Enterovirus infections with special reference to enterovirus 71.

The enteroviruses comprise a large group of immunologically distinct serotypes of viruses belonging to the family of Picornaviridae. Many enteroviruses cause diseases in human, but the infections are generally mild as asymptomatic, therefore, enteroviruses are considered to be unimportant as human pathogens. However, enteroviruses may also result in serious or even fatal disease (as shown in the enterovirus 71 (EV71) epidemic in Taiwan in 1998). There are three types of polioviruses, Coxsackievirus group A and group B viruses, and echoviruses group. All together a total of 67 types are available. Starting from enterovirus type 68 to 71, they are named as enterovirus types. Enterovirus type 72 is hepatitis A virus. Paralytic disease of poliomyelitis was recorded in ancient time but characterization of poliovirus was not reported until the turn of the 19th century that poliomyelitis was a viral disease. The major breakthrough for diagnosing and controlling of poliomyelitis was the discovery that poliovirus can be propagated in human embryonic tissues in cultures. As soon as cultures of human and monkey cells began to use for isolating polioviruses in stool specimen of patients, more unknown viruses were isolated which unlike polioviruses nor Coxsackie viruses; they were called "orphan" viruses or human enteric viruses, name later simplified to "echoviruses". Morphologically all enteroviruses are alike. They are small, ether insensitive viruses with an RNA genome. Their nucleic acid is single stranded, and the nucleocapsid has a cubic (icosahedral) symmetry, and is naked. The host ranges of enteroviruses vary greatly from one type to the next and even among strains of the same type. Polioviruses have a very restricted host range among laboratory animals. Virus isolation is the best method for diagnosis of enterovirus infection, but infection in the central nervous system (CNS) may be detected by polymerase chain reaction (PCR). Currently final identification and serotyping of enteroviruses are by indirect immunofluorescent tests using monoclonal antibody or by neutralization test using antiserum pools described by Lim and Benyesh-Melnick. The incidence and prevalence of diseases associated with the enterovirus infections are varied. The circulation of enteroviruses recently in Tainan and the epidemic of EV71 in Taiwan in 1998 are described in this review. Although poliovirus infection may be eradicated from the world due to the efficient vaccination program, there is no specific antiviral agents for either treatment or prevention for other enterovirus infections. In 1991, a new antiviral "pleconaril" which is a novel orally bioavailable and systematically acting small molecule inhibitor for picornaviruses. "Pleconaril" is currently in clinical trials for treatment of enterovirus meningitis and respiratory infections.

Characterization of conditions for the activation of endogenous guinea pig retrovirus in cultured cells by 5-bromo-2'-deoxyuridine.

Human endogenous retroviral sequences recently have been shown to be associated with breast cancer and some leukemias. These retroviral sequences have similarities to an endogenous retrovirus expressed in guinea pigs. The conditions for activation of this guinea pig retrovirus (GPRV) in cultured guinea pig embryo (GPE) cells using 5-bromo-2'-deoxyuridine (BrdU) was investigated. These studies employed the reverse transcriptase activity (RT) assay and electron microscopy (EM), in conjunction with Northern blot analysis that utilized a 2.6 kb GPRV-specific cDNA probe. Contrary to published studies, dexamethasone at concentrations ranging from 10(-8) to 10(-5) M appeared to play a minimal role in enhancing the production of GPRV. Following a 6 hr incubation with BrdU, GPRV mRNA was present in cultured GPE cells. Extracellular virion release was also observed by EM 12 hr later, although RT activity was not detected. All three methods detected viral expression at 48 hr after the addition of the drug. Additionally, after 6 hr exposure to BrdU, detectable RT and mRNA levels were maintained through 44 days after the removal of BrdU in a stationary culture condition and through 31 days in cultures that were subcultured weekly in media not containing BrdU. Low levels of extracellular viruses were detected in these cultures by electron microscopy through 49 days. Therefore, after only a 6 hr exposure to BrdU was extracellular GPRV detected 12 hr after drug removal and virus production continued for up to 49 days. This study provides information about an animal endogenous retroviral system that may be used as a model for the study of human endogenous retroviruses.

Comparison of mitochondrial morphology, mitochondrial DNA content, and cell viability in cultured cells treated with three anti-human immunodeficiency virus dideoxynucleosides.

The toxic effects of various concentrations of 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), and 2',3'-dideoxyinosine (ddI) on CEM cells after 4 days of culture were assessed by measuring cell viability, mitochondrial DNA (mtDNA) content, and mitochondrial morphology. Cell viability and mtDNA content in drug-treated cultures were significantly reduced in a concentration-dependent fashion in comparison with cell viability and mtDNA content in untreated cultures. Cells in the treated cultures also showed significant changes in their mitochondrial morphologies which included distortion and reduction of the cristae and numerous vesicles. Unique features of the morphological changes were associated with each drug. The decrease in cell viability and mtDNA content and the increase in mitochondrial ultrastructural changes were directly related to the concentrations of the drugs used. The potencies of these compounds in reducing cell viability, mtDNA content, and normal mitochondria were in the order ddC > D4T > ddI. Comparison of the three assays used demonstrated that mtDNA content is a significantly more sensitive measure of drug toxicity than cell viability and mitochondrial morphology for the three compounds studied.

Isolation of St. Louis encephalitis virus from a killer whale.

We report the isolation of St. Louis Encephalitis (SLE) virus from a mature male killer whale (Orcinus orca). This represents the first isolation of SLE virus from a marine mammal. The animal presented with reduced appetite, rapidly became lethargic and subsequently died. Virus-induced CPE was observed in a dolphin cell line, SP-1K (ATCC CCL 78), inoculated with brain, kidney, and lung tissues obtained at necropsy. Electron microscopy of infected SP-1K cells revealed the presence of virions having morphology and size resembling members of the Flaviviridae. Final identification as SLE virus was made by neutralization and immunofluorescence staining tests.

Interactions between HIV-1 and cytomegalovirus in human osteosarcoma cells carrying both viruses.

Cytomegalovirus (CMV) and the human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. We compared CMV replication in human osteosarcoma (HOS) cells to that in HOS cells genetically engineered to contain an envelope-deficient HIV-1 proviral construct (designated HOS-HXG). Following acute CMV infection of each cell line, HOS-HXG cells contained higher numbers of intranuclear CMV nucleocapsids than did HOS cells. Infectious CMV could be persistently detected in culture supernatant fluids of the CMV-infected HOS-HXG cells, whereas CMV was lost over several weeks from HOS cells infected with CMV in parallel. HIV-1 CMV pseudotypes were not detected in supernatant fluids from CMV-infected HOS-HXG cells. On day 119 after CMV infection, these cultures were superinfected with HIV-1. These dually infected HOS-HXG cells produced infectious HIV-1 and exhibited markedly enhanced CMV replication compared to parental CMV-infected HOS-HXG cells. Two different HIV-1 tat gene function antagonists, Ro24-7429 and chemically modified antibodies to the Tat protein, did not inhibit the replication of CMV in either acute or persistent infections of HOS-HXG cells at concentrations that inhibited HIV-1 replication.

Combined treatment with 2'-nor-cGMP and ganciclovir against cytomegalovirus infection in a guinea pig model.

The combination 2'-nor-cGMP/DHPG at fixed ratios 1:5, 1:10 and 1:20 showed synergistic antiviral effects against GPCMV replication in vitro with CI value < 1. In vivo, a fixed ratio of 1:10 at three different dosage levels of 1.25/12.5 mg, 2.5/25 mg and 5/50 mg/kg/day 2'-nor-cGMP/DHPG combination showed only additive results when compared with each drug alone. However, synergistic antiviral effects were obtained when infected guinea pigs were treated with 2'-nor-cGMP/DHPG combination 2.5/10 mg/kg/day (1:4). A significantly lower GPCMV infectivity titer was noted in the salivary gland, lung and spleen of infected guinea pigs treated with the combination of 2'-nor-cGMP/DHPG 2.5/10 mg/kg/day, as compared to animals treated with a corresponding dose of each drug alone. In addition, GPCMV-infected animals treated with the latter combination showed increased body weight than when either drug was used alone. Histopathologically, each drug alone reduced the viral induced changes in the lung and spleen, but the combination therapy reduced these changes still further. Toxic changes seen in the kidney and bone marrow of infected animals treated with 2'-nor-cGMP, 2.5 mg/kg/day were not significantly increased when DHPG 10 mg/kg/day was added to the regimen. Therefore, combined treatment with 2'-nor-cGMP/DHPG in appropriate concentration is more helpful for acute cytomegalovirus infection in guinea pigs than when either drug was used alone.

Ganciclovir antagonizes the anti-human immunodeficiency virus type 1 activity of zidovudine and didanosine in vitro.

In studies examining potential interactions between ganciclovir (GCV) and either zidovudine (AZT) or didanosine (DDI) in H9 cells, GCV was found to consistently reduce the anti-human immunodeficiency virus type 1 potency of both AZT and DDI. In the presence of GCV, the 50% effective doses of AZT and DDI were increased three- to sixfold, depending on the molar ratio of drugs and the measure of human immunodeficiency virus type 1 replication (p24 antigen, reverse transcriptase activity, or infectious virus yield). Multiple dose-effect analysis revealed strong antagonism between GCV and either AZT or DDI (combination indices, 2.2 to 6.7). This antagonistic effect occurred at drug concentrations that were well below the cytotoxic range. At higher drug concentrations, the combination of GCV and AZT was synergistically cytotoxic (combination indices, less than 1.0), whereas GCV and DDI were only additively cytotoxic (combination indices, ca. 1.0). Thus, the combination of GCV with AZT or DDI may result in antiviral antagonism and either synergistic (AZT-GCV) or additive (DDI-GCV) cytotoxicity.

Demonstration of cytomegalovirus and retrovirus pseudotypes in cultured guinea pig cells.

A mixed viral infection with a cytomegalovirus and a retrovirus in cultured guinea pig embryo (GPE) cells was investigated. The expression of an endogenous guinea pig retrovirus (GPRV) in cultured guinea pig cells was induced by a medium containing 5-bromo-2'-deoxyuridine and dexamethasone. When the induced GPE cells were superinfected with a guinea pig cytomegalovirus (GPCMV), pseudotype virions were observed. Morphological characterization of both viruses and their locations within infected cells was achieved by examination of thin sections of infected cells with transmission electron microscopy. Immunolabeling with colloidal gold particles, 5 or 15 nm in size, permitted the identification of each virus type using GPCMV- or GPRV-specific polyclonal antibodies and the detection of a population of GPCMV and GPRV particles which expressed antigens of both viruses on their envelopes. Enhanced reverse transcriptase activity of GPRV and reduced infectivity titers of GPCMV was noted in dually infected cultures. These data suggest that interaction between GPCMV and GPRV had occurred in dually infected GPE cells and that expression of GPRV was enhanced.

Comparative effects of two acycleonucleoside derivatives on ultrastructure and replication of guinea pig cytomegalovirus in cultured cells.

Two antiviral agents, compound 164, also known as 2'-nor-cGMP, 9-[(2-hydroxy-1-3-2-dioxophosphorinan-5-yl)-oxymethyl]-guani ne P-oxide, and compound 102, 4-amino-5-bromo-7-(2-hydroxyethoxymethyl)-pyrrolo-(2-3-d)-pyrimidine, together with their parental drugs, 2-deoxyguanosine (DHPG) and acyclovir (ACV), were evaluated for their antiviral activities against guinea pig cytomegalovirus (GPCMV) infection in cultured guinea pig embryo (GPE) cells. The two compounds were demonstrated to be more potent against GPCMV replication when compared to their parental drugs DHPG and ACV. Compound 164 was the most potent of the four compounds tested, with ED50 values being approximately 20-, 80-, and 180-fold more potent than those of DHPG, compound 102, and ACV, respectively. Compounds 164 and 102 were slightly more cytotoxic to uninfected GPE cells, but their selectivity (therapeutic) indexes were higher than those of their parental drugs. Of the four antiviral agents tested, compound 164 had the highest selectivity index which was 8-, 30-, and 50-fold higher than that of DHPG, compound 102, and ACV, respectively. Ultrastructural studies suggest that compounds 164 and 102 have different mechanisms of inhibiting GPCMV replication in cultured GPE cells.

Combined antiviral effects of paired nucleosides against guinea pig cytomegalovirus replication in vitro.

Several promising antiviral nucleosides have been tested in paired combinations against guinea pig cytomegalovirus (GPCMV) replication in guinea pig embryo (GPE) cells by plaque reduction assay; these are [9-(2-hydroxy-1-3-2-dioxaphosphorinan-5-yl)oxymethyl]-guanin e P-oxide (2'nor-cGMP, compound 164), [4-amino-5-bromo-7-(2-hydroxyethoxymethyl)-pyrrolo(2,3-d)pyrimidine] (compound 102), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV) and 3'-azido-3'-deoxythymidine (zidovudine, AZT). Various degrees of interactions were observed; i.e. synergistic reactions were noted in the presence of compound 164/compound 102 and compound 164/DHPG combinations at all concentrations tested. HPMPC/DHPG combinations were synergistic at relatively lower concentrations of DHPG, but became antagonistic as the concentration of DHPG increased. Combinations of compound 164/ACV and DHPG/AZT were antagonistic.

Activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) against guinea pig cytomegalovirus infection in cultured cells and in guinea pigs.

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC, and two HPMPC-related nucleoside analogs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, HPMPA, and (2-phosphonylmethoxyethyl)guanine, PMEG, were evaluated for their antiviral activities against guinea pig cytomegalovirus (GPCMV) infection in guinea pig embryo (GPE) cells and human cytomegalovirus (HCMV) infection in human diploid fibroblast (MRC-5) cells. DHPG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, was used for comparison. The antiviral activity of HPMPC against GPCMV infection in vivo and its toxicity to Hartley guinea pigs were also evaluated. The 50% antiviral effective doses (ED50) of HPMPC, HPMPA, PMEG and DHPG against GPCMV infection in GPE cells were 0.22, 1.4, 0.07 and 62 microM, respectively; and against HCMV infection in MRC-5 cells, the ED50s were 0.51, 0.72, 0.01 and 17.5 microM, respectively. Their cytotoxic doses (CyD50) in GPE replicating cells were 84, 35, 1.4 and 700 microM, respectively and in MRC-5 cells were approximately 114, 31, 0.86 and 750 microM, respectively. Based on their calculated therapeutic indexes, HPMPC was the most potent and selective of the four compounds tested. In vivo, during acute infection, the spleen indexes of all infected animals that were treated with 1.25 to 5.0 mg/kg/day of HPMPC for 5 days were significantly reduced as compared with sham-treated animals. Virus infectivity titers in blood and various tissues of infected animals treated with HPMPC, 2.5 or 1.25 mg/kg/day were not significantly lower than those of the infected, sham-treated animals; with 5 mg/kg/day, infectivity titers in the blood, spleen, and salivary gland were significantly lower in HPMPC-treated than in sham-treated animals. However, HPMPC was toxic to guinea pigs especially at doses of 5 to 10 mg/kg/day. These data showed that HPMPC was highly active and selective in cultured guinea pig cells and human fibroblast cells against CMV infection but did not effectively inhibit GPCMV infection in guinea pigs at minimum toxic concentrations.

Evaluation of new antiviral agents: II. The use of animal models.

Antiviral chemotherapy has become a reality in the 1980s. Since the use of animal models in the testing of new antiviral agents is an inevitable step prior to clinical trial in human patients, it is important to understand the basic principles of using model systems. Briefly reviewed in this paper are the heterologous and homologous animal models which have been used for studies of various herpesvirus infections in humans. Discussions of the use of the guinea pig models mainly, for members of the Herpesviridae are presented in more detail. Precautions needed for the development of new animal models, and suggestions proposed for the use of animal models for testing new antiviral agents are outlined. It is hoped that new animal models will be developed in the foreseeable future for evaluating the much needed effective but less toxic antiviral agents for a variety of human viral diseases.

Effect of 2'-nor-cyclic GMP against guinea pig cytomegalovirus infection.

Cyclic phosphate derivative of DHPG, 2'-nor-cGMP [9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]-guani ne phosphate-oxide] was evaluated for activity against guinea pig cytomegalovirus (GPCMV) infection in cultured guinea pig embryo cells and in guinea pigs. By virus yield reduction and plaque reduction assays, 2'-nor-cGMP was demonstrated to be 15- to 20-fold more potent against GPCMV infection than its parental drug DHPG. The selectivity index of 2-nor-cGMP was 110, which was 10-fold higher than that of DHPG. In cultured cells, 2'-nor-cGMP attained maximal antiviral activity when added to the cells within 12 h postinfection. In the studies on GPCMV infection in guinea pigs, 2'-nor-cGMP administered subcutaneously once daily (5 mg/kg per day) for 8 days, starting 24 after virus inoculation, significantly suppressed GPCMV infectivity titers in the blood, spleen, lung, and salivary gland during acute infection (10 days postinfection) as compared with sham-treated infected animals. A greater reduction of GPCMV infectivity titers in the salivary gland was noted during chronic infection (i.e., 24 days postinfection). Clinically, splenomegaly and peripheral lymphocytosis were significantly modified as compared with the sham-treated animals (P less than 0.05). The drug, administered at this dosage, was reasonably tolerated by the guinea pigs and showed clinical benefit.

Activities of two new antiviral agents against guinea pig lymphotropic herpesvirus infection in vitro.

Two new antiviral agents, compound 164, also known as 2'-nor-cGMP (9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]-guani ne P-oxide), and compound 102 [4-amino-5-bromo-7-(2-hydroxyethoxymethyl)-pyrrolo(2,3-d)pyrimidine], together with acyclovir for comparison, were evaluated for activities against the guinea pig lymphotropic herpesvirus infection in vitro by plaque reduction and virus yield reduction assays in guinea pig embryo cells. The two new compounds were demonstrated to be more potent against guinea pig lymphotropic herpesvirus infections than acyclovir. Compound 164 was the most potent of the three; drug concentrations required to reduce the number of plaques by 50% were 2, 35.5, and 144.5 microM for compounds 164, 102, and acyclovir, respectively. The two new compounds were cytostatic but not cytotoxic to guinea pig embryo cells in cultures. Attempts were made to investigate the inhibition of viral replication by these compounds, and the influence of test conditions on antiviral evaluations is discussed.

Evaluation of new antiviral agents: I. In vitro perspectives.

A variety of different methods for the evaluation of antiviral agents in cell culture systems are briefly reviewed. It has been repeatedly noted that many test conditions such as the cell culture system, virus strain, virus challenge dose, virus input multiplicity of infection, and time of harvesting, etc., can substantially affect or even alter the test results, thus making comparative studies and unambiguous evaluations very difficult. Attempts are made to discuss previous test methods together with our recent studies with the aim to simplify test procedures and assay methods. Suggestions are proposed for in vitro evaluation of new antiviral agents. It is hoped that this review will alarm investigators to the problems of assaying new antiviral agents. If the suggestions made in this review can be followed, the screening of the enormous number of promising antiviral compounds may be made more efficiently in the near future.

Dual infection of retina with human immunodeficiency virus type 1 and cytomegalovirus.

We examined retinal tissue from eight human immunodeficiency virus type 1 (HIV-1) seropositive patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex for evidence of dual infection with HIV-1 and cytomegalovirus. Culture demonstrated simultaneous infection with HIV-1 and cytomegalovirus in two of 13 retinal specimens. This was confirmed by both immunofluorescence and immunohistochemical staining. Moreover, coinfection of individual cells with cytomegalovirus and HIV-1 was observed by immunohistochemical staining. Infection of retina with cytomegalovirus or HIV-1 alone occurred in one and six of the 13 retinal specimens, respectively. HIV-1 antigens were present on scattered cells in all layers of the retina and on retinal vascular endothelium. HIV-1 was isolated from retinal tissue derived from eyes both with and without gross ocular lesions. Cytomegalovirus antigens were found in all layers of the retina, but not on vascular endothelial cells. The atypically rapid clinical progression of retinitis in one of the patients with dual HIV-1 and cytomegalovirus infection suggests the possibility that interactions between these two viruses may influence retinal disease in patients with AIDS.