ICTV Virus Taxonomy Profile: Picornaviridae.

The family Picornaviridae comprises small non-enveloped viruses with RNA genomes of 6.7 to 10.1 kb, and contains >30 genera and >75 species. Most of the known picornaviruses infect mammals and birds, but some have also been detected in reptiles, amphibians and fish. Many picornaviruses are important human and veterinary pathogens and may cause diseases of the central nervous system, heart, liver, skin, gastrointestinal tract or upper respiratory tract. Most picornaviruses are transmitted by the faecal-oral or respiratory routes. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Picornaviridae, which is available at www.ictv.global/report/picornaviridae.

Use of nucleotide composition analysis to infer hosts for three novel picorna-like viruses.

Nearly complete genome sequences of three novel RNA viruses were acquired from the stool of an Afghan child. Phylogenetic analysis indicated that these viruses belong to the picorna-like virus superfamily. Because of their unique genomic organization and deep phylogenetic roots, we propose these viruses, provisionally named calhevirus, tetnovirus-1, and tetnovirus-2, as prototypes of new viral families. A newly developed nucleotide composition analysis (NCA) method was used to compare mononucleotide and dinucleotide frequencies for RNA viruses infecting mammals, plants, or insects. Using a large training data set of 284 representative picornavirus-like genomic sequences with defined host origins, NCA correctly identified the kingdom or phylum of the viral host for >95% of picorna-like viruses. NCA predicted an insect host origin for the 3 novel picorna-like viruses. Their presence in human stool therefore likely reflects ingestion of insect-contaminated food. As metagenomic analyses of different environments and organisms continue to yield highly divergent viral genomes NCA provides a rapid and robust method to identify their likely cellular hosts.

Cultivation and serological characterization of a human Theiler's-like cardiovirus associated with diarrheal disease.

Cardioviruses (e.g., Theiler's murine encephalomyelitis virus [TMEV]) are members of the Picornaviridae family that cause myocarditis and encephalitis in rodents. Recently, several studies have identified human cardioviruses, including Saffold virus (SAFV) and a related virus named human TMEV-like cardiovirus (HTCV). At least eight cardiovirus genotypes are now recognized, with SAFV and most strains of HTCV belonging to genotypes 1 and 2, respectively; genotype 2 strains are the most common in the population. Although a genotype 3 cardiovirus has recently been cultured (SAFV-3), the genotype 1 and 2 cardioviruses have been difficult to propagate in vitro, hindering efforts to understand their seroprevalence and pathogenicity. Here we present the isolation and characterization of a genotype 2 human cardiovirus (HTCV-UC6). Notably, successful cultivation of HTCV-UC6 from stool required the addition of cytokine-blocking antibodies to interrupt downstream antiviral pathways. Unlike SAFV-3, HTCV-UC6 exhibited slow replication kinetics and demonstrated only a moderate cytopathic effect. Serologic assays revealed that 91% of U.S. adults carry antibodies to the genotype 2 cardioviruses, of which 80% generate neutralizing antibodies, in agreement with previous data showing that cardiovirus infection is widespread in humans. We also demonstrate an acute cardiovirus seroconversion event in a child with diarrhea and vomiting, thus reporting for the first time evidence linking cardiovirus infection to diarrheal disease in humans.

Multiple novel astrovirus species in human stool.

Diarrhoea remains a significant cause of morbidity and mortality in developing countries where numerous cases remain without identified aetiology. Astroviruses are a recently identified cause of animal gastroenteritis which currently includes two species suspected of causing human diarrhoea. Using pan-astrovirus RT-PCR, we analysed human stool samples from different continents for astrovirus-related RNA sequences. We identified variants of the two known human astrovirus species plus, based on genetic distance criteria, three novel astrovirus species all distantly related to mink and ovine astroviruses, which we provisionally named HMOAstV species A-C. The complete genome of species A displayed all the conserved characteristics of mammalian astroviruses. Each of the now three groups of astroviruses found in human stool (HAstV, AstV-MLB and HMOAstV) were more closely related to animal astroviruses than to each other, indicating that human astroviruses may periodically emerge from zoonotic transmissions. Based on the pathogenic impact of their closest phylogenetic relatives in animals, further investigations of the role of HMOAstV, so far detected in Nigeria, Nepal and Pakistan, in human gastroenteritis are warranted.

Frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the United States.

Untreated sewage samples from 12 cities in the United States were screened for the presence of recently characterized RNA and DNA viruses found at high prevalence in the stool specimens of South Asian children. Genetic variants of human cosaviruses and cardioviruses in the Picornaviridae family and of DNA circoviruses and human bocaviruses were detected, expanding the known genetic diversity and geographic range of these newly identified viruses. All four virus groups were detected in sewage samples of less than a milliliter from multiple U.S. cities. PCR screening of particle-protected viral nucleic acid in sewage samples could therefore rapidly establish the presence and determine the diversity of four newly described enteric viruses in large urban populations. More frequent and deeper sampling of viral nucleic acids in sewage samples could be used to monitor changes in the prevalence and genetic composition of these and other novel enteric viruses.

A highly divergent picornavirus in a marine mammal.

Nucleic acids from an unidentified virus from ringed seals (Phoca hispida) were amplified using sequence-independent PCR, subcloned, and then sequenced. The full genome of a novel RNA virus was derived, identifying the first sequence-confirmed picornavirus in a marine mammal. The phylogenetic position of the tentatively named seal picornavirus 1 (SePV-1) as an outlier to the grouping of parechoviruses was found consistently in alignable regions of the genome. A mean protein sequence identity of only 19.3 to 30.0% was found between the 3D polymerase gene sequence of SePV-1 and those of other picornaviruses. The predicted secondary structure of the short 506-base 5'-untranslated region showed some attributes of a type IVB internal ribosome entry site, and the polyprotein lacked an apparent L peptide, both properties associated with the Parechovirus genus. The presence of two SePV-1 2A genes and of the canonical sequence required for cotranslational cleavage resembled the genetic organization of Ljungan virus. Minor genetic variants were detected in culture supernatants derived from 8 of 108 (7.4%) seals collected in 2000 to 2002, indicating a high prevalence of SePV-1 in this hunted seal population. The high level of genetic divergence of SePV-1 compared to other picornaviruses and its mix of characteristics relative to its closest relatives support the provisional classification of SePV-1 as the prototype for a new genus in the family Picornaviridae.

Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes.

The genetic diversity of human immunodeficiency virus (HIV) is a major concern thought to impact on immunologic escape and eventual vaccine efficacy. Here, simple and rapid methods are described for the detection and estimation of genetic divergence between HIV strains on the basis of the observation that DNA heteroduplexes formed between related sequences have a reduced mobility in polyacrylamide gels proportional to their degree of divergence. Reliable phylogenetic subtypes were assigned for HIV-1 strains from around the world. Relationships between viruses were closest when derived from the same or epidemiologically linked individuals. When derived from epidemiologically unlinked individuals, the relationships between viruses in a given geographic region correlated with the length of time HIV-1 had been detected in the population and the number of strains initiating widespread infection. Heteroduplex mobility analysis thus provides a tool to expedite epidemiological investigations by assisting in the classification of HIV and is readily applicable to the screening and characterization of other infectious agents and cellular genes.

Identification of low-level contamination of blood as basis for detection of human immunodeficiency virus (HIV) DNA in anti-HIV-negative specimens.

The significance of detection of human immunodeficiency virus (HIV) DNA by the polymerase chain reaction (PCR) in seronegative or seroconverting (SC) subjects remains controversial. In a previously reported study, we identified a case in which a specimen collected 12 months before seroconversion (pre-SC) was found repeatedly to be PCR positive in three experienced laboratories, while the 6-month pre-SC bleed was PCR-negative; PCR-based human leukocyte antigen (HLA)-DQA and -DRB typing of serial peripheral blood mononuclear cell (PBMC) samples from this case did not indicate a specimen mix-up or labeling error. To further investigate this case, we used HIV env sequence and DNA heteroduplex gel-shift analyses to characterize HIV quasispecies present in serial pre- and post-SC specimens. HIV env sequences and gel-shift pattern analyses from the 12-month pre-SC versus post-SC samples indicated that markedly distinct quasispecies were present, suggesting possible abortive infection followed by reinfection and subsequent seroconversion. However, the HIV burden of this pre-SC sample was very low (1 provirus/10(6) PBMCs), and the quasispecies was highly heterogeneous, findings suggesting long-term rather than recent HIV infection. To test the hypothesis that the index pre-SC sample was PCR positive owing to trace blood contamination during initial processing, we analyzed the three seropositive samples collected on the same date in 1985. One of these samples was highly related to the index pre-SC sample by env sequence and gel-shift methodologies.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution analysis of T-cell receptor beta-chain repertoires using DNA heteroduplex tracking: generally stable, clonal CD8+ expansions in all healthy young adults.

The accurate measurement of T-cell receptor (TCR) repertoire changes requires the analysis of a representative sampling of complex T-cell populations. The number and frequency of clonally expanded TCR beta-chain transcripts bearing distinct CDR3 sequences were accurately determined using a simple DNA heteroduplex tracking assay. This method allowed major and minor clonal expansions (> or = 1% of a Vbeta subfamily's transcripts) to be rapidly and reproducibly quantified. Oligoclonal CD8 + cell expansions were detected in all young adults tested, while CD4 + cells generally expressed more polyclonal beta-chain repertoires. The same pattern of CD8 + cells oligoclonality and CD4 + cells polyclonality was observed in asymptomatic HIV-1 infected individuals with high CD4 + cell counts. CD8 + CD45RA + and CD8 + CD45RO + cell fractions both displayed oligoclonal, although distinct, TCR beta chain repertoires while CD8 + cells from umbilical cord blood were generally polyclonal. Oligoclonal CD8 + cell repertoires from young adults were generally stable over a period of weeks, although minor, transient, clonal expansions could also be detected in the absence of symptomatic infections. DNA heteroduplex tracking analysis provided a higher level of sensitivity for the detection of TCR beta chain transcript expansions than CDR3 length (spectrotyping/immunoscope) analysis. DNA heteroduplex tracking of TCR beta-chain transcripts is therefore a simple and sensitive method for assessing the level of clonality and for measuring changes in the TCR beta chain repertoire of different T-cell populations.

HIV type 1 envelope quasispecies in the thymus and lymph Nodes of AIDS patients.

To test for the presence of HIV syncytium-inducing (SI) strains in the thymus in vivo we sequenced HIV envelope V3 variants from thymic and peripheral lymph node tissues of three subjects who died of AIDS. Phylogenetic analysis of proviral sequences derived by direct sequencing of multiple independent PCRs showed that the HIV-1 quasispecies did not segregate into distinct clusters in the thymus versus lymph nodes. Examination of env sequences for V3 loop amino acids associated with the SI phenotype did not show its preferential localization in either thymus or lymph node. One subject harbored only putative SI variants, another only putative NSI variants, and the third subject carried a mixture of genotypes in both tissues. The thymus and lymph nodes of terminal AIDS patients therefore appeared to harbor closely related proviral envelope quasispecies.

Apparent founder effect during the early years of the San Francisco HIV type 1 epidemic (1978-1979).

HIV-1 envelope sequence variants were RT-PCR amplified from serum samples cryopreserved in San Francisco in 1978-1979. The HIV-1 subtype B env V3-V5 sequences from four homosexual men clustered phylogenetically, with a median nucleotide distance of 2.8%, reflecting a recent common origin. These early U.S. HIV-1 env variants mapped close to the phylogenetic root of the subtype B tree while env variants collected in the United States throughout the 1980s and 1990s showed, on average, increasing genetic diversity and divergence from the subtype B consensus sequence. These results indicate that the majority of HIV-1 currently circulating in the United States may be descended from an initial introduction and rapid spread during the mid- to late 1970s of subtype B viruses with limited variability (i.e., a founder effect). As expected from the starburst-shaped phylogeny of HIV-1 subtype B, contemporary U.S. strains were, on average, more closely related at the nucleic acid and amino acid levels to the earlier 1978-1979 env variants than to each other. The growing levels of HIV-1 genetic diversity, one of multiple obstacles in designing a protective vaccine, may therefore be mitigated by using epidemic founding variants as antigenic strains for protection against contemporary strains.

Analysis of HIV-1 envelope mutants and pseudotyping of replication-defective HIV-1 vectors by genetic complementation.

Infectious HIV-1 particles containing replication-defective vectors that express the hygromycin B phosphotransferase gene were generated by transient complementation in COS-1 cells. A defective vector dependent only on trans-complementation with an env gene and a small vector containing a deletion of almost all of the trans region were used to examine pseudotyping of HIV-1 by an amphotropic murine retrovirus. Although pseudotyping by the heterologous envelope glycoprotein occurred with efficiency, no pseudotyping at the RNA level was observed. Genetic complementation was used to rapidly analyze the effect of env mutations in the V3, proteolytic processing site, fusion domain, and cytoplasmic tail on viral infectivity. Mutations decreasing syncytium formation usually also lowered infectivity. However, a mutation in the cytoplasmic tail and a separate mutation adjacent to the fusion domain dramatically decreased viral particle infectivity but did not appreciably decrease envelope glycoprotein-mediated cell-to-cell fusion. These results may indicate that these regions of the transmembrane peptide are necessary for acquisition of envelope glycoprotein by budding virus particles or for virus entry.

Rapid molecular epidemiology of human immunodeficiency virus transmission.

Close sequence homology between strains of HIV-1 have been used to corroborate cases of epidemiologically identified transmission. As an alternative to extensive DNA sequence analysis, genetic relateness between pairs of HIV quasispecies was estimated using the reduced electrophoretic mobilities of HIV-1 envelope DNA heteroduplexes through polyacrylamide gels. All six infections acquired in a dental practice in the late 1980s and four of six infections acquired through blood product transfusions and sexual contact in 1984-1985 could be rapidly identified. A rising level of genetic diversity within HIV-1 subtype B facilitated the detection of later transmission events. Transmission linkages could be detected up to 4 years following infection. The simple and rapid technique of DNA heteroduplex tracking can therefore assist epidemiological investigations of HIV transmission and potentially of other genetically variable infectious agents.

Genetic analysis of HIV-1 isolates from Brazil reveals presence of two distinct genetic subtypes.

The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. Here we report the genetic characteristics of 21 HIV-1 isolates from Brazil. The isolates were initially characterized using a heteroduplex mobility assay. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B. Four isolates belonged to a more recently identified genotype, termed subtype F. The subtype F sequences from Brazil are distinguishable in both gag and env from five other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.

PIP: The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. The genetic characteristics of HIV-1 isolates from whole blood samples of 21 HIV-1-seropositive Brazilian patients collected during 1989 and 1990 are reported. Virus was isolated by cocultivation of patient peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA)-stimulated donor PBMCs. The isolates were initially characterized using a heteroduplex mobility assay, which estimates the DNA sequence homology of a selected genomic region from different HIV-1 isolated from the migration of heteroduplexes in polyacrylamide gels. Five distinct HIV-1 envelope subtypes were identified, and a sixth subtype, termed F, was identified using isolates from Brazil and Romania. One Brazilian isolate was identified as subtype B by the more rapid relative migration of heteroduplexes. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B, whereas 4 isolates belonged to subtype F, a more recently identified genotype. The gag and env genes of several Brazilian isolates belonging to subtypes B and F were cloned and sequenced to allow a detailed analysis of their phylogenic relationships. This further established the existence of two distinct and well-separated genetic subtypes among Brazilian HIV-1 isolates. The subtype F sequences from Brazil are distinguishable in both gag and env from 5 other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.

Rapid genetic characterization of HIV type 1 strains from four World Health Organization-sponsored vaccine evaluation sites using a heteroduplex mobility assay. WHO Network for HIV Isolation and Characterization.

To assist in the preparation for the testing of vaccines against human immunodeficiency virus (HIV) we, as part of the World Health Organization Network for HIV Isolation and Characterization (WHO-NHIC), evaluated the genotypic variation of HIV-1 in cohorts from Brazil, Rwanda, Thailand, and Uganda. Here we report the results from a pilot study of 65 HIV-1-infected individuals. In all cases in which viral envelope gene fragments could be amplified by polymerase chain reaction, subtypes could be assigned using a heteroduplex mobility assay (HMA)1 by comparison with HIV-1 strains representing six HIV-1 envelope subtypes. All subtype classifications matched those found by envelope gene sequencing. Phylogenetic relationships were further clarified by heteroduplex formation between samples within each subtype. A relatively homogeneous subtype E virus population predominated over subtype B viruses in the sample set from Thailand. Viruses from the other countries were also limited to one or two subtypes but were more divergent within each subtype. All samples from Rwanda (13/13) and some from Uganda (3/16) were of subtype A; all Brazilian samples were of subtype B, except for one belonging to subtype C; most samples from Uganda (13/16) clustered with the subtype D. Analysis by HMA is therefore applicable for screening of HIV-1 genotypes in countries under consideration for large-scale vaccine trials. It should be generally useful when samples containing at least one variable genetic locus need to be rapidly classified by genotype and/or analyzed for epidemiological clustering.

Importation of multiple HIV type 1 strains into West Papua, Indonesia (Irian Jaya).

HIV-1 from 16 sexually transmitted disease clinic patients in Timika, West Papua, Indonesia was amplified by RT-PCR and subtyped by a combination of envelope and gag region heteroduplex mobility analysis (HMA) and direct PCR DNA sequencing. HMA showed the presence of 14 subtype E (CRF01_AE) and 2 subtype B HIV-1. Phylogenetic analysis of a 540-bp V3-V4 region of gp120 showed that 9 of 10 CRF01_AE variants clustered tightly with a median distance of 1.3% (range, 0.5 to 2.2%) whereas 1 CRF01_AE variant diverged significantly from the others (median distance, 10.7%; range, 10.1 to 11.8%). One subtype B virus envelope was typical of United States/European strains whereas the other appeared to be related to Thai subtype B' variants. These results reflect the independent introduction of multiple HIV-1 strains into West Papua, with the rapid spread in the majority of infected patients tested of a single strain of HIV-1E (CRF01_AE).

Conserved V3 loop sequences and transmission of human immunodeficiency virus type 1.

The third variable region (V3) of the surface glycoprotein (gp120) of envelope sequence subtype B, type 1 human immunodeficiency virus (HIV-1B), is highly variable among T cell line-adapted viruses and syncytium-inducing HIV-1-B isolates. Here we analyze the V3 region sequences from 93 individuals close to the time of seroconversion and show that the cysteine-bridged V3 loop, which also encompasses a major neutralizing determinant, is highly conserved, whereas sequences immediately surrounding the loop are similarly divergent in all HIV-1-B strains. Viruses with this conserved V3 loop have been reported to be more resistant to antibody-mediated neutralization than T cell-adapted viruses with divergent V3 sequences. We hypothesize, therefore, that on transmission from a donor to a recipient, virions inherently more resistant to neutralization by donor antibodies have a greater chance of initiating infection than those more sensitive to neutralization. This might explain the conservation of V3 early in infection and has implications for the design of HIV vaccines.

Detection of HIV-1 in alternative specimen types using the APTIMA HIV-1 RNA Qualitative Assay.

Peripheral blood mononuclear cells (PBMCs), saliva, seminal plasma, and dried blood spots were evaluated as specimen types for the APTIMA HIV-1 RNA Qualitative Assay (APTIMA HIV-1 Assay), which employs a target capture step to recover HIV-1-specific sequences from complex specimen types. Analytical sensitivity studies were carried out using samples that were either diluted or eluted with a buffered detergent and spiked with different concentrations of HIV-1 ranging from 1 to 10,000 copies/mL. PBMC samples spiked with HIV-1 had comparable analytical sensitivity to HIV-1 spiked plasma with a 95% limit of detection of 13.1 and 17.2 copies/mL, respectively. Analytical sensitivity in seminal plasma specimens diluted 1:5 and saliva diluted 1:2 was comparable to HIV-1 spiked dilution buffer alone. Whole blood and dried blood spot specimens spiked with HIV-1 had equivalent reactivity at 250 copies/spot (5000 copies/mL). However, the 95% limit of detection values were significantly different (293.7 copies/mL for whole blood and 2384 copies/mL for dried blood spot specimens). No significant effect on analytical sensitivity was observed when one HIV-1 positive dried blood spot punch was pooled with up to 9 HIV-1 negative dried blood spot punches. Together, these studies demonstrate that the APTIMA HIV-1 RNA Qualitative Assay can be used to process a diverse array of specimen types with minimal impact on analytical sensitivity for most specimen types.