Use of in vivo infected spleen cells to study Fv-2r-mediated resistance of mice to Friend spleen focus-forming virus.

In vivo infection of DBA/2 and D2.Fv-2r mice with 10(5) FFU of FV resulted in a rapid adsorption of virus onto the spleen cells. Thus, these cells could be used in the analysis of Fv-2 expression on FV target cells without any difficulty in avoiding extracellular FV particles. Higher frequencies of cells capable of forming IC could be detected in the spleen than in the bone marrow at 2 and 48 h post infection and levels were very low in D2.Fv-2r mice. When recently infected cells were transferred to secondary Fv-2s recipients shortly after infection had been established, the virus-replicating cells released SFFV equally well regardless of whether they originated from Fv-2s or Fv-2r mice. This suggests that suppression of SFFV replication may not take place in Fv-2r cells at an early stage of infection.

Evaluation of anti-HSV-2 activities of Barleria lupulina and Clinacanthus nutans.

Barleria lupulina Lindl. and Clinacanthus nutans (Burm. f.) Lindau, both belonging to the family Acantaceae, are well-known medicinal plants used in Thai folklore medicine. Virucidal effects of organic extracts of these two plants against herpes simplex virus type 2 strain G, HSV-2 (G), the standard HSV-2 strain were noted. The extracts were assessed for intracellular activities against HSV-2 (G) and five clinical HSV-2 isolates. B. lupulina extract exhibited activity against all five isolates but not the standard strain while that of C. nutans did not show any activity against these viruses as determined by plaque inhibition assay. When the activities were verified by yield reduction assay, anti-HSV-2 activities of B. lupulina extract were observed against HSV-2 (G) as well. The results suggest a therapeutic potential of B. lupulina but not C. nutans against HSV-2.

Prevalence of genital herpes simplex infection and abnormal vaginal cytology in late pregnancy in asymptomatic patients.

A study was undertaken to determine the prevalence of genital herpes simplex virus (HSV) infection in 809 asymptomatic, late pregnant women attending the antenatal clinic (ANC) at Ramathibodi Hospital. There was only one case where the cervico-vaginal swab specimen was positive for HSV by cultivation and the biotin-streptavidin enzyme-linked immunosorbent assay (B-SA ELISA), for detection of viral antigens. This gave a prevalence rate of 0.12%. The anti-HSV IgM in cord blood of the infant born to this mother was negative. The Papanicolaou (Pap) smear was performed in 554 cases, including the above patient, but none showed evidence of HSV infection. The high prevalence of lower genital tract infection in these subjects was noted in both Pap smear (31.05%) and wet preparation of the cervico-vaginal discharge (46.72%). Thus, examination of the cervico-vaginal discharge during late pregnancy should be of benefit to the patients, in revealing asymptomatic infections of the lower genital tract.

A sensitive assay for anti-HIV-1 drug discovery in a biological safety level-2 laboratory.

Studies involving infectious, wild type HIV-1 must be performed under strict BSL-3 practice. We have employed a defective (deltaTat/Rev)MC99 and cloned 1A2 line, ie, mutated HIV-1 and Tat/Rev transfected cells to verify anti-HIV-1 activity in a BSL-2 laboratory. A number of extracts from various parts of 11 species of plants were studied. Results were correlated with those of an anti-HIV-1 reverse transcriptase (RT) assay.

Encapsidation defectiveness of herpes simplex virus type 2 during replication at acid pH condition.

The maximal yield of herpes simplex virus type 2 (HSV-2) grown at pH 6.5 decreased 10(2)-10(3) fold compared to that recovered at pH 7.5. Electron microscopic observation of the infected cells maintained at these 2 pH conditions indicated that approximately equal amounts of immature virions were synthesized 6 hours after infection. However, at 18 hours post infection the majority of viruses present in the nucleus of infected cells maintained at pH 6.5 were empty or partially cored capsids with some particles enveloped and present in the cytoplasm, whereas at pH 7.5 mature virions already appeared at the cytoplasmic membrane. Analysis of viral polypeptides by radioimmunoprecipitation indicated that the synthesis of p40, a family of polypeptides closely involved in viral DNA encapsidation, was significantly impaired in infected cells maintained at pH 6.5.

Evaluation of biotin-streptavidin enzyme-linked immunosorbent assay for detection of genital herpes simplex virus infection.

A biotin-streptavidin enzyme-linked immunosorbent assay (B-SA ELISA) was evaluated for detection of herpes simplex virus (HSV) in clinical specimens which were cervico-vaginal swabs from 205 asymptomatic women and swabs from the genital lesions of 163 suspected patients. All specimens were also subjected to a conventional virus isolation in cell culture. A blocking B-SA ELISA had 100% specificity and 98% sensitivity compared with viral isolation from patients, but had only 40% sensitivity using specimens from asymptomatics. The conventional B-SA ELISA might also be used; it gave results corresponding to B-SA ELISA blocking test except for a single specimen which was considered a false positive.

Recovery frequencies of herpes simplex virus types 1 and 2 from symptomatic and asymptomatic genital herpes cases and antiviral sensitivities of isolates.

Herpes simplex viruses were isolated from 40.8 to 56.0 per cent of the patients with genital herpes. The frequency of recovery seemed to be higher in females than in males, particularly during the first episode of infection. Asymptomatic shedding of the virus from female genitalias was approximately 0.7 per cent. Herpes simplex virus type 2 represented 98.4 per cent of all isolates and the remaining isolates were type 1. These isolates exhibited a wide range of sensitiveness to 9-(2-hydroxyethoxymethyl) guanine (acyclovir) as demonstrated by antiviral inhibition assay and no single strain exhibited high in vitro drug ID50 value.

Epidemiological study of enteric coronavirus excretion by an enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay with a blocking test was used to determine the prevalence of enteric coronavirus excretion among the population of Bangkok and Khon Kaen province. The results indicated that coronaviruses were present in fecal samples from both children and adults with or without diarrhea. It is interesting that the viruses were more frequently observed in the apparently healthy individuals in Bangkok than in the patients with diarrhea whereas the excretion rates were approximately alike in both groups in Khon Kaen province. Among children aged less than 5 years (with or without diarrhea) the viruses were more frequently observed in stools from young children (1-5 years old) than from the newborn (less than 1-year-old) particularly those living in the Bang Khen slum area and in the rural Khon Kaen community. The rate of coronavirus excretion did not increase with age and some apparently healthy individuals continued to excrete the viruses for several months. The results also suggested that chronic parasitic infection associated with low socioeconomic status might influence the excretion of enteric coronaviruses in the feces.

Shut-down of virus synthesis during progression of erythroleukemia induced by Friend virus in mice.

Infection of (C57BL/6 X DBA/2)F1 (BDF1) mice with 2,500 FFU of Friend virus complex (FV) resulted in erythroleukemia followed by recovery, at which time virus could not be detected in the spleens of mice, and with splenomegaly (progressor mice) or without splenomegaly (regressor mice). Progressor and regressor mice developed equally high amounts of FV-neutralizing activity in their sera. Progressor mice contained cells capable of producing virus, despite the lack of viral envelope antigen(s) in their spleens. The tropism of FV recoverable from the spleens of secondary recipients, injected with spleen cells from progressor mice, did not show any change, although the viral genome was present in Fv-I-restrictive host for at least 7 weeks. When the number of spleen colony-generating cells was enumerated, by the spleen colony assay, the frequency in normal syngeneic and in allogeneic hosts was approximately the same at the early stage of erythroleukemia but was about 1,000 times higher in the syngeneic recipients during leukemia progression. These spleen colony-generating cells were considered to be FV-transformed cells capable of self-renewal and capable of generating infectious centers (IC), respectively. Most of these transformed cells might be non-producer leukemia cells.

Restriction endonuclease analysis of deoxyribonucleic acid (DNA) from genital herpes simplex virus type 2 isolates in Thailand.

Local herpes simplex virus (HSV) isolates were characterized by the use of BamHI, KpnI, and EcoRI restriction endonucleases. Analysis of 120 clinical isolates by observation of presence or absence of cleavage sites and of mobility variation in deoxyribonucleic acid (DNA) fragments from the terminal and joint regions of the HSV genome revealed that 112 (93.3%) were type-2 (HSV-2) and the others were type-1 (HSV-1), mixed HSV-2 and HSV-1, and mixed HSV-2 strains. The genomes of HSV-2 isolates were highly conserved and could be classified into 25 distinct restriction endonuclease (RE) cleavage patterns, which differed from those obtained with isolates of other countries.

Fv-2r-mediated resistance of mouse bone-marrow cells to Friend spleen focus-forming virus infecton.

A target cell assay based on the formation of infectious centers (IC) was used to characterize the Fv-2resistance gene of mice. Dose-response curves were obtained for the number of IC generated from incubated mixtures of bone-marrow cells and Friend virus (FV). The hemopoietic stimulus of bleeding increased the frequency of potential "target" cells capable of forming IC in DBA/2 but not in D2.Fv-2r mice; however, even without the bleeding stimulus D2.Fv-2r mice contained fewer target cells in their bone marrow than DBA/2 mice. During the first 48 h postinfection a massive dose of FV overcame the inhibitory effect of the FV-2r gene as measured by the release of FV from the spleen. However, the concentration of IC recoverable from the spleens of these infected mice was still much lower in the D2.Fv-2r strain. When irradiated F1 hybrid hosts were used as recipients for the proliferation of infected donor cells, splenic IC were not generated as efficiently from infected bone-marrow cells of D2.Fv-2r origin as from those of DBA/2 origin. However, the amounts of SFFV recoverable were approximately the same. Similarly, after in vitro infection there was no great difference between the amounts of SFFV released from bone-marrow cells of DBA/2 and D2.Fv-2r mice. We conclude that the primary effect of the Fv-2r gene may be to inhibit the formation and proliferation of IC in vivo, and that SFFV replication could be inhibited secondarily.

Intratypic variations in neutralizable epitopes among herpes simplex virus type 2 isolates.

Intratypic variation among 94 isolates of herpes simplex virus type 2 (HSV-2) was investigated using 4 different monoclonal antibodies (MAbs). By neutralization test, these MAbs appeared to be directed to at least 2 distinct epitopes on the viral glycoprotein D (gD), i.e., 6G6.G9 and 6E8.F11 which did not require complement (C-MAb) and gD-105 and gD-110 whose neutralizing activities could be enhanced by complement (C+MAb). The C-MAb pairs each separately could detect significant intratypic variations among the isolates. Whether these variations also existed in the gD epitope(s) recognized by C+MAbs remains to be elucidated. The results suggested that intratypic variation occurred on at least one of the neutralizable (thus related to protective immunity) epitopes on gD of HSV-2.

Intratypic variation of herpes simplex virus type 2 isolates detected by monoclonal antibodies against viral glycoproteins.

Monoclonal antibodies (MAbs) to herpes simplex virus (HSV) glycoproteins gD, gG, gB, and gE were used to analyze antigenic variations of 128 genital HSV-2 isolates by an indirect enzyme-linked immunosorbent assay (ELISA). Isolates were considered significantly different from the standard HSV-2 strain 186 when their optical density (OD) in ELISA was less than half that of strain 186. This criterion gave 30 patterns of reactivity among the genital HSV-2 isolates. The MAbs to gB, gG, and 2 of the gD antibodies reacted with more than 90% of the isolates, suggesting that these MAbs recognized highly conserved epitopes. However, the gE MAb reacted with only 47% of the isolates, and one of the gD antibodies with only 39%. Thus, HSV-2 can readily tolerate modifications in some parts of the gD and gE molecules while remaining infectious.

Rapid detection of herpes simplex virus DNA by in situ hybridization with photobiotin-labelled double-stranded DNA probes.

An assay for rapid detection of herpes simplex virus in infected cells is described. The assay utilizes in situ hybridization with photobiotin-labelled double-stranded DNA probes prepared from HSV-1 DNA cloned in plasmid vectors. The assay provided an alternative method for earlier detection of virus in cell cultures with the ease of preparation of photobiotin-labelled double-stranded DNA.

Specific neutralization of defective spleen focus-forming virus in Friend virus complex by rat antiserum.

The spleen focus-forming virus (SFFV), a rapidly transforming, replication-defective virus in Friend virus (FV) complex that is readily neutralized by antisera directed against its helper virus, was examined for the presence of SFFV-specific antigens. Antisera prepared in Fisher rats against an SFFV-infected Fisher rat embryo fibroblast line (SFFV-FRE) neutralized SFFV effectively, but not Friend-associated murine leukemia virus (F-MuLV) whether the latter was tested alone or was mixed with SFFV in the FV complex. In contrast, serum from mice immunized with SFFV-infected nonproducer mouse cells had little or no neutralizing activity against SFFV. Both absorption and immunoprecipitation studies indicate that the SFFV-specific antigen is immunologically related to xenotropic murine leukemia virus antigens. The role of both SFFV- and F-MuLV-specific antigens in the neutralization of SFFV suggests that this defective virus could be an antigenic mosaic and that viruses in the FV complex may participate in a undirectional form of phenotypic mixing.

Anti-herpes simplex virus activities of crude water extracts of Thai medicinal plants.

A number of Thai medicinal plants, recommended as remedies for herpesvirus infection and have been used in primary health care were investigated for their intracellular activities against herpes simplex viruses (HSV). Centella asiatica L., Maclura cochinchinensis Cornor, and Mangifera indica L. contained both anti-HSV-1 and -2 activities, as determined by plaque inhibition assay. An inhibition of the production of infectious HSV-2 virions from infected Vero cells could also be demonstrated. Combinations of each of these reconstituted extracts with 9-(2-hydroxyethoxymethyl) guanosine (acyclovir; ACV) resulted either in subadditive, additive, or synergistic interaction, against HSV-2, depending on the dose of ACV used; mixture of C. asiatica and M. indica exerted an additive effect in a similar assay. Furthermore, the inhibitory effects of these plant extracts were also substantiated by flow cytometric analysis of virus-specific antigens in the infected cells. The active constituent present in C. asiatica extract was determined to be asiaticoside while in M. indica was mangiferin. Thus, these data suggest therapeutic potential of these plant extracts.

Detection of herpes simplex virus type 2 Bgl II N fragment in paraffin-embedded cervical tissue sections using nested polymerase chain reaction.

To study the presence of transforming sequence Bgl II N of HSV-2 in cervical cancer tissues, we developed the nested polymerase chain reaction (PCR) for detecting such a sequence in paraffin-embedded cervical tissue sections. Samples derived from 46 patients with premalignant and malignant lesions were tested. The sequence was found in 20-25% of total cases tested but not observed in any of the normal healthy controls. This study also indicates that for the detection of HSV-2 Bgl II N sequence in cervical tissue, the nested PCR may be more reliable than the in situ hybridization method.

Anti-herpes simplex virus component isolated from Maclura cochinchinensis.

The powerful anti-herpes simplex virus (HSV) activity of Maclura cochinchinensis in vitro prompted us to carry out biologically-guided separation of the active component(s). Ethyl acetate and methanol extracts exhibited anti-HSV-2 activity at EC50 values of 38.5 micrograms/ml and 50.8 micrograms/ml, respectively. Although petroleum ether extract was inactive, a chloroform extract was too toxic to the test cell culture to perform the test. Biologically-guided chromatographic separation of the ethyl acetate extract yielded compound A, identified as morin using a spectroscopic method. Morin exhibited anti-HSV-2 activity at an EC50 value of 53.5 micrograms/ml. Morin pentaacetate was synthetized, but was inactive. This result suggested that free hydroxyl groups are required for anti-HSV-activity, as demonstrated previously for the antiviral activity of other flavonoids.

2-substituted furans from Polyalthia suberosa.

Two new 2-substituted furans, 1-(2-furyl)pentacosa-16,18-diyne and 23-(2-furyl)tricosa-5,7-diynoic acid, have been isolated from stems of Polyalthia suberosa. The structures were assigned by spectroscopic methods. The two compounds together with the previously reported kalasinamide, N-trans-feruloyltyramine and N-trans-coumaroyltyramine showed anti-HIV activities.