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Biomedical subjects

S M Desai

Publications and source records attributed to S M Desai.

At least 37 records · Page 2Linked to original sources
PubMed1997

Impact of pretransplantation GB virus C infection on the outcome of renal transplantation.

Among renal transplant recipients with posttransplantation liver disease, the etiology remains unknown in 10 to 16% of patients. The discovery of yet another parenterally transmitted hepatitis virus, GB virus C (GBV-C), has opened avenues to study the prevalence and risk factors for GBV-C infection among patients undergoing renal transplantation and its impact on posttransplantation clinical outcomes. A cohort of 103 randomly selected recipients of kidneys were examined from anti-hepatitis C virus (HCV)-negative donors between 1986 and 1990. Pretransplantation sera were available in 99 of 103 (96%) recipients and were tested for anti-HCV, using a second-generation ELISA, and for GBV-C RNA by reverse transcription PCR. Pretransplantation GBV-C RNA was present in 18 of 99 (18%, 95% confidence interval [CI], 17.2 to 18.8%) recipients. GBV-C RNA was present in 5 of 22 (23%) anti-HCV-positive recipients compared with 13 of 77 (17%) anti-HCV-negative recipients (P = 0.53). The median number of pretransplantation blood transfusion among recipients with GBV-C RNA before transplantation was significantly higher than among recipients without GBV-C RNA (10 versus 7, P = 0.05). Posttransplantation liver disease and non-A, non-B hepatitis (NANBH) was observed in 35 and 18%, respectively, of GBV-C RNA-positive recipients compared with 28 and 10%, respectively, of GBV-C RNA-negative recipients. Using Cox regression analysis, the relative risk (RR) of posttransplantation liver disease among recipients with GBV-C RNA before transplantation was 1.37 (95% CI, 0.55 to 3.41), and posttransplantation NANBH was 2.09 (95% CI, 0.64 to 6.79). The RR of graft loss and death were not increased (0.88 and 0.92, respectively). When adjusted for pretransplantation anti-HCV, the RR of posttransplantation liver disease, NANBH, graft loss, and death did not change appreciably. In summary, although a higher risk of posttransplantation liver disease was observed among recipients with pretransplantation GBV-C infection, the analyses presented here do not allow for a precise estimate of this risk.

Adult↗
PubMed1996

Expression of the GB virus C E2 glycoprotein using the Semliki Forest virus vector system and its utility as a serologic marker.

A 336-amino-acid segment of the GB virus C second envelope protein (E2) has been produced in BHK-21 cells using the Semliki Forest virus vector system. Secretion of this protein was facilitated by deletion of a hydrophobic region at the C-terminus that may represent the membrane anchoring domain. The E2 protein recovered from the culture supernatant exhibited a molecular mass of approximately 52 kDa, with the increase in size relative to the polyprotein backbone being contributed by N-linked glycosylation. A radioimmunoprecipitation assay using GBV-C E2 was developed to test for the presence of antibodies against this protein in human sera. The prevalence of antibodies to E2 was high among injection drug users and other individuals at risk for acquiring parenterally transmitted agents. There was a much higher percentage of anti-E2 seropositivity in GBV-C RT-PCR negative compared to GBV-C RT-PCR positive samples from these populations. In addition, serial samples from patients transfused with blood containing GBV-C showed seroconversion to anti-E2 positivity and loss of GBV-C viremia as measured by RT-PCR within 11 months of transfusion in five of seven individuals. Thus, this system provided a rapid means to identify GBV-C E2 as a useful antigen for the study of GBV-C exposure.

Base Sequence↗
PubMed1996

Sequence and genomic organization of GBV-C: a novel member of the flaviviridae associated with human non-A-E hepatitis.

Recently, sequences from a novel virus, termed GB virus C (GBV-C), were identified in serum from several patients with cryptogenic hepatitis. In the present study, the nucleotide sequence of this virus has been extended to near-genome length. GBV-C encodes a putative single large polyprotein in which the structural proteins are positioned at the N-terminal end, with the non-structural proteins located at the C-terminal end. Amino acid sequence analysis of this large polyprotein reveals the presence of protease, helicase, and replicase motifs. Sequence alignments of the polyprotein followed by phylogenetic analyses suggest that GBV-C is a member of the Flaviviridae, most closely related to the recently described GB virus A.

Amino Acid Sequence↗
PubMed1996

Identification of antigenic regions in the GB hepatitis viruses GBV-A, GBV-B, and GBV-C.

The genomes of two novel members of the Flaviviridae associated with GB agent hepatitis (GB viruses A and B) were cloned and sequenced recently. The genome of a third novel virus (GB virus C), related to but distinct from GB viruses A and B, has also been identified and characterized. Overlapping clones encompassing the large open reading frames of these three viruses have been expressed in E. coli as CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS) fusion proteins. Bacterial lysates were subjected to Western blot analyses using sera from GB agent-infected tamarins and human sera from various individuals with or "at risk" for non-A, non-B, non-C, non-D, non-E hepatitis. Antigenic regions were identified in the putative NS3, NS4, and NS5 proteins from all three viruses. An antigenic region was also identified in the putative core protein of GB virus B. Many of the clones identified originally as encoding antigenic proteins were quite large. To map these regions more narrowly, smaller overlapping clones were generated by polymerase chain reaction (PCR), expressed as recombinant CKS fusion proteins and tested by Western blot. Additionally, a lambda gt11 expression library was generated from infectious tamarin sera and immunoscreened. These studies have identified at least three epitopes in GB virus A, five epitopes in GB virus B and four epitopes in GB virus C.

Animals↗
PubMed1996

Consensus oligonucleotide primers for the detection of GB virus C in human cryptogenic hepatitis.

Recently, sequences from a putative member of the Flaviviridae, GB virus C (GBV-C), were isolated from the serum of patients with cryptogenic hepatitis. These sequences were 83-99% identical at the nucleotide level. Because of the divergence between these GBV-C isolates, it is likely that the PCR-based detection assay yields false negatives, underestimating dramatically the true prevalence of GBV-C in human hepatitis. We report the design of a GBV-C consensus oligonucleotide primer pair that is superior to those originally described. These primers identify GBV-C sequences in cases of cryptogenic hepatitis, allowing a better estimation of the prevalence of this virus in human populations.

Base Sequence↗
PubMed1996

Identification of conserved nucleotide sequences within the GB virus C 5'-untranslated region: design of PCR primers for detection of viral RNA.

Recently, the discovery of a new human RNA virus, GB virus C (GBV-C), was reported. GBV-C was isolated from the serum of a West African individual using degenerate oligonucleotide PCR primers designed from a consensus sequence of the NS3 helicase genes of hepatitis C virus (HCV), GBV-A, and GBV-B. Seven other individuals were shown to be infected with GBV-C via RT-PCR using these primers. Subsequently, degenerate PCR primers based upon a consensus sequence of the eight original isolates were designed. These primers were shown to be superior to the original set. However, since they were derived from a region of the viral genome exhibiting up to 17% nucleotide sequence divergence, mismatch between the primers and template may result in an underestimation of the true GBV-C prevalence. To overcome this potential problem, we obtained the sequences at the 5'-untranslated region (UTR) of the GBV-C genome from 35 infected individuals and identified regions of high sequence conservation among the isolates. We describe the design and testing of PCR primers derived from conserved sequences within the 5'-UTR of the GBV-C genome. These primers were shown to be as effective as the helicase-derived primers in detecting GBV-C RNA in human sera.

Base Sequence↗
PubMed1996

Sequence heterogeneity within the 5'-terminal region of the hepatitis GB virus C genome and evidence for genotypes.

BACKGROUND: GB virus C is a positive-strand RNA virus that is associated with hepatitis in humans. GB virus C bears some resemblance to hepatitis C virus in its genomic sequence and organization. However, unlike hepatitis C virus, an open reading frame possessing a complete core protein was not identified in the original isolate. METHODS: To verify the sequence at the 5'-end of the GB virus C genome, we amplified approximately 600 nucleotides from this region from 35 globally distributed individuals. The nucleotide sequences were translated in all possible reading frames and then examined for conserved motifs indicative of nucleocapsid or core-like peptides. RESULTS: Forty-two unique GB virus C sequences were obtained from the 35 individuals. The deduced amino acid sequences upstream of the putative E1 gene from each isolate varied in length and composition, such that a conserved core-like sequence was not apparent. No core-like sequences were evident in the other reading frames. There was, however, a single methionine codon held in common among all isolates, although it was located very near the presumed amino-terminus of the putative E1 protein. Further analysis of the sequences for their evolutionary relatedness demonstrated the existence of five GB virus C subtypes that demonstrated a significant correlation with geographic distribution. CONCLUSIONS: GB virus C differs from hepatitis C virus and GB virus B in that it does not encode a nucleocapsid or core protein. The existence of GB virus C subtypes emphasizes the importance of investigating the correlation between infecting subtype and the severity of liver disease and/or responsiveness to treatment of GB virus C-associated hepatitis.

Base Sequence↗
PubMed1996

Molecular cloning and characterization of a GB virus C isolate from a patient with non-A-E hepatitis.

Recently, the isolation of a novel virus, GB virus C (GBV-C), associated with cryptogenic hepatitis has been reported. Following the molecular cloning of this virus genome, it became apparent that the genomic sequence did not encode a protein resembling a nucleocapsid or core-like protein similar to those observed in other flaviviruses, pestiviruses, hepatitis C virus (HCV) and GB virus B. Similar findings were subsequently observed in the cloning of two viral genomes representing isolates of GBV-C, namely hepatitis G virus (HGV). To verify the presence or absence of a viral nucleocapsid protein, identify conserved protein motifs and determine the overall genomic variability, an additional virus isolate has been characterized. Here we report the full-length genomic sequence of GBV-C(EA), isolated from an East African suffering from acute non-A-E hepatitis. GBV-C(EA) was compared with the prototype West African isolate (GBV-C) and the two HGV isolates from the United States. The analyses demonstrate several characteristics of these novel viruses. (1) The degree of variability within the 5' nontranslated region (NTR) approximates that observed between HCV isolates. (2) The nucleotide sequence of the coding region and the 3' NTR is highly conserved between these isolates, in contrast to the extensive variability observed between HCV isolates from distinct geographical locations. (3) There is a high degree of amino acid conservation across the precursor polyproteins of these isolates; most striking is the lack of 'hypervariable' regions within the envelope proteins. (4) There appears to be no nucleocapsid protein near the amino terminus of the GBV-C/HGV polyproteins.

Acute Disease↗
PubMed1996

Species-specific variants of GB virus A in captive monkeys .

Sequences from the putative 5' nontranslated region of GB virus A were isolated from mystax, owl monkeys, and tamarins. Though sequences of isolates from each animal species are virtually identical at the nucleotide level (95%), isolates from different species are dramatically different (52 to 79% identical) and genetically cluster on this basis.

Animals↗
PubMed1996

Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization.

GB viruses A and C (GBV-A and GBV-C) are two recently described RNA viruses which appear to be members of the Flaviviridae. Although both viruses appear to contain long 5' nontranslated regions, the sites of polyprotein initiation and the presence of core-like proteins remain to be determined. Translation studies were undertaken to determine the mechanism and sites of polyprotein initiation in GBV-A and GBV-C. Rabbit reticulocyte lysates programmed with monocistronic RNAs containing 5' ends of GBV-A or GBV-C fused in-frame with the chloramphenicol acetyltransferase (CAT) open reading frame generated GBV-CAT fusion proteins in vitro. Site-specific mutagenesis and N-terminal sequencing located the sites of translation initiation immediately upstream of the putative signal sequence for the GBV E1 envelope glycoproteins. Efficient translation of the monocistronic GBV-CAT RNAs required the inclusion of GBV coding sequences. This, coupled with the presence of at least 523 nucleotides of 5' nontranslated RNA containing multiple AUG codons, suggests that translation initiation of these RNAs did not utilize a ribosome scanning mechanism. Translation of bicistronic RNAs containing 5' nontranslated sequences within the intercistronic space was consistent with the presence of a weakly active internal ribosome entry site in both GBV-A and GBV-C. Secondary structure predictions indicate that the 5' ends of these viruses assume similar complex structures distinct from those identified in the internal ribosome entry site-containing picornaviruses, pestiviruses, and hepatitis C viruses. The data indicate that GBV-A and GBV-C are unique members of the Flaviviridae that do not contain core-like proteins at the N termini of their putative polyproteins.

Amino Acid Sequence↗
PubMed1996

Improved management of abdominal undescended testicular tumors with bulky confluent retroperitoneal nodal metastases.

PURPOSE: Germ cell tumors of the abdominal undescended testis associated with confluent bulky retroperitoneal metastases are challenging problems. We report the results of neoadjuvant cisplatin based chemotherapy after diagnosis of germ cell tumors by fine needle aspiration cytology of the abdominal testicular mass. After chemotherapy all patients underwent abdominal orchiectomy with retroperitoneal lymph node dissection for residual nonseminomatous germ cell tumors or radiotherapy for pure seminomas. MATERIALS AND METHODS: Between 1980 and 1991, 57 of 425 patients (13.4%) with germ cell tumors of the testicle had malignancy in an undescended testis, while 39 (68.4%) had tumor in an abdominal testis with confluent bulky metastasis. Metastatic evaluation included tumor marker studies, chest x-ray and computerized tomography of the abdomen. Among the tumors 29 (74.4%) were large volume seminomas (stages IIc, III and IV) and 10 (25.6%) were large volume nonseminomas. All 39 patients received 3 cycles of induction chemotherapy, and orchiectomy was deferred until its completion (14 received vinblastine, actinomycin D and bleomycin-6, and 25 received bleomycin, etoposide and cisplatin). After evaluation of response, the testis was excised. Overall followup was 2 to 12 years (median 4.6). RESULTS: Of 29 seminomas 14 (48.3%) showed a complete and 11 (37.9%) showed a partial response. The latter tumors were treated subsequently with radiotherapy. Four patients with progressive disease died, for an actuarial survival rate of 86%. Of the 10 patients with nonseminomatous germ cell tumor 2 (20%) had a complete response and 4 had a partial response. All patients with a partial response underwent retroperitoneal lymph node dissection. Overall, 4 patients with progression and 2 with a partial response died, for an actuarial survival rate of 39%. Of 39 post-chemotherapy orchiectomy specimens 24 (61.5%) showed viable tumor cells. Furthermore, 16 of 39 patients (41%) had additional ilioinguinal metastases requiring adjuvant radiotherapy or surgery. CONCLUSIONS: Surgical removal of the primary tumor in an undescended testis with bulky metastasis is difficult. We believe that initial chemotherapy followed by 1-stage surgical removal of the primary and residual metastasis is a favorable option to improve compliance and decrease the incidence of loss to followup. Atypically altered ilioinguinal metastases may necessitate a change in radiotherapy ports and/or retroperitoneal lymph node dissection boundaries. The significantly poorer survival with nonseminomatous germ cell tumor could be due to the fact that 50% of the lesions were stage IV at presentation. However, multivariate analysis showed only tumor histology to be the significant parameter and not initial stage at presentation.

Actuarial Analysis↗
PubMed1995

Identification of two flavivirus-like genomes in the GB hepatitis agent.

A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.

Acute Disease↗
PubMed1995

Isolation of novel virus-like sequences associated with human hepatitis.

Two viruses, GB virus A (GBV-A) and GB virus B (GBV-B), were recently identified in the GB hepatitis agent. Human sera containing antibodies that recognize GBV-A and/or GBV-B recombinant proteins were subjected to polymerase chain reaction studies with degenerate oligonucleotides capable of amplifying a segment of the putative helicase genes from GBV-A, GBV-B or hepatitis C virus. Novel sequences related to members of the Flaviviridae were identified in sera from 12 individuals including 4 individuals with hepatitis. The limited nucleotide sequence identity between GBV-A, GBV-B and HCV sequences suggests that a novel virus, tentatively named GB virus C, may be responsible for some cases of non-A, non-B, non-C, non-D, non-E hepatitis.

Adult↗
PubMed1995

Genomic organization of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis.

The genomes of two positive-strand RNA viruses have recently been cloned from the serum of a GB agent-infected tamarin by using representational difference analysis. The two agent, GB viruses A and B (GBV-A and GBV-B, respectively), have genomes of 9,493 and 9,143 nucleotides, respectively, and single large open reading frames that encode potential polyprotein precursors of 2,972 and 2,864 amino acids, respectively. The genomes of these agents are organized much like those of other pestiviruses and flaviviruses, with genes predicted to encode structural and nonstructural proteins located at the 5' and 3' ends, respectively. Amino acid sequence alignments and subsequent phylogenetic analysis of the RNA-dependent RNA polymerases (RdRps) of GBV-A and GBV-B show that they possess conserved sequence motifs associated with supergroup II RNA polymerases of positive-strand RNA viruses. On the basis of similar analyses, the GBV-A- and GBV-B-encoded helicases show significant identity with the supergroup II helicases of positive-strand RNA viruses. Within the supergroup II RNA polymerases and helicases, GBV-A and GBV-B are most closely related to the hepatitis C virus group. Across their entire open reading frames, the GB agents exhibit 27% amino sequence identity to each other, approximately 28% identity to hepatitis C virus type 1, and approximately 20% identity to either bovine viral diarrhea virus or yellow fever virus. The degree of sequence divergence between GBV-A and GBV-B and other Flaviviridae members demonstrates that the GB agents are representatives of two new genera within the Flaviviridae family.

Amino Acid Sequence↗
PubMed1993

Hypervariable 5'-terminus of hepatitis C virus E2/NS1 encodes antigenically distinct variants.

Synthetic peptides representing sequences encoded at the 5'-terminus of E2/NS1 in hepatitis C virus (HCV) were constructed. Peptides synthesized based on the sequences of four distinct HCV isolates were used to develop enzyme immunoassays (EIAs) for detection of antibodies in chronic HCV patients and in HCV-infected plasma donors. HCV sequence-specific antibodies were detected among patients with chronic HCV from the United States and Italy at frequencies of 22.2% and 55.8%, respectively. Similarly, sequence-specific antibodies were detected in 54.6% of U.S. and 55.6% of Japanese commercial plasma donors who had previous evidence of HCV exposure. Our data support earlier findings of geographic variability among HCV variants. The region encoded by amino acids (aa) 380-436 was shown to contain at least one variant-specific and one conserved epitope. The data further indicate that a majority of patients chronically infected with HCV (58.1% U.S., 68.8% Italy) have antibodies directed to the 5'-terminus of the E2/NS1 gene product. We conclude that genotypic variability within the E2/NS1 gene of HCV results in antigenically distinct variants.

Amino Acid Sequence↗
PubMed1992

Lack of protective immunity against reinfection with hepatitis C virus.

Some individuals infected with hepatitis C virus (HCV) experience multiple episodes of acute hepatitis. It is unclear whether these episodes are due to reinfection with HCV or to reactivation of the original virus infection. Markers of viral replication and host immunity were studied in five chimpanzees sequentially inoculated over a period of 3 years with different HCV strains of proven infectivity. Each rechallenge of a convalescent chimpanzee with the same or a different HCV strain resulted in the reappearance of viremia, which was due to infection with the subsequent challenge virus. The evidence indicates that HCV infection does not elicit protective immunity against reinfection with homologous or heterologous strains, which raises concerns for the development of effective vaccines against HCV.

Acute Disease↗
PubMed1991

Evidence for persistent hepatitis C virus (HCV) infection in hemophiliacs.

Hepatitis C virus (HCV) is the major etiologic agent associated with non-A, non-B hepatitis. This study was designed to assess virologic and serologic markers in hemophiliacs exposed to non-heat-treated and/or virus-inactivated plasma derivatives. Serial bleeds from 48 hemophilic patients were analyzed for the presence of HCV viral RNA sequences as detected by polymerase chain reaction (PCR) and antibodies to structural (core) and nonstructural (C-100 and 33C) proteins by specific dot immunoblot assay. All patients exposed to non-heat-treated products, and four of six patients exposed only to virus inactivated products, had evidence of HCV infection. However, over the 5-yr study period, six exposed patients (13%) consistently lacked detectable anti-C-100 and seven (15%) lost this antibody. HCV viremia (PCR positive) was found in 91% of exposed patients, and was significantly more frequent in HIV seropositive hemophiliacs (P less than 0.05). Six patients had high antibody level to HCV and elevated ALT, but appeared to clear viremia. Four hemophiliacs were HCV seropositive but lacked detectable viremia. These data indicate that hemophiliacs remain persistently infected by HCV and that antibody to the core antigen of HCV is a reliable marker of this transfusion transmissible agent.

Adolescent↗