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Isolation and characterization of acyclovir-resistant strains of herpes simplex virus.

A number of clinical studies have documented herpes simplex infections which appear to be resistant to nucleoside analogs; these include idoxuridine [1,2] and acyclovir [3]. Few, if any of the viruses isolated from such patients have yet been thoroughly characterized. We have isolated a number of acyclovir-resistant mutants by selection for resistance in tissue culture. The study of the biochemical and biological properties of these mutants has given some insight into the likely nature of resistant clinical strains. We have devised a number of simple tests to allow classification of laboratory mutants. We also draw attention to some of the difficulties the clinical virologist may encounter when analyzing putative resistant virus isolated from treated patients.

Acyclovir↗

Systematic classification of HIV biological subtypes on lymphocytes and monocytes/macrophages.

The growth properties and cytopathic effects of several HIV-1 and HIV-2 strains were compared between cultures on human lymphocytes and monocytes/macrophages, respectively. For some isolates (among these three paired isolates from blood and cerebrospinal fluid) replication and cytopathogenicity were comparable between lymphocytes and monocytes/macrophages (dual tropic viruses), while others showed a very specific tropism for only one cell type. Yet another subtype grew neither well on lymphocytes nor on macrophages. Taking into account the growth properties in monocytes/macrophages we propose a classification system for HIV subtypes on these cells (alpha-delta), in analogy to the nomenclature for HIV-subtyping on lymphocytes (a-d). Using this system, some prototypic viruses (LAV/HTLV-IIIB, HIV-2ROD, SIVBK28, HIV-2ALT) as well as several other HIV-1 and HIV-2 isolates were subtyped.

Cells, Cultured↗

Isolation and characterization of a novel single-stranded RNA Virus infectious to a marine fungoid protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea).

Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 x 10(3) to 6.4 x 10(4) infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms.

Animals↗

Vaginal infections in human immunodeficiency virus-infected women.

OBJECTIVE: This study was undertaken to compare the frequencies of vaginal infections among human immunodeficiency virus-infected women with those among human immunodeficiency virus-seronegative women. STUDY DESIGN: Human immunodeficiency virus-seropositive women attending a comprehensive care center for human immunodeficiency virus disease at the outpatient department of an inner-city hospital in Houston underwent rigorous gynecologic evaluation for sexually transmitted diseases, including evidence of vaginal infections such as bacterial vaginosis, vulvovaginal candidiasis, and trichomonal vaginitis. Demographic information was collected, as was information regarding disease classification and degree of immunosuppression. Data regarding sexually transmitted diseases, data regarding vaginal infections, and demographic information were collected from a cohort of human immunodeficiency virus-seronegative women attending a sexually transmitted disease and family planning clinic at the same institution. The two groups were compared to determine whether there were any differences between them in the frequencies of sexually transmitted diseases and vaginal infection. Data analysis used the t test for parametric data and the Fisher exact test for nonparametric data where appropriate. P <.05 was considered significant. Statistical analysis was performed with the SAS (SAS Institute, Inc, Cary, NC) statistical software package. RESULTS: There was no difference in age between the 2 groups. The human immunodeficiency virus-infected group was predominantly African American (80.5%), whereas the human immunodeficiency virus-seronegative control group was more evenly divided between African American women (45.8%) and Latinos (41.7%; P <.0001). Although there was no significant association between HIV infection and sexually transmitted diseases in general, there were significant associations between human immunodeficiency virus infection and bacterial vaginosis (P =.02), vulvovaginal candidiasis (P =.001), and trichomonal vaginitis (P =. 003). CONCLUSION: Human immunodeficiency virus-infected women had increased frequencies of vaginal infections, including bacterial vaginosis, vulvovaginal candidiasis, and trichomonal vaginitis, with respect to human immunodeficiency virus-seronegative control subjects. No differences between the 2 groups were seen in the frequencies of sexually transmitted diseases.

Adult↗

Possible members of the potyvirus group transmitted by mites or whiteflies share epitopes with aphid-transmitted definitive members of the group.

There are at least ten viruses identified in the literature that resemble definitive potyviruses in having flexuous filamentous particles and inducing the formation of "pinwheel" cytoplasmic inclusions in infected cells but that are transmitted by eriophyid mites, whiteflies or soil fungi and not by aphids, the vectors of the definitive potyviruses. The taxonomic status of these viruses is uncertain at present. Using a broadly cross-reactive antiserum raised against the dissociated coat protein core (residues 68-285) of a definitive potyvirus (Johnsongrass mosaic virus), we have shown that wheat streak mosaic virus which is transmitted by mite and sweet potato mild mottle virus which is transmitted by whitefly have coat proteins that share epitopes with definitive potyviruses. This finding further supports their classification as definitive members of the potyvirus group. The cross-reactive antiserum used here had been shown previously to react with coat proteins of fifteen different definitive potyviruses. The antiserum did not react with coat proteins of potexviruses and tobamoviruses.

Animals↗

Direct sequencing of genomic cDNA fragments amplified by the polymerase chain reaction for molecular epidemiology of dengue-2 viruses.

A nucleotide fragment encoding amino acids 29 to 94 in the E-protein of 28 dengue-2 isolates of diverse geographic and host origins was examined by direct sequencing of a polymerase chain reaction (PCR)-amplified product, and compared to six previously published sequences. Nucleotide divergence ranged from 0 to 19.8% corresponding to a maximum of 9% divergence in the amino acid sequence. Taking a divergence of 6% between the nucleotide sequence as a cut off for genotype classification, six groups have been established. Southeast Asian and the Jamaican 1983 genotypes show a high rate of similarity (> 95.2%). Our results suggest that virus of this group is now circulating as the dominant topotype in Brazil (1990) and in French Guyana (1986-1991). African strains fall into two groups, one endemic group (1970-1990) and one epidemic group (1986-1987). The three other groups correspond to viruses from Sri Lanka (1982) and the Seychelles (1977), from Puerto Rico (1973) and from Tahiti (1975). Our approach appears to be valuable characterizing dengue isolates, easily and rapidly.

Aedes↗

Classification and management of necrotising vasculitides.

Systemic vasculitides are a heterogeneous group of diseases. Having only a partial understanding of the aetiologies and pathogenetic mechanisms of these disorders explains the difficulties encountered in classifying and treating patients. Nevertheless, some important points have been established. Classification is mainly based on the size of vessels affected and, from the polyarteritis nodosa group, microscopic polyangiitis (MPA) has been separated from classic polyarteritis nodosa (c-PAN). The latter is a rare disease which is, in a small number of cases, the consequence of hepatitis B or C virus (HBV/HCV) infection. In the other cases of c-PAN and in MPA, the aetiology is unknown as for Churg-Strauss syndrome (CSS) and Wegener's granulomatosis (WG). MPA, CSS and WG are mainly antineutrophil cytoplasmic antibodies (ANCA)-related vasculitides. ANCA play a part in the pathogenesis of diseases and are sometimes useful markers for diagnosis and follow-up. Vasculitis treatments should be chosen according to classification, aetiology, pathogenetic mechanisms, severity and predictable outcome. In virus-associated vasculitides, treatment is based on the combination of antiviral agents and symptomatic or immunomodulating therapies. HBV-related PAN and HCV-related cryoglobulinaemia respond to interferon-alpha and to plasma exchange. Responses are excellent in HBV-PAN but usually partial in HCV-cryoglobulinaemia, and relapses occur in the majority of cases. MPA, c-PAN, WG and other vasculitides respond to corticosteroids and cytotoxic agents, mainly cyclophosphamide. Treatment duration and ways of administration can vary from one disease to another. Plasma exchange is not recommended as the first-line treatment. Immunoglobulins and other immunomodulating treatments are indicated in limited cases and their indications necessitate further prospective studies.

Blood Vessels↗

Measles- or mumps virus-infected cells forming rosettes with lymphocytes from patients with multiple sclerosis.

Peripheral blood lymphocytes (PBLs) from patients with multiple sclerosis (MS) were studied to determine the frequency at which they formed rosettes with target cells persistently infected with measles or mumps virus. Results were compared with (1) the rosette-forming capability of lymphocytes from age- and sex-matched normal control subjects and (2) the rosette-forming capability of lymphocytes with uninfected target cells from patients with MS. Comparison of mean measles antibody titers in patients with MS was significantly higher than in control subjects. A similar comparison for mumps antibodies showed a significant differences. There was no significant difference between patients and control subjects in the frequency of lymphocytes that formed rosettes, no matter which target cell was used. When data obtained using target cells infected with measles were analyzed according to sex or clinical classification, no significant difference was observed. Lymphocytes from patients or control subjects formed significantly more rosettes when reacted with virus-infected rather than uninfected target cells. These data suggest that PBL rosette formation with measles- or mumps-infected cells may represent nonspecific adherence rather than specific adherence.

Adult↗

[Cardiotropic DNA viruses and bacteria in the pathogenesis of dilated cardiomyopathy with or without inflammation].

In the report of the 1995 WHO/ISFC task force on the definition and classification of cardiomyopathies a new entity within the dilated cardiomyopathies was introduced as "inflammatory cardiomyopathy". It is defined as myocarditis associated with cardiac dysfunction. Idiopathic, autoimmune and infectious forms of inflammatory cardiomyopathy are now recognized through this definition. Dilated cardiomyopathy with inflammation (DCMi, chronic myocarditis) was also defined by a recent ISFC task force as > 14 lymphocytes/macrophages/mm3. Enteroviruses, adenoviruses and cytomegaloviruses are considered as main etiopathogenetic factors in the pathogenesis of inflammatory heart disease and have been demonstrated as important trigger for inflammatory cardiac disease. They may also cause dilated cardiomyopathy by viral persistence or secondary immunopathogenesis due to antigenic or molecular mimicry. For the detection of viral persistence the investigation of endomyocardial biopsies in patients with cardiomyopathy by the use of polymerase chain reaction and southern blot analysis is an important step for the standardization of diagnostic criteria on virally induced inflammatory cardiomyopathy. Present studies indicate an incidence of cytomegalovirus-DNA in patients with inflammatory cardiomyopathy in 10%, adenoviral-DNA in 17% and borreliosis only in rare cases (< 1%). In dilated cardiomyopathy without inflammation the respective incidences were for cytomegalovirus 12%, 15% for adenovirus and only 0.5% of cases for borreliosis. In addition the results of immunohistochemical analysis and molecular biological investigations of endomyocardial biopsies may have implications for future therapeutic studies. Depending on the etiology of the disease, immunosuppression may have benefit for patients with virus-negative cardiomyopathy with inflammation in contrast to patients with cytomegalo-, adenovirus-DNA or enteroviral persistence, in whom immunomodulation with hyperimmunoglobulins or immunoglobulins may be a feasible therapeutic option. Patients with a positive PCR for Borrelia burgdorferi should be treated with 3rd generation cephalosporines and/or sublactam.

Biopsy↗

Classification of organisms: living and fossil.

The classification advanced herein provides a framework to which any organism can be readily placed and offers comprehensive coverage of the entire biological spectrum. The viruses are incorporated as a kingdom of organisms and a rationale advanced for a 6-kingdom system. Many seldom-classified fossil organisms are included, with a significant number of them classified at an advanced level. Individual phyla are recognized for the fossil genera Tribrachidium, Amiskwia, Dinomischus, Anomalocaris, Tullimonstrum, Hallucigenia, Opabinia and Pikaia. Other seldom-recognized phyla include viruses, conularids, loriciferans, tentaculites, hyoliths, scleritophores, myzostomans, acorn worms, conodonts, monoblastozoans and volborthellids. The notion that failures (geologically) cannot be phyla is rejected. A system of kingdom-related and rank-related suffixes is incorporated with the flexible opportunity, if deemed necessary, to advance or reduce taxa by re-suffixing. Other features of the complete classification of Anderson (Classification of Organisms: Living and Fossil, Golden Crowns Press, Lancaster, OH, 1992) include the elimination of duplicate taxa, incorporation of geologic ranges and number of species for many groups, and extensive usage of common names, which are completely indexed.

Animal Population Groups↗

The nucleotide sequence of coxsackievirus A9; implications for receptor binding and enterovirus classification.

The complete nucleotide sequence of the genome of coxsackievirus A9 (CAV-9) has been determined from cDNA cloned in Escherichia coli. Excluding the 3' poly(A) stretch, the RNA genome is 7452 nucleotides long and encodes a single polyprotein of 2201 amino acids. Comparison of the nucleotide and predicted amino acid sequences with those of the coxsackieviruses B1, B3 and B4 reveals a surprising degree of homology, with overall amino acid homologies of 86.9%, 86.2% and 87.0%, respectively. In contrast, there is much less homology to another coxsackie A virus, CAV-21, 60.4% overall amino acid homology. This demonstrates the high degree of diversity within the CAV group and indicates that the current classification does not directly correlate with molecular genetic properties. One major feature of CAV-9 is an insertion, relative to all other enteroviruses sequenced to date, which is located at the C terminus of VP1, and includes an arginine-glycine-aspartic acid tripeptide. Such sequences in a number of other proteins are known to have activity in promoting attachment to cell receptors and the implications for CAV-9 receptor binding are discussed.

Amino Acid Sequence↗

Genetic polymorphism across regions of the three open reading frames of "Norwalk-like viruses".

Genomic characterization of Norwalk-like human caliciviruses (NLVs) originating from outbreaks and sporadic cases of acute gastroenteritis has revealed surprisingly high levels of diversity, even in the RNA polymerase gene, which is anticipated to be highly conserved. Since information on antigenic relationship is limited, due to the lack of a tissue culture system for these viruses, strains mostly are described on the basis of their genetic relatedness. However, the lack of uniformly applied criteria has led to a confusing array of strains with different groups employing different names for similar genetic lineages. Our goal was to conduct a structured analysis of genomic relationships among NLV strains in an attempt to provide an interim framework for genotyping. We assembled a panel of 31 potentially distinct genogroup I (GGI) and genogroup II (GGII) NLVs that reflected the diversity seen in strains detected by our laboratories and in published sequences. Phylogenetic analysis of sequences from regions of the open reading frames (ORF) 1, 2 and 3 was performed in order to investigate genomic relationships. The strains sequenced fell into seven phylogenetic groups in GGI and at least five phylogenetic groups in GGII, based on greater than 80% nucleotide identity in the region of ORF2 encoding the N-terminus of the capsid protein, and consistent clustering with high bootstrap values irrespective of the method used. Analysis of the ORF1 and ORF3 regions supported for most strains the clustering as established for those derived from ORF2. In the ORF1 region, used by most laboratories for diagnostic RT-PCR, clustering was consistent when a putative genotype border was set at 15% nucleotide mismatches for viruses in GGI and at 10% for viruses in GGII. Two strains grouped within different clusters based on ORF1 and ORF2 indicating that recombination may have occurred. We discuss the implications of these observations for the classification and typing of NLVs.

Caliciviridae↗

Nucleotide sequence analysis of RNA-2 of a flat apple isolate of Cherry rasp leaf virus with regions showing greater identity to animal picornaviruses than to related plant viruses.

RNA-2 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) appears to consist of 3274 nucleotides, excluding a 3' poly (A) tail. The data supports re-classification of CRLV in a new genus in the family Comoviridae. A single open reading frame (ORF) encoding a putative 108 kDa polyprotein was identified. Potential protease cleavage sites were identified which would result in the production of a putative movement protein (41 kDa), and 3 capsid protein subunits (24, 20, and 22 kDa, respectively). A 5'-UTR and 3'-UTR were identified, 248 nt and 146 nt long, respectively. The genome organisation of CRLV-FA RNA-2 is similar to that of Apple latent spherical virus (ALSV) RNA-2, a new member of the family Comoviridae. The Vp25 amino acid sequences were unique to CRLV-FA and ALSV (54% identity), with no relationship identified to any other virus. CRLV-FA Vp20 and Vp24 amino acid sequences were closely related to ALSV (59 and 65%, respectively) but the only other relationships identified were with a range of animal ssRNA positive-strand viruses.

Amino Acid Sequence↗

DNA polymerase gene sequences indicate western and forest tent caterpillar viruses form a new taxonomic group within baculoviruses.

Baculoviruses infect larval lepidopterans, and thus have potential value as microbial controls of agricultural and forest pests. Understanding their genetic relatedness and host specificity is relevant to the risk assessment of viral insecticides if non-target impacts are to be avoided. DNA polymerase gene sequences have been demonstrated to be useful for inferring genetic relatedness among dsDNA viruses. We have adopted this approach to examine the relatedness among natural isolates of two uncharacterized caterpillar-infecting baculoviruses, Malacosoma californicum pluviale nucleopolyhedrovirus (McplMNPV) and Malacosoma disstria nucleopolyhedrovirus (MadiMNPV), which infect two closely related host species with little to no cross-infectivity. We designed two degenerate primers (BVP1 and BVP2) based on protein motifs conserved among baculoviruses. McplMNPV and MadiMNPV viral DNA was obtained from naturally infected caterpillars collected from geographically distinct sites in the Southern Gulf Islands and Prince George regions of British Columbia, Canada. Sequencing of 0.9 kb PCR amplicons from six McplMNPV and six MadiMNPV isolates obtained from a total of eight sites, revealed very low nucleotide variation among McplMNPV isolates (99.2-100% nucleotide identity) and among MadiMNPV isolates (98.9-100% nucleotide identity). Greater nucleotide variation was observed between viral isolates from the two different caterpillar species (only 84.7-86.1% nucleotide identity). Both maximum parsimony and maximum likelihood phylogenetic analyses support placement of McplMNPV and MadiMNPV in a clade that is distinct from other groups of baculoviruses.

Amino Acid Sequence↗