Sequence and phylogenetic analysis of the S-class genome segments of a duck orthoreovirus

In spite of common properties duck orthoreoviruses (DRVs) are antigenically different from other avian orthoreoviruses (ARVs). We analyzed the S-class genome segments of the DRV S12 and compared them with S-class genome segments of other orthoreoviruses compared to those of ARV S1133. The full-length S-class sigmaA, sigmaB, sigmaNS, and sigmaC genes were determined and compared with other ARVs to study the degree of genetic divergence and evolution of DRVs. The alignment of the DRV S12 sigmaA, sigmaB, sigmaNS, and sigmaC genes with DRV 89026 showed 90.0%, 93.6%, 88.0%, and 93.1% nucleotide identity and 97.1%, 94.3%, 95.8%, and 93.7% amino acid identity, respectively. The alignment of the DRV S12 sigmaA, sigmaB, sigmaNS, and sigmaC genes with other ARVs revealed 76.0-77.1%, 52.5-55.1%, 78.4-79.6%, and 2.7-9.9% nucleotide identity and 89.5-91.2%, 61.4-62.0%, 91.6-92.7% and 22.6-26.7% amino acid identity, respectively. Phylogenetic analyses demonstrated that DRVs were quite different from other ARVs and provided the evidence for the diversity among avian orthoreoviruses.     

Sequences of avian reovirus M1, M2 and M3 genes and predicted structure/function of the encoded mu proteins

We report the first sequence analysis of the entire complement of M-class genome segments of an avian reovirus (ARV). We analyzed the M1, M2 and M3 genome segment sequences, and sequences of the corresponding muA, muB and muNS proteins, of two virus strains, ARV138 and ARV176. The ARV M1 genes were 2,283 nucleotides in length and predicted to encode muA proteins of 732 residues. Alignment of the homologous mammalian reovirus (MRV) mu2 and ARV muA proteins revealed a relatively low overall amino acid identity ( approximately 30%), although several highly conserved regions were identified that may contribute to conserved structural and/or functional properties of this minor core protein (i.e. the MRV mu2 protein is an NTPase and a putative RNA-dependent RNA polymerase cofactor). The ARV M2 genes were 2158 nucleotides in length, encoding predicted muB major outer capsid proteins of 676 amino acids, more than 30 amino acids shorter than the homologous MRV mu1 proteins. In spite of the difference in size, the ARV/MRV muB/mu1 proteins were more conserved than any of the homologous proteins encoded by other M- or S-class genome segments, exhibiting percent amino acid identities of approximately 45%. The conserved regions included the residues involved in the maturation- and entry- specific proteolytic cleavages that occur in the MRV mu1 protein. Notably missing was a region recently implicated in MRV mu1 stabilization and in forming "hub and spokes" complexes in the MRV outer capsid. The ARV M3 genes were 1996 nucleotides in length and predicted to encode a muNS non-structural protein of 635 amino acids, significantly shorter than the homologous MRV muNS protein, which is attributed to several substantial deletions in the aligned ARV muNS proteins. Alignments of the ARV and MRV muNS proteins revealed a low overall amino acid identity ( approximately 25%), although several regions were relatively conserved.     

Extensive Sequence Divergence and Phylogenetic Relationships between the Fusogenic and Nonfusogenic Orthoreoviruses: A Species Proposal

The orthoreoviruses can be divided into subgroups based on either their restricted host range or the unusual ability of certain members of this group of nonenveloped viruses to induce cell-cell fusion from within. Phylogenetic relationships cannot be inferred based on these biological properties because fusogenic reoviruses are present in both the avian and mammalian subgroups. To address this issue, the complete nucleotide sequences of the three S-class genome segments encoding the major sigma-class core, outer capsid, and nonstructural proteins of four fusogenic reoviruses were determined and used to establish the phylogeny of the orthoreoviruses. The viruses analysed included two strains of avian reovirus and the only known fusogenic mammalian reoviruses, Nelson Bay virus and baboon reovirus. Comparative sequence analysis of these fusogenic reoviruses and the prototypical nonfusogenic mammalian reoviruses indicated a highly diverged genus with both conserved and unique sequence-predicted structural motifs in the major sigma-class proteins. Phylogenetic analysis provided the basis for the first taxonomic subdivision of the orthoreoviruses into species classes based on inferred evolutionary relationships. It is proposed that the orthoreoviruses consist of at least four species that separate into three clades. The nonfusogenic mammalian reovirus species represent a single clade, and the fusogenic reoviruses separate into two distinct clades. The first clade of fusogenic reoviruses contains the avian reovirus- and Nelson Bay virus-type species, with the second clade being occupied by the single baboon reovirus isolate that represents a fourth orthoreovirus species.     

Reptilian reovirus: a new fusogenic orthoreovirus species

The fusogenic subgroup of orthoreoviruses contains most of the few known examples of non-enveloped viruses capable of inducing syncytium formation. The only unclassified orthoreoviruses at the species level represent several fusogenic reptilian isolates. To clarify the relationship of reptilian reoviruses (RRV) to the existing fusogenic and nonfusogenic orthoreovirus species, we undertook a characterization of a python reovirus isolate. Biochemical, biophysical, and biological analyses confirmed the designation of this reptilian reovirus (RRV) isolate as an unclassified fusogenic orthoreovirus. Sequence analysis revealed that the RRV S1 and S3 genome segments contain a novel conserved 5'-terminal sequence not found in other orthoreovirus species. In addition, the gene arrangement and the coding potential of the bicistronic RRV S1 genome segment differ from that of established orthoreovirus species, encoding a predicted homologue of the reovirus cell attachment protein and a unique 125 residue p14 protein. The RRV S3 genome segment encodes a homologue of the reovirus sigma-class major outer capsid protein, although it is highly diverged from that of other orthoreovirus species (amino acid identities of only 16-25%). Based on sequence analysis, biological properties, and phylogenetic analysis, we propose this python reovirus be designated as the prototype strain of a fifth species of orthoreoviruses, the reptilian reoviruses.     

Molecular evolution of avian reovirus: evidence for genetic diversity and reassortment of the S-class genome segments and multiple cocirculating lineages

Nucleotide sequences of the S-class genome segments of 17 field-isolates and vaccine strains of avian reovirus (ARV) isolated over a 23-year period from different hosts, pathotypes, and geographic locations were examined and analyzed to define phylogenetic profiles and evolutionary mechanism. The S1 genome segment showed noticeably higher divergence than the other S-class genes. The sigma C-encoding gene has evolved into six distinct lineages. In contrast, the other S-class genes showed less divergence than that of the sigma C-encoding gene and have evolved into two to three major distinct lineages, respectively. Comparative sequence analysis provided evidence indicating extensive sequence divergence between ARV and other orthoreoviruses. The evolutionary trees of each gene were distinct, suggesting that these genes evolve in an independent manner. Furthermore, variable topologies were the result of frequent genetic reassortment among multiple cocirculating lineages. Results showed genetic diversity correlated more closely with date of isolation and geographic sites than with host species and pathotypes. This is the first evidence demonstrating genetic variability among circulating ARVs through a combination of evolutionary mechanisms involving multiple cocirculating lineages and genetic reassortment. The evolutionary rates and patterns of base substitutions were examined. The evolutionary rate for the sigma C-encoding gene and sigma C protein was higher than for the other S-class genes and other family of viruses. With the exception of the sigma C-encoding gene, which nonsynonymous substitutions predominate over synonymous, the evolutionary process of the other S-class genes can be explained by the neutral theory of molecular evolution. Results revealed that synonymous substitutions predominate over nonsynonymous in the S-class genes, even though genetic diversity and substitution rates vary among the viruses.     

The genomic constellation of a novel<Emphasis Type="Italic"> avian orthoreovirus</Emphasis> strain associated with runting-stunting syndrome in broilers

Avian orthoreoviruses (ARVs) are responsible for considerable economic losses in broiler chickens; yet, the genetic characterization of most ARV strains is limited to a few genes, and the full coding region has been determined for only S1133 and 138, two ARV strains associated with tenosynovitis. Recently, in parts of the United States, ARVs with novel neutralization antigen type were isolated from chickens afflicted with runting-stunting syndrome. One such strain, AVS-B, was selected for full genome sequencing and phylogenetic analysis. The complete genome was 23,494 bp in size and included 12 open reading frames. The lengths of the coding regions, as well as those of the 5′ and 3′ ends, were fairly well conserved between AVS-B and other reference strains. In pairwise comparisons to the S1133 and 138 strains, the AVS-B strain shared a wide range of sequence identities along each genome segment, i.e., a range of 54–55% for the σC coding region of S1 genome segment and 91–93% for the S2 genome segment. Phylogenetic analyses of individual genes of AVS-B did not identify any single common ancestor among more completely characterized ARV strains for which sequence data are available. One exception to this lack of identity was strain 138, which shared 90–93% nt identity with AVS-B along seven of ten genome segments; only M2, M3, and S1 segments of these strains shared lower sequence identities. Collectively, our analyses indicated that multiple reassortment events and strong divergence caused by the accumulation of point mutations could have led to the observed assortment and genetic heterogeneity of the AVS-B genome.     

Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat

This report describes the discovery and characterization of a new fusogenic orthoreovirus, Broome virus (BroV), isolated from a little red flying-fox (Pteropus scapulatus). The BroV genome consists of 10 dsRNA segments, each having a 3′ terminal pentanucleotide sequence conserved amongst all members of the genus Orthoreovirus, and a unique 5′ terminal pentanucleotide sequence. The smallest genome segment is bicistronic and encodes two small nonstructural proteins, one of which is a novel fusion associated small transmembrane (FAST) protein responsible for syncytium formation, but no cell attachment protein. The low amino acid sequence identity between BroV proteins and those of other orthoreoviruses (13-50%), combined with phylogenetic analyses of structural and nonstructural proteins provide evidence to support the classification of BroV in a new sixth species group within the genus Orthoreovirus.     

Characterization of the σC-encoding Gene from Musocvy Duck Reovirus

The σC-encoding gene of two muscovy duck reovirus (DRV) S14 and C4 strains were cloned and completely sequenced. The open reading frame (ORF) comprised 810 bp and encoded 269 amino acids with a predicated molecular mass of 29.5 kDa. Expressed σC fusion protein in Escherichia coli BL21 strain could be detected by Western blotting under duck anti-reovirus polyclonal serum. There are two large gap insertions at the N-terminal part of the DRV σC when necessary to optimize the alignment of the amino acid sequences of the DRV σC had a heptapeptide repeat and leucine zipper patterns structurally related to ARV σC. All DRVs grouped into one specified genogroup within Orthoreoviruses genus subgroup II. The degree of differences between the S14/C4 and ARV was only 23–24%, and 21–22%, respectively, at both the nucleotide and deduced amino acid levels, suggested that DRVs are quite different from ARVs and should give a precise classification for DRVs in Orthoreovirus genus.     

Development of TaqMan real-time RT-PCR for detection of avian reoviruses

Avian reoviruses (ARVs) are an important cause of economic losses in commercial poultry. A TaqMan real-time RT-PCR assay for detecting of ARVs was developed. The primer-probe set was from the conserved region of ARV S4 genome segment. Real-time RT-PCR detected ARV strains including CO8 and ss412 strains, which belonged to different serological subgroups, and the test had no cross-reaction with other avian viruses. The detection limit of this assay was 5 ARV genome copies per 5 μl and was 150 times more sensitive than traditional RT-PCR. Statistical analyses indicated excellent reproducibility. For ARV strain 2408, a titer of 50% embryo infection dose and 50% tissue culture infectious dose equivalent to 3.9 ± 0.8, and 2.9 ± 0.3 ARV genome copies, respectively. This test was rapid, specific, and sensitive for the detection of ARVs and will be useful in veterinary diagnostic laboratories and for the quantitation of vaccine viruses for pharmaceutical companies.     

Three-dimensional electron cryo-microscopy as a powerful structural tool in molecular medicine

Electron cryo-microscopy has established itself as a valuable method for the structure determination of protein molecules, protein complexes, and cell organelles. This contribution presents an introduction to the various aspects of three-dimensional electron cryomicroscopy. This includes the need for sample preservation in the microscope vacuum, strategies for minimizing radiation damage, methods of improving the poor signal-to-noise ratio in electron micrographs of unstained specimens, and the various methods of three-dimensional image reconstruction from projections. The various specimen types (e.g., flat and tubular two-dimensional crystals, protein filaments, individual protein molecules, and large complexes) require different means of three-dimensional reconstruction, and we review the five major reconstruction techniques (electron crystallography, helical reconstruction, icosahedral reconstruction, single-particle reconstruction, and electron tomography), with an emphasis on electron crystallography. Several medically relevant three-dimensional protein structures are chosen to illustrate the potential of electron cryo-microscopy and image reconstruction techniques. Among the structural methods, electron cryo-microscopy is the only tool for studying objects that range in size from small proteins over macromolecular complexes to cell organelles or even cells.     

Sequence and phylogenetic analysis of the S1 genome segment of turkey-origin reoviruses

Based on previous reports characterizing the turkey-origin avian reovirus (TRV) σB (σ2) major outer capsid protein gene, the TRVs may represent a new group within the fusogenic orthoreoviruses. However, no sequence data from other TRV genes or genome segments has been reported. The σC protein encoded by the avian reovirus S1 genome segment is the cell attachment protein and a major antigenic determinant for avian reovirus. The chicken reovirus S1 genome segment is well characterized and is well conserved in viruses from that species. This report details the amplification, cloning and sequencing of the entire S1 genome segment from two and the entire coding sequences of the σC, p10 and p17 genes from an additional five TRVs. Sequence analysis reveals that of the three proteins encoded by the TRV S1 genome segment, σC shares at most 57% amino acid identity with σC from the chicken reovirus reference strain S1133, while the most similar p10 and p17 proteins share 72% and 61% identity, respectively, with the corresponding S1133 proteins. The most closely related mammalian reovirus, the fusogenic Nelson Bay reovirus, encodes a σC protein that shares from 25% to 28% amino acid identity with the TRV σC proteins. This report supports the earlier suggestion that the TRVs are a separate virus species within the Orthoreovirus genus, and may provide some insight into TRV host specificity and pathogenesis. NC/SEP–R44/03 S1 genome segment DQ525419 NC/98 S1 genome segment DQ995806 TRV sigmaC sequences DQ996601–DQ996605 TRV p10 sequences DQ996606–DQ996610 TRV p17 sequences DQ996611–DQ996615     

Development and application of monoclonal antibodies for detection and analysis of aquareoviruses

Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.     

Traditional Complementary and Alternative Medicine and Antiretroviral Treatment Adherence Among HIV Patients in Kwazulu-Natal, South Africa

Adherence to antiretroviral medication in the treatment of HIV is critical, both to maximize efficacy and to minimize the emergence of drug resistance. The aim of this prospective study in three public hospitals in KwaZulu-Natal, South Africa, is to assess the use of Traditional Complementary and Alternative Medicine (TCAM) by HIV patients and its effect on antiretroviral (ARV) adherence 6 months after initiating ARVs. 735 (29.8% male and 70.2% female) patients who consecutively attended three HIV clinics completed assessments prior to ARV initiation and 519 after six months on antiretroviral therapy (ART) Results indicate that the use of herbal therapies for HIV declined significantly from 36.6% prior to antiretroviral treatment (ART) initiation to 7.9% after being on ARVs for 6 months. Faith healing methods, including spiritual practices and prayer for HIV declined from 35.8% to 22.1% and physical/body-mind therapy (exercise and massage) declined from 5.0% to 1.9%. In contrast, the use of micronutrients (vitamins, etc.) significantly increased from 42.6% to 87.4%. In multivariate regression analyses, ARV non-adherence (dose, schedule and food) was associated with the use of herbal treatment, not taking micronutrients and the use of over-the-counter drugs. The use of TCAM declined after initiating ARVs. As herbal treatment for HIV was associated with reduced ARV adherence, patients'' use of TCAM should be considered in ARV adherence management.     

Methadone,Buprenorphine, and Street Drug Interactions with Antiretroviral Medications

While street drugs appear unlikely to alter the metabolism of antiretroviral (ARV) medications, several ARVs may induce or inhibit metabolism of various street drugs. However, research on these interactions is limited. Case reports have documented life-threatening overdoses of ecstasy and gamma-hydroxybutyrate after starting ritonavir, an ARV that inhibits several metabolic enzymes. For opioid addiction, methadone or buprenorphine are the treatments of choice. Because a number of ARVs decrease or increase methadone levels, patients should be monitored for methadone withdrawal or toxicity when they start or stop ARVs. Most ARVs do not cause buprenorphine withdrawal or toxicity, even if they alter buprenorphine levels, with rare exceptions to date including atazanavir/ritonavir associated with significant increases in buprenorphine and adverse events related to sedation and mental status changes in some cases. There are newer medications yet to be studied with methadone or buprenorphine. Further, there are many frequently used medications in treatment of complications of HIV disease that have not been studied. There is need for continuing research to define these drug interactions and their clinical significance.     

Characterization of Two Avian Reoviruses That Exhibit Strain-Specific Quantitative Differences in Their Syncytium-Inducing and Pathogenic Capabilities

We previously proposed that the conservation of the nonessential syncytium-inducing phenotype among all reported avian reovirus (ARV) isolates may reflect a mechanism for enhanced virus disseminationin vivo, which in turn could contribute to the natural pathogenicity of ARV. Direct testing of this hypothesis has been hampered by the lack of available virus strains with defined differences in their fusion-inducing capability. We now report on the characterization of two ARV strains, ARV-176 and ARV-138, that exhibited strain-specific differences in their fusogenic properties, which correlated with their pathogenic potential in embryonated eggs. Moreover, both virus strains possessed similar replicative abilities in cell culture, suggesting that the weakly fusogenic ARV-138 virus is specifically inhibited in its syncytium-inducing ability. To test the use of these viruses for reassortant studies aimed at assessing the role of cell fusion in viral pathogenesis, a preliminary genetic analysis was undertaken using a monoreassortant that contained nine genome segments from the parental ARV-138 virus and the S1 genome segment from the highly fusogenic and pathogenic ARV-176 parental virus. The monoreassortant possessed the full fusogenic potential of the ARV-176 parental virus and displayed enhanced embryo pathogenicity, providing the first genetic evidence implicating the ARV S1 genome segment in both syncytium formation and viral pathogenesis.     

Protease inhibitor and nonnucleoside reverse transcriptase inhibitor concentrations in the genital tract of HIV-1-infected women

The pharmacokinetics of antiretrovirals (ARVs) in the female genital tract (FGT) are likely to influence vertical and sexual transmission of HIV, the development of viral resistance, and post-exposure prophylaxis regimens. This study is the first to compare ARV concentrations in direct aspirates of cervicovaginal fluid (CVF) and blood plasma (BP). This unique method provides direct assessment of concentrations without the confounding of cervicovaginal lavage dilution. Of 8 ARVs, CVF concentrations ranged from <10% to >100% of BP concentrations. These large differences in CVF penetration suggest that further research into ARV pharmacokinetics and drug efficacy in the FGT is necessary.     

Modeling trends in HIV incidence among homosexual men in Australia 1995-2006

BACKGROUND: Previous mathematical models have indicated that any decrease in HIV incidence in homosexual men due to decreased infectiousness from antiretroviral treatment (ARV) may be offset by modest increases in unsafe sex. The aims of this study were to assess the effects of ARV use and increasing unprotected anal intercourse with casual partners (UAIC) in homosexual men on HIV incidence during 1995-2001 and to project HIV incidence depending on trends in ARV use and UAIC. METHODS: A mathematical model of HIV transmission among homosexual men in Australia was developed. HIV incidence during 1995-2001 was estimated assuming that 70% of men in whom HIV was diagnosed received ARVs and assuming a 10% annual increase in UAIC. For 2001-2006, scenarios included ARV levels remaining at 70% or declining to 50% by 2006, combined with UAIC levels remaining at the 2001 level or continuing to increase annually by 10%. FINDINGS: The number of incident HIV cases per year was predicted to have declined during 1996-1998 due to the introduction of effective ARVs, with a slow increase during 1998-2001 due to increased levels of UAIC when use of therapies was fairly stable. From 2001, a continued increase in UAIC was predicted to lead to a rise in HIV incidence. A rise in UAIC combined with a moderate decline in ARV use could lead to a 50% increase in HIV incidence by 2006. INTERPRETATION: These models suggest that widespread ARV use has had some effect in reducing HIV incidence among homosexual men in Australia. However, if current trends in UAIC and ARV use continue, a resurgent HIV epidemic is predicted.     

Comparative costs of inpatient care for HIV-infected and uninfected children and adults in Soweto, South Africa

BACKGROUND: HIV/AIDS creates a massive burden of care for health systems. A better understanding of the impact of HIV infection on health care utilization and costs may enable better use of limited resources. METHODS: We compared public sector inpatient costs of HIV-infected versus uninfected adults and children at a large hospital in Soweto, South Africa. Daily hotel costs estimated from hospital financial data and total patient visits were combined with utilization, abstracted from patients' charts, and costed using government price lists to estimate total inpatient costs. RESULTS: A total of 1185 eligible records were included over a 6-week period in 2005. Eight hundred twelve were from HIV-infected patients, and of these, 77 were on antiretroviral (ARV) therapy. The mean length of stay (LOS) and mean drug and intravenous fluid utilization of HIV-infected adults not on ARVs was greater than those of uninfected adults, resulting in a $200 higher total average admission cost. Patients on ARVs had longer LOS and incurred a total average admission cost of $750 more than HIV-infected adults not on ARVs. CONCLUSIONS: Inpatient costs were greater for this selected group of HIV-infected adults, and even higher for the small proportion of individuals receiving ARVs. Budget allocations should incorporate case mix by HIV and ARV status as a key determinant of hospital expenditure.