Molecular epidemiology of human rotaviruses: reassortment in vivo as a mechanism for strain diversity?
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Humans↗
Biomedical subjects
U Desselberger
Publications and source records attributed to U Desselberger.
Concurrent evolution of regions of the envelope and polymerase genes of human immunodeficiency virus type 1 during zidovudine (AZT) therapy.
Nucleotide sequences of regions of the envelope (env) and polymerase (pol) genes of human immunodeficiency virus type 1 (HIV-1) proviral DNA were obtained from sequential blood and autopsy samples from an AIDS patient who had been treated with zidovudine for 9 months. Phylogenetic analyses showed that a reduction in genetic heterogeneity of the env regions of viruses present in the proviral blood population occurred during therapy, and this coincided with an increased pol gene heterogeneity. Differences were observed in different organs obtained post mortem for both the env and pol coding regions. The cardiac blood proviral population consisted mainly of variants which possessed sequences containing mutations at position 215 of the pol gene, associated with drug resistance. By contrast, the brain population consisted entirely of zidovudine sensitive genotypes, and this organ also harboured variants with genetically distinct env sequences. The lymph tissues obtained after death held more diverse proviral env and pol populations, containing both zidovudine sensitive and resistant genotypes.
Amino Acid Sequence↗
Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry.
A panel of primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates that infected several CD4+ T-cell lines, including MT-2 and C8166, were tested for infection of blood-derived macrophages. Infectivity titers for C8166 cells and macrophages demonstrated that primary SI strains infected macrophages much more efficiently than T-cell line-adapted HIV-1 strains such as LAI and RF. These primary SI strains were therefore dual-tropic. Nine biological clones of two SI strains, prepared by limiting dilution, had macrophage/C8166 infectivity ratios similar to those of their parental viruses, indicating that the dual-tropic phenotype was not due to a mixture of non-SI/macrophage-tropic and SI/T-cell tropic viruses. We tested whether the primary SI strains used either Lestr (fusin) or CCR5 as coreceptors. Infection of cat CCC/CD4 cells transiently expressing Lestr supported infection by T-cell line-adapted strains including LAI, whereas CCC/CD4 cells expressing CCR5 were sensitive to primary non-SI strains as well as to the molecularly cloned strains SF-162 and JR-CSF. Several primary SI strains, as well as the molecularly cloned dual-tropic viruses 89.6 and GUN-1, infected both Lestr+ and CCR5+ CCC/CD4 cells. Thus, these viruses can choose between Lestr and CCR5 for entry into cells. Interestingly, some dual-tropic primary SI strains that infected Lestr+ cells failed to infect CCR5+ cells, suggesting that these viruses may use an alternative coreceptor for infection of macrophages. Alternatively, CCR5 may be processed or presented differently on cat cells so that entry of some primary SI strains but not others is affected.
CD4-Positive T-Lymphocytes↗
Molecular epidemiology of hepatitis C virus infection amongst intravenous drug users in rural communities.
The prevalence of hepatitis C virus (HCV) infection amongst a group of intravenous drug users (IVDUs) resident in West Suffolk (East Anglia, England) was investigated and compared with the prevalence of infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV). In addition, both the level of HCV persistence, as defined by detection of viral RNA, and the HCV genotypes present in this population were determined. It was found that HCV antibodies were present in 59% of those tested; by comparison 22% had antibodies to HBV and 1% antibodies to HIV. HCV RNA was found in 44% of those with HCV antibody. HCV genotype 1 was the most prevalent within this population although both genotypes 2 and 3 were also represented.
Adolescent↗
Construction of gag, pol, and env specific riboprobes for confirmation of HIV-1 specific polymerase chain reaction products.
Conserved regions of the gag, pol, and env genes of HIV-1 pBH10 DNA (gag nucleotides(nt)1508-1652, pol nt 2811-3118, env nt 7792-7934; Ratner et al., 1985) were amplified by the polymerase chain reaction (PCR) using oligonucleotides complementary to the termini of these regions as primers. Primer areas of the amplified DNA were then removed by digestion with restriction endonucleases, and the internal fragments purified and cloned in both orientations into the 'riboprobe' transcription vector pGEM-5Z. Riboprobes made from these plasmids did detect the specific sequences of pBH10 DNA and of HIV-1 DNA amplified by PCR from clinical material. The riboprobes will be useful to confirm the specificity of PCR-amplified fragments of lymphocyte DNA obtained from infants of HIV-infected mothers and from high risk, but seronegative contacts of HIV-1 infected individuals.
Cloning, Molecular↗
Differential Tax expression in HTLV type I-infected asymptomatic carriers.
tax gene expression in a family cluster of three HTLV-I-infected asymptomatic individuals was investigated. Two carriers had normal tax mRNA, Tax-specific humoral antibody, and cell-mediated immune (CMI) response. In one carrier who had only weak Tax-specific humoral and no Tax-specific CMI response, an abnormal Tax-related mRNA product was detected. This product was sequenced and found to consist of two exons derived from the LTR gag and pX regions. The abnormal mRNA has an ORF predicting a 17-kDa protein, the translation of which is initiated in the first exon. The presence of this protein, of antibody to it, and of its function remain to be elucidated.
Amino Acid Sequence↗
Temporal correlation between a single amino acid change in the VP4 of a porcine rotavirus and a marked change in pathogenicity.
We previously described a marked increase in the pathogenicity of a cell culture grown porcine rotavirus, PRV 4F, during serial passage in gnotobiotic piglets (Bridger et al., 1992). Here we report close temporal correlation between this change in pathogenicity and an amino acid change within a highly conserved hydrophobic domain of VP4 at position 469. Cell culture grown PRV 4F is unique in having a hydrophilic residue, glutamine, at amino acid 469; all previously sequenced VP4s have hydrophobic leucine or phenylalanine residues at the corresponding position. The detection of a point mutation causing a deduced amino acid change from glutamine to leucine at amino acid 469 of PRV 4F VP4 in virus obtained from one piglet at the second serial passage correlated exactly with the emergence of viral pathogenicity. However, given the multifactorial nature of virus pathogenicity, genetic studies are required to ascertain the degree to which this mutation is responsible for the observed change in pathogenicity.
Amino Acid Sequence↗
Hepatitis C virus infections in transplant patients: serological and virological investigations.
Hepatitis C virus (HCV) is transmitted by organs of HCV antibody-positive donors to transplant recipients. This study investigated the serological and virological responses of 14 initially HCV antibody-negative transplant patients who received organs from four HCV antibody-positive donors (A-D) (before donor screening for HCV infection was introduced in 1991). Second generation HCV enzyme immunoassay (Abbott HCV EIA) was used to detect anti-HCV antibody. Recombinant immunoblot (RIBA-2; Chiron Corporation) and Wellcozyme Western blot (Wwb) assays were compared as confirmatory assays of positive EIA results. Reverse transcription (RT) followed by "nested" polymerase chain reaction (PCR) was performed to detect viral RNA. HCV RNA was only found in the sera of donors B and C, however, transplantation of organs from all donors resulted in infection of all recipients. HCV RNA was found in recipient sera within 30 days after transplant and remained detectable throughout the period of sampling. An anti-HCV antibody response was found in only 6 (of the 14) recipients and only after 300 days. Much longer periods passed before detection of HCV antibody in six recipients. For detection of HCV infection in transplant recipients it is essential that testing for HCV RNA by RT-PCR is carried out.
Antibodies, Viral↗
The rotavirus genome.
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Amino Acid Sequence↗
Molecular epidemiology of human rotaviruses in Manipur: genome analysis of rotaviruses of long electropherotype and subgroup I.
In 1987/88 a winter outbreak of infantile gastroenteritis occurred in Manipur, India which was mainly due to rotaviruses of long electropherotype and subgroup (SG) I. The VP7 gene of one of these viruses (M48) has been cloned and sequenced. It was found to be very closely related to the VP7 genes of the G2 serotype human rotaviruses RV-5 and S2. Follow-up epidemiology of this event in Manipur during 1989-1992 yielded mainly rotaviruses of more conventional characteristics (94 isolates of SG II and long electropherotype, and 90 isolates of SG I and short electropherotype), but also 6 isolates of SG I with long electropherotype, indicating that these viruses continue to circulate in the Manipur community. One isolate of short electropherotype was of subgroup II, and one long electropherotype isolate reacted with the group A but not with either the subgroup I or subgroup II monoclonal antibodies.
Amino Acid Sequence↗
Construction of gag, pol, and env specific riboprobes for confirmation of HIV-1 specific polymerase chain reaction products.
Conserved regions of the gag, pol, and env genes of HIV-1 pBH10 DNA (gag nucleotides(nt)1520-1665, pol nt 2326-2663, env nt 7192-7333; Ratner et al., 1985) were amplified by the polymerase chain reaction (PCR) using oligonucleotides complementary to the termini of these regions as primers. Primer areas of the amplified DNA were then removed by digestion with restriction endonucleases, and the internal fragments purified and cloned in both orientations into the 'riboprobe' transcription vector pGEM-5Z. Riboprobes made from these plasmids did detect the specific sequences of pBH10 DNA and of HIV-1 DNA amplified by PCR from clinical material. The riboprobes will be useful to confirm the specificity of PCR-amplified fragments of lymphocyte DNA obtained from infants of HIV-infected mothers and from high risk, but seronegative contacts of HIV-1-infected individuals.
DNA, Viral↗
Human parvovirus B19 serology with recombinant VP1 and VP2 antigens: diagnosis of acute infections by detecting B19-specific IgM and IgA antibodies.
BACKGROUND: The availability of immunoassays for the laboratory diagnosis of human parvovirus B19 (B19) infection, which is commonly associated with erythema infectiosum in children and arthropathy and arthralgia in adults has been hampered by the lack of native B19 antigen. The production of abundant supplies of recombinant (r) B19 proteins, through the cloning of the B19 genome into expression vectors, has led to a proliferation of assays for detecting B19-specific antibodies. OBJECTIVES: This study was undertaken to evaluate serological assays for detecting B19-specific IgM and IgA antibodies using rVP1 and rVP2 B19 viral coat proteins. Their suitability in the diagnosis of acute B19 infections and the incidence of non-specific reactivity were determined. STUDY DESIGN: A panel of sera consisting of B19-specific IgM-positive and -negative samples was tested for B19-specific IgM and IgA antibodies in an indirect IFA using rVP1 antigen. These samples and a further panel collected from patients with other virus infections and samples containing rheumatoid factor were tested for B19-specific IgM in an antibody-capture ELISA and an indirect ELISA, both of which utilized rVP2 antigen. RESULTS: Data from the two ELISAs using rVP2 antigen and the IFA with rVP1 antigen all showed significant correlation (P >/= 0.001) with a reference RIA using native B19 antigen. Non-specific reactions were observed with Paul-Bunnell-positive and rubella virus-specific IgM antibody-positive sera in the ELISAs but not in the IFA. B19-specific IgA antibodies were detected in all sera containing B19-specific IgM antibodies but were also found in a small number of sera collected from healthy blood donors with no history of recent B19 infection. CONCLUSION: This study demonstrates the usefulness of assays employing rVP1 and rVP2 B19 antigens for detecting B19-specific antibodies. The use of IgM-specific ELISAs allows the processing of large numbers of samples and the absence of non-specific reactivity in the IFA may indicate a role for this assay as a confirmatory test.
Journal Article↗
Genomic variation of human immunodeficiency virus type 1 (HIV-1): molecular analyses of HIV-1 in sequential blood samples and various organs obtained at autopsy.
Length polymorphisms and partial nucleotide sequences were determined for the hypervariable regions, V1 to V5, of the human immunodeficiency virus type 1 (HIV-1) env gene obtained from proviral DNA of sequential peripheral blood samples, from viral RNA in plasma, and from proviral DNA obtained from different organs of individuals at autopsy. The lengths of several env regions of HIV-1 proviral DNA differed markedly when obtained from different organs of an individual. Nucleotide sequences of the hypervariable V3 region of HIV-1 obtained from different organs of one patient demonstrated distinct viral variants. Most proviral DNA sequences found in organs were also present in viral RNA obtained from plasma. The majority of HIV-1 V3 variants present in the lymph tissue could be found in the plasma viral population obtained at autopsy and in the sequential blood samples obtained before death, but were absent from the cardiac blood provirus population obtained at autopsy. However, sequence variants found in the brain proviral DNA were not detected in either plasma or the sequential blood samples. Sequence differences were observed at the apex of the V3 loop between HIV-1 variants present in sequential blood samples and in blood lymphocytes and nervous tissue, lymph tissue and plasma obtained post-mortem. The potential effect of lymph tissue on the long-term persistence of different viral variants is discussed. Virus obtained from the two sequential blood samples produced syncytia in primary cultures and was easily transmitted to the continuous JM cell line. Consensus (majority) V3 loop sequences determined for the adapted viruses demonstrated that some, but not all, sequences were represented within the in vivo viral population.
Acquired Immunodeficiency Syndrome↗
Sequence analysis of two porcine rotaviruses differing in growth in vitro and in pathogenicity: distinct VP4 sequences and conservation of NS53, VP6 and VP7 genes.
The VP4, VP7, NS53 and VP6 genes of two porcine rotavirus variants which differ in their in vitro growth properties and pathogenicity have been cloned and sequenced. The VP4 genes show only 67.2% nucleic acid and 70.6% amino acid identity. The VP4 gene of one variant (4S) is closely related to that of the bovine UK rotavirus strain, whereas the VP4 gene of the other variant (4F) is only distantly related to known VP4 genes and is likely to represent a new P serotype. In contrast the NS53 (VP5), VP6 and VP7 genes of the 4F and 4S variants show greater than 99% nucleotide and amino acid identity, indicating that the two viruses are genetically related by a reassortment event. The implications for the role of VP4 in the determination of in vitro growth characteristics and pathogenicity are discussed.
Amino Acid Sequence↗
Detection of immunoglobulin M (IgM), IgA, and IgG Norwalk virus-specific antibodies by indirect enzyme-linked immunosorbent assay with baculovirus-expressed Norwalk virus capsid antigen in adult volunteers challenged with Norwalk virus.
Pre- and postexposure sera collected from 17 adult volunteers challenged with Norwalk virus as described previously (D. Y. Graham, X. Jiang, T. Tanaka, A. Opekun, P. Madore, and M. K. Estes, J. Infect. Dis. 170:34-43, 1994) were examined for Norwalk virus-specific immunoglobulin M (IgM), IgA, and IgG by indirect enzyme-linked immunosorbent assays with recombinant Norwalk virus antigen bound to the solid phase. Sixteen of the 17 volunteers had evidence of past infection, all presenting with preexisting IgG antibody of high avidity; only one volunteer had no evidence of previous infection. Virus infection was detected in 14 of the 16 volunteers with evidence of past infection, and 9 of the infected volunteers had symptomatic illness. A significant rise in both virus-specific IgA and IgG titers was detected after challenge in all of the volunteers who became ill. Five of the asymptomatic volunteers who were infected had rising titers of virus-specific IgG, but only two of the five had a concomitant rise in their virus-specific IgA antibody titers. Antibody rises were detectable in eight of nine ill volunteers 8 to 11 days after challenge but in the asymptomatic volunteers only after more than 15 days had elapsed. Virus-specific IgM was detected after challenge in all 14 infected volunteers. Between symptomatic and asymptomatic volunteers there were no significant differences in titers of virus-specific IgG and IgA in serum before challenge; however, there were significantly higher titers in symptomatic volunteers between 8 and > 90 days after challenge for virus-specific IgG and 8 and 24 days after challenge for virus-specific IgA.
Adult↗
Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability.
A group A rotavirus isolated from a lamb with diarrhea in Qinhai province, China, was serially passaged in fetal calf kidney cells. In passage 96, rearrangements of RNA segments 5 and 6 of the viral genome were found. Here we report the nucleotide and predicted amino acid sequences of normal and rearranged RNA 6, coding for the major inner capsid protein VP6. In comparison with the normal gene (N6), the rearranged RNA 6 (R6) contained the normal open reading frame followed by a 473-nucleotide (nt) duplication of the gene beginning 23 nt after the termination codon. The duplicated region starts at nt 768 and runs through to the 3' end of the gene. In accordance with the nucleotide sequence of the rearranged RNA 6, a normal-length VP6 product was found in cells infected with the mutant. However, a single-amino-acid change from proline to glutamine at position 309 slightly affected the electrophoretic mobility of the VP6 monomer of the R6 mutant and reduced the stability of VP6 trimers on gels and at low pH values compared with the normal gene product. The degree of relatedness of VP6 of the Chinese lamb rotavirus Lp14 to those of other group A rotaviruses was determined.
Animals↗
Sequence diversity of small, round-structured viruses in the Norwalk virus group.
We have determined the nucleotide sequences of a highly conserved region of the RNA-dependent RNA polymerase of the prototype Snow Mountain agent (SMA) and of four other small, round-structured viruses (antigenically Norwalk virus [NV]-like or SMA-like) following reverse transcription-PCR amplification of viral RNA obtained from human stools. The stool samples were either from volunteers administered SMA or from sporadic cases and outbreaks of gastroenteritis that occurred in Japan and the United Kingdom between 1984 and 1992. The GLPSG and YGDD RNA polymerase motifs were in the proper locations in the sequences of the five SRSVs, but each sequence was distinct from the 8FIIa prototype NV sequence and from each other. Analysis of the sequences and reactivities in a new NV antigen enzyme-linked immunosorbent assay showed that the five viruses could be divided into two groups (serogroups) with NV and SMA, respectively, being the prototypes. The sequences of the capsid region and a nonstructural region (2C) were determined from one strain from each group. One virus (SRSV-KY-89/89/J), isolated in Japan and antigenically similar to the prototype NV (isolated 21 years earlier in Ohio), showed a remarkable level of sequence similarity to NV. KY-89 and the 8FIIa NV showed 87.2% nucleotide similarity over 2,516 continuous nucleotides amounting to 96 to 98.9% amino acid similarity in three distinct domains in two open reading frames. Between the prototype SMA and NV, the polymerase region showed 63% nucleotide and 59% amino acid similarity, respectively. Two other antigenically SMA-like isolates (SRSV-925/92/UK and SRSV-OTH-25/89/J), from the United Kingdom and Japan, showed 80% nucleotide and 88 to 92% amino acid similarity in the polymerase region to the prototype SMA isolated 16 and 13 years earlier in the United States. The capsid region of the antigenically SMA-like OTH-25 virus showed 53% nucleotide and 65% amino acid similarity to the prototype NV capsid region. Domains of sequence diversity and conversation were identified within the capsid protein of these two distinct prototype serotypes of virus. These results indicate that NV-like and SMA-like agents are still circulating, and sequence comparisons will be useful to identify and classify distinct viruses in the NV group.
Base Sequence↗
Nucleotide sequences of the VP4 and VP7 genes of a Chinese lamb rotavirus: evidence for a new P type in a G10 type virus.
The VP7 and VP4 genes of a group A rotavirus (Lp14) isolated from a lamb with diarrhea in Qinhai province, China, in 1981 were reverse transcribed, amplified by PCR, cloned, and sequenced. The virus was found to possess a VP7 gene, classifying it as a G10 type, and a VP4 gene which showed only limited homology with any other known VP4 gene, suggesting its classification as a new P type and providing further evidence for the vast diversity of group A rotaviruses.
Amino Acid Sequence↗