enzyme analysis and Southern blot hybridizations revealed that FLDV isolates can be classified into two different strains: FLDV strain 1 occurs in flounders and plaice, whereas strain 2 is usually found in lesions of dabs.

The susceptibility of juvenile Tupaias to several strains of temperature-sensitive mutants of HSV-1 and 2 was tested. It was found that in all cases the animal survived an infection of 1 X 10(7) PFU of temperature-sensitive mutants which were administered intravenously. In contrast, an infection dose of 1 X 10(3) PFU of wild-type HSV-2 and 1 X 10(2) PFU or HSV-1 was fatal for juvenile Tupaias. Those animals which had initially been infected with the ts-mutants of HSV-1 and/or 2 were protected against a superinfection of a lethal dose of HSV-1 or HSV-2. The state of HSV latency in surviving animals was investigated. The infectious virus was recovered from the spinal cords of those animals which had been initially infected with wild-type HSV-1 or 2 and/or superinfected with HSV-1 or 2. In contrast, the infectious viruses were recovered only from the spleens of those animals which had initially been infected with ts-mutants of HSV-2 and superinfected with HSV-1. These recovered viruses were plaque-purified and analysed in detail. It was found, firstly, that recovered viruses from the spleens of Tupaias lost their pathogenicity and tropism in Tupaias. Secondly, significant changes in the genome of the recovered viruses from the spleen of Tupaias were detected. The results indicate that an intertypic interaction between HSV-1 and 2 occurred in vivo which is responsible for the observed change in organotropism and pathogenicity of the recovered viruses.

The polypeptide composition of four Tupaia herpesvirus strains--of which two were isolated from malignant tumors--was analysed by one- and two-dimensional gel analysis. It was found that purified virions of the tree shrew consist of a least 37 viral polypeptides when determined by SDS polyacrylamide gel electrophoresis. The number of viral polypeptides increased to 49 when analysed by the two-dimensional technique with the molecular weights ranging from 12.000 to 230.000. Variations in the viral polypeptide patterns made it possible to differentiate between the different virus isolated even by the one-dimensional method. The two-dimensional technique allows the identification of virus-specific spots among the polypeptides of virus-infected cells. Radiolabeling experiments identified at least five glycoproteins which seem to reside on the surface of the virus envelope. In addition, neutralisation and immunodiffusion tests were performed which revealed cross-reactivities among the tupaia herpesvirus strains. Rabbit anti-tupaia herpesvirus sera did not neutralize other animal or human herpesvirus.

A survey of the occurrence of Ornithodoros ticks in animal burrows was conducted in Haiti and the Dominican Republic. Vacuum sampling techniques were used. Four of 8 sample sites in Haiti and 10 of 70 sample sites in the Dominican Republic were positive for O. puertoricensis Fox. Positive sample sites in Haiti were usually near swine. Sites in the Dominican Republic were in drier regions of the country and were not directly associated with previous swine locations. The determination that O. puertoricensis, a potential vector and reservoir of African swine fever (ASF), is present in this region may pose a serious problem for eradication of ASF from the island of Hispaniola.

Five Tupaia herpesviruses have been isolated until now: four in our laboratory which were termed THV-2, 3, 4, and 5, whereas THV-1 has been isolated by Melnick and his colleagues. THV-2 was isolated from tumour cell culture of a high-grade malignant lymphoma of a Tupaia, THV-3 was released from a cell culture of another Tupaia lymphoma, THV-4 from a spleen tissue culture of a moribund animal with finely granulated liver cirrhosis, and THV-5 from cultured spleen cells of an apparently healthy tree shrew. THV-1 to 5 were efficiently propagated, plaque-purified and cloned on Tupaia embryonic fibroblasts. The five isolates of Tupaia herpesviruses are easily distinguished from each other by restriction enzyme analysis of their genomes. THV-1 to 4 are highly pathogenic (lethality 100%) for juvenile Tupaias by intravenous inoculation. In contrast, only 25% lethality was found by intraperitoneal administration. THV-1 to 4 can persist as a latent infection in spleens of Tupaias and rabbits, which allows the recovery of infectious virus from cultured spleens of both animals. THV-2 and 3 induced hyperplasia of the thymus of rabbits which developed malignant thymoma in a few cases. The biological properties and genomic size and structure indicate that THV cannot be considered to belong to one of the three existing subfamilies of herpesviruses.

To define the degree of renal insufficiency at which the immune response to vaccination against hepatitis B is impaired, anti-HB concentrations after vaccination with 20 micrograms HB-Vax at 0, 1 and 6 months were examined in 76 dialysis patients, 24 patients with incipient renal failure (S-creatinine 1.4-3.5mg/dl) and in 43 controls. Compared with controls, seroconversion rate and anti-HB concentrations were significantly (p less than 0.02) lower in patients with incipient renal failure. The time course of anti-HBs in dialysis patients with successful vaccination, either with three doses (0, 1, 6 months) or with five doses (0, 1, 2, 4, 6 months) of HB-Vax was compared with healthy controls. The proportion of patients losing anti-HBs and the decrease of antibody concentration was significantly greater in dialysis patients immunised with three doses of the vaccine. In dialysis patients vaccinated with five doses, the percentage losing HB antibodies was slightly higher than in controls, but final titres after 24 months were comparable.