We describe a technique for the systematic inactivation of nonessential genes within the genome of a herpesvirus without the requirement for phenotypic selection. This technique is based on the insertion of an oligonucleotide containing translational stop codons at a random site within a large cloned viral DNA fragment. Mutant virus is then reconstituted by cotransfection with overlapping viral clones, together comprising the entire viral genome, as described previously (M.
Pseudorabies virus (PRV) expressing the envelope glycoprotein E1 (E1) of hog cholera virus (HCV) was used as a model to study the potential risks connected with the use of a live herpesvirus vaccine expressing a foreign gene. The gene encoding E1 was inserted into the glycoprotein X (gX) locus of both a virulent PRV strain and a non-virulent PRV strain in which the virulence genes encoding glycoprotein I (gI) and thymidine kinase (TK) had been inactivated. We investigated whether strain M205 (gI-, TK-, gX-, E1+) had a changed cell or host tropism or virulence compared with strain M206 (gI-, TK-, gX-) in pigs, rabbits, hamsters, rats, mice and rhesus monkeys. The insertion of E1 into this non-virulent PRV strain caused no change in cell or host tropism. However, pigs inoculated with M205 shed less virus over a shorter period than pigs inoculated with M206. Theoretically, virulent PRV strains expressing E1 (gX-, E1+) could arise through transfer of the E1 gene of M205 to a virulent PRV strain. Therefore, we inoculated pigs with strain M12 (gX-, E1+) or the control strain M104 (gX-) and compared the virulence and pathogenesis. M12 and M104 were of approximately equal virulence and the pathogenesis of both strains was similar. We concluded that incorporating E1 of HCV into the gX locus of PRV did not change cell or host tropism, nor did it change the virulence of either non-virulent or virulent PRV.
Fragments of glycoprotein G (gG-2 281-594His ), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1 t26-189His ), and glycoprotein D of HSV-1 (gD-1 1-313 ), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1 t26-189His and gG-2 281-594His fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1 t26-189His and the fragment of gG-2 281-594His were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1 t26-189His , sera positive for HSV-2 reacted with the gG-2 281-594His fragment, and sera positive for both types reacted with gG-1 t26-189His and gG-2 281-594His in Western blotting. The human sera recognized polypeptides of gG-2 281-594His with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1 t26-189His and the recombinant gG-2 281-594His fragment were used as type-specific antigens for the detection of HSV-1-and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1 1-313 were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2 281-594His fragment can be obtained in relatively large quantities at low cost.Herpes simplex virus type 1 (HSV-1) and HSV-2 are well known as similar subtypes of HSV (38). HSV-2 is the main cause of recurrent genital infections (8). Most primary infections are asymptomatic, and people silently shed virus (14,30,51,57,58). After primary infection, the virus establishes latent infections in the local ganglia and is reactivated frequently, and antibody titers against HSV become detectable in serum samples. Many of the HSV-2 reactivations are asymptomatic and clinically not recognized (15,30,39,40,57). Most HSV infections are transmitted in the absence of lesions (17,57,58). Severe, generalized infections are seen particularly in neonates and immunocompromised (human immunodeficiency virus-infected) patients. Epidemiological studies indicate that in developed countries there is an ongoing HSV-2 epidemic with a significant rise in HSV-2 prevalence. Risk factors for acquiring HSV-2 infections are related to sexual life style, gender, race, and socioeconomic status. HSV type-specific antibody testing may be considered in a number of at-risk situations. Thus, identification of HSV-2-infected individuals may be important to reduce the risk of transmission in these groups.Laboratory diagnosis of HSV infections is based on direct detection of the virus and on serology (2, 16). Viru...
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