Detection of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae merozoite surface protein 1-p19 antibodies in human malaria patients and experimentally infected nonhuman primates.

Approximately 3.2 billion people live in areas where malaria is endemic, and WHO estimates that 350 to 500 million malaria cases occur each year worldwide. This high prevalence, and the high frequency of international travel, creates significant risk for the exportation of malaria to countries where malaria is not endemic and for the introduction of malaria organisms into the blood supply. Since all four human infectious Plasmodium species have been transmitted by blood transfusion, we sought to develop an enzyme-linked immunosorbent assay (ELISA) capable of detecting antibodies elicited by infection with any of these species. The merozoite surface protein 1 (MSP1), a P. falciparum and P. vivax vaccine candidate with a well-characterized immune response, was selected for use in the assay. The MSP1 genes from P. ovale and P. malariae were cloned and sequenced (L. Birkenmeyer, A. S. Muerhoff, G. Dawson, and S. M. Desai, Am. J. Trop. Med. Hyg. 82:996-1003, 2010), and the carboxyl-terminal p19 regions of all four species were expressed in Escherichia coli. Performance results from individual p19 ELISAs were compared to those of a commercial test (Lab 21 Healthcare Malaria enzyme immunoassay [EIA]). The commercial ELISA detected all malaria patients with P. falciparum or P. vivax infections, as did the corresponding species-specific p19 ELISAs. However, the commercial ELISA detected antibodies in 0/2 and 5/8 individuals with P. malariae and P. ovale infections, respectively, while the p19 assays detected 100% of individuals with confirmed P. malariae or P. ovale infections. In experimentally infected nonhuman primates, the use of MSP1-p19 antigens from all four species resulted in the detection of antibodies within 2 to 10 weeks postinfection. Use of MSP1-p19 antigens from all four Plasmodium species in a single immunoassay would provide significantly improved efficacy compared to existing tests.

Isolation and characterization of the MSP1 genes from Plasmodium malariae and Plasmodium ovale.

The merozoite surface protein 1 (MSP1) is the principal surface antigen of the blood stage form of the Plasmodium parasite. Antibodies recognizing MSP1 are frequently detected following Plasmodium infection, making this protein a significant component of malaria vaccines and diagnostic tests. Although the MSP1 gene sequence has been reported for Plasmodium falciparum and Plasmodium vivax, this gene has not been identified for the other two major human-infectious species, Plasmodium malariae and Plasmodium ovale. MSP1 genes from these two species were isolated from Cameroon blood donor samples. The genes are similar in size to known MSP1 genes and encode proteins with interspecies conserved domains homologous to those identified in other Plasmodium species. Sequence and phylogenetic analysis of all available Plasmodium MSP1 amino acid sequences clearly shows that the Po and Pm MSP1 sequences are truly unique within the Plasmodium genus and not simply Pf or Pv variants.

Microheterogeneous monoclonal antibody subspecies with differential hepatitis C virus core antigen binding properties identified by SEC-HPLC.

A monoclonal antibody directed against the core protein of hepatitis C virus was characterized for its utility in a sandwich antigen immunoassay wherein the mAb was used as the conjugate. Analysis of unconjugated and acridinium-conjugated monoclonal IgG using a silica-based HPLC size exclusion column revealed the existence of a single, symmetrical peak. Subsequent analysis of unconjugated IgG using a methacrylate-based HPLC size exclusion column revealed the presence of two species of IgG, but only by using a low ionic strength mobile phase buffer. Independent conjugation and testing of the two species showed significant differential reactivity towards HCV core antigen. Isoelectric focusing gels indicated subtle differences in the subspecies composition. Measurement of target peptide dissociation constants using fluorescence correlation spectroscopy indicated that the two HPLC column fractions exhibited a two-fold difference in Kd in low salt buffer that disappeared in high salt buffer. ESI-MS analysis of the fractionated IgG peaks revealed a reduction sensitive modification of the IgG and F(ab')2 of approximately 674 Da. In addition, both IgG and F(ab')2 contained two major heavy chain subspecies differing by about 1216 Da that was reduction insensitive. These modifications were present in only the one of the two SEC-HPLC peaks. These results suggest that this monoclonal antibody consists of microheterogeneous subspecies that exhibit different antigen binding properties associated with differences in post-translational modification of the heavy chain variable region. The choice of size exclusion column matrix and buffer composition was critical to the identification of these monoclonal IgG subspecies.

Mutations in the hepatitis C virus core gene are associated with advanced liver disease and hepatocellular carcinoma.

PURPOSE: Hepatitis C virus (HCV) infection can promote the development of hepatocellular carcinoma (HCC). Published data implicate the HCV core gene in oncogenesis. We tested the hypothesis that core gene sequences from HCC patients differ from those of patients without cirrhosis/HCC. EXPERIMENTAL DESIGN: Full-length HCV sequences from HCC patients and controls were obtained from the investigators and GenBank and compared with each other. A logistic regression model was developed to predict the HCC risk of individual point mutations and other sequence features. Mutations in partial sequences (bases 36-288) from HCC patients and controls were also analyzed. The first base of the AUG start codon was designated position 1. RESULTS: A logistic regression model developed through analysis of full-length core gene sequences identified seven polymorphisms significantly associated with increased HCC risk (36G/C, 209A, 271U/C, 309A/C, 435A/C, 481A, and 546A/C) and an interaction term (for 209A-271U/C) that had an odds ratio <1.0. Three of these polymorphisms could be analyzed in the partial sequences. Two of them, 36G/C and 209A, were again associated with increased HCC risk, but 271U/C was not. The odds ratio of 209A-271U/C was not significant. CONCLUSIONS: HCV core genes from patients with and without HCC differ at several positions. Of interest, 209A has been associated with IFN resistance and HCC in previous studies. Our findings suggest that HCV core gene sequence data might provide useful information about HCC risk. Prospective investigation is needed to establish the temporal relationship between appearance of the viral mutations and development of HCC.

Internal initiation stimulates production of p8 minicore, a member of a newly discovered family of hepatitis C virus core protein isoforms.

The hepatitis C virus (HCV) core gene is more conserved at the nucleic acid level than is necessary to preserve the sequence of the core protein, suggesting that it contains information for additional functions. We used a battery of anticore antibodies to test the hypothesis that the core gene directs the synthesis of core protein isoforms. Infectious viruses, replicons, and RNA transcripts expressed a p8 minicore containing the C-terminal portion of the p21 core protein and lacking the N-terminal portion. An interferon resistance mutation, U271A, which creates an AUG at codon 91, upregulated p8 expression in Con1 replicons, suggesting that p8 is produced by an internal initiation event and that 91-AUG is the preferred, but not the required, initiation codon. Synthesis of p8 was independent of p21, as shown by the abundant production of p8 from transcripts containing an UAG stop codon that blocked p21 production. Three infectious viruses, JFH-1 (2a core), J6/JFH (2a core), and H77/JFH (1a core), and a bicistronic construct, Bi-H77/JFH, all expressed both p8 and larger isoforms. The family of minicores ranges in size from 8 to 14 kDa. All lack the N-terminal portion of the p21 core. In conclusion, the core gene contains an internal signal that stimulates the initiation of protein synthesis at or near codon 91, leading to the production of p8. Infectious viruses of both genotype 1 and 2 HCV express a family of larger isoforms, in addition to p8. Minicores lack significant portions of the RNA binding domain of p21 core. Studies are under way to determine their functions.

Genotype dependence of peptide-based immunoassays for the detection of HCV core antibodies.

Detection of hepatitis C virus (HCV) antibodies is partially influenced by the genotype of the infecting isolate. Immunoassays using genotype-1a-derived recombinants or peptides results in diminished reactivity among individuals infected with heterologous genotypes. We examined the magnitude of this effect on detection of core antibodies by using genotype-1a-derived core peptide immunoassays to test 254 HCV anti-core-positive individuals infected with genotypes 1-4 or 6. Peptides corresponding to amino acids 1-18, 10-24, and 11-28 reacted with 60%, 89%, and 85% of all samples, respectively. Peptide 1-18 detected 78% of individuals infected with genotype-1 or 2 but only 43% of those infected with genotypes 3, 4, or 6. Genotype-dependent reactivity was also observed for peptides 10-24 and 11-28. The use of a 34-mer peptide (encompassing amino acids 10-43) within the immunodominant region detected antibodies in 100% of specimens, thereby eliminating the genotype-dependent antibody detection observed with shorter peptides. Sequence differences between peptides and core of the infecting isolate did not entirely account for the genotype-dependent reactivity since some individuals displayed reactivity to peptides containing up to seven amino acid differences relative to the sequence of the infecting isolate, while others with identical core sequences had little or no reactivity. Thus, HCV core sequence divergence accounts for only a portion of the differential core antibody detectability observed when non-type-specific peptides are used. Differences in immune response between individuals infected with identical isolates also plays a significant role in core antibody detection using short peptides.

A previously unrecognized sixth genotype of GB virus C revealed by analysis of 5'-untranslated region sequences.

GB virus C (GBV-C) is a positive-strand RNA virus that infects a large proportion of the world's human population. It has been classified tentatively as a member of the Flaviviridae family and has been shown to exist as a group of five closely related genotypes. Recently, we reported the first full-length genome sequence of a genotype 5 isolate from South Africa. As part of the analysis of that sequence, a phylogenetic tree was elucidated from the 5'-untranslated region (UTR) that showed excellent congruence to the tree produced by analysis of complete open reading frame sequences. When 5'-UTR analysis was broadened subsequently to include additional isolates from around the globe, a heretofore unrecognized GBV-C genotype was discovered in Indonesia. When first reported in 2000, these isolates were described as constituting a novel fifth genotype. However, comparison to isolates from the then-known fourth and fifth genotypes (from Myanmar/Vietnam and South Africa, respectively) was not performed. A dataset of 121 GBV-C 5'-UTR sequences was complied and included representatives of the fourth and fifth genotypes as well as the "novel" Indonesian sequences and demonstrated, with strong support via bootstrap analysis, the existence of a sixth GBV-C genotype among infected individuals in Indonesia. The discovery of this sixth genotype emphasizes the diverse nature of GBV-C isolates and may have important implications for the interpretation of studies involving GBV-C/HIV co-infected individuals.

African origin of GB virus C determined by phylogenetic analysis of a complete genotype 5 genome from South Africa.

GB virus C (GBV-C), a positive-strand RNA virus, currently infects approximately one-sixth of the world's population. This virus exists as a collection of genotypes whose global distribution correlates with geographical origin. Genotyping of GBV-C isolates by phylogenetic analysis has relied upon the use of 5'-untranslated region (5'-UTR) sequences, however, complete genome sequences are used to demonstrate definitively their existence and geographical correlation. Initial identification of the fifth genotype from South Africa was based upon phylogenetic analysis of the 5'-UTR. It was sought to confirm this classification by analysis of full-length E2 genes from South African isolates and by analysis of a complete genotype 5 genome. Analysis of full-length E2 genes from 28 GBV-C-infected South African individuals revealed the existence of a unique group of 18 isolates, distinct from the other four genotypes. Bootstrap analysis provided strong support (95 %) for this fifth group. The remaining isolates were either genotype 1 (n=8) or 2 (n=2). Analysis of human E2 gene sequences, with the E2 gene from the chimpanzee variant GBV-Ctro included as the outgroup, produced a tree rooted on the genotype 1 branch. The complete genome nucleotide sequence of South African genotype 5 isolate D50 was determined. Phylogenetic analysis of the 5'-UTR and open reading frame produced congruent trees that grouped the sequences into five major genotypes. Inclusion of the corresponding region of the chimpanzee isolate GBV-Ctro in the analysis produced trees rooted on the branch leading to the genotype 5 isolate D50, suggesting an ancient African origin of GBV-C.

Enzyme-linked immunosorbent assays using recombinant envelope protein expressed in COS-1 and Drosophila S2 cells for detection of West Nile virus immunoglobulin M in serum or cerebrospinal fluid.

Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.

A non-isotopic method for the determination of activity of the thermostable NAD-dependent DNA ligase from Thermus thermophilus HB8.

A simple and accurate method for determination of enzymatic activity of the NAD-dependent DNA ligase of Thermus thermophilus HB8 has been developed that requires no radiolabeled substrates. lambda-DNA digested with BstEII provides two substrate DNA molecules (fragments 1 and 4) containing 12 base pair cohesive ends that are stably annealed at the assay temperature of 45 degrees C. One cohesive end unit is defined as the amount of enzyme required to achieve 50% ligation of fragment 1 in 15 min at 45 degrees C. Percent ligation is determined by analysis of reaction products, produced in reactions containing serial dilutions of enzyme, separated by agarose gel electrophoresis and photographed using a digital imaging device. Imaging software quantifies the amounts of fragment 1 and non-substrate fragment 7 present in the each lane (reaction). The latter is used to normalize the amount of fragment 1. This normalization process corrects for variations in sample loading, electrophoretic artifacts, and optical distortion of the gel image. A negative control containing no enzyme allows calculation of percent substrate ligated into product. Unit activity is then calculated from a dose-response curve in which percent of fragment 1 ligated is plotted against the log(10) of the enzyme dilution factor.

Experimental infection of rhesus macaques with West Nile virus: level and duration of viremia and kinetics of the antibody response after infection.

Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1-2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors.

Combination HCV core antigen and antibody assay on a fully automated chemiluminescence analyzer.

BACKGROUND: HCV exposure among blood donors is serologically determined by detection of antibodies to HCV (anti-HCV); however, the recent development of an assay for the detection of HCV core antigen identifies infection before anti-HCV development. Simultaneous detection of HCV core antigen and anti-HCV would shorten the window period before seroconversion over conventional HCV antibody screening assays. STUDY DESIGN AND METHODS: A prototype chemiluminescent immunoassay was developed for simultaneous detection of HCV core antigen and anti-HCV in human sera and plasma. The assay was performed on a single-channel instrument representing an automated serologic analyzer (PRISM, Abbott Laboratories) system. Sensitivity and specificity were evaluated by testing 23 HCV seroconversion panels and plasma or sera from volunteer blood donors. RESULTS: The prototype HCV core antigen and antibody combination assay detected 80 of 89 (89.9% ) HCV RNA-positive and antibody-negative specimens from 23 panels, thereby reducing the seroconversion window period by an average of 34.3 days compared to PRISM HCV antibody detection. All PRISM HCV antibody-positive specimens were detected by the combination assay for a relative sensitivity of 100 percent. The repeatedly reactive rate was 0.20 percent based on testing of 3017 screened anti-HCV-negative sera and plasma. CONCLUSIONS: The prototype combination assay was shown to detect HCV core antigen and anti-HCV simultaneously and significantly closed the time gap between the initial detection of HCV RNA and the first appearance of detectable antibodies to HCV.

Selectively primed adaptive driver RDA (SPAD-RDA): an improved method for subtractive hybridization.

A modification of the Representational Difference Analysis (RDA) method for subtractive hybridization, termed Selectively Primed Adaptive Driver (SPAD) RDA, is described. It differs from conventional RDA primarily in the manner by which initial driver (D) and tester (T) amplicon complexities are determined, and by optimizing the composition of D with respect to T for each round of subtraction. Total nucleic acid is extracted from serum or plasma and converted to double-stranded DNA/cDNA. A polymerase chain reaction (PCR) primer containing a selective nucleotide(s) at its 3'-end is used to generate amplicons of reduced complexity. Parallel subtractions are carried out, D vs. T (DT) for enrichment of tester-unique sequences and D vs. D (Driver Control or DC) to generate an optimized driver for use in the subsequent round. Following each round, agarose gel electrophoresis is used to visually identify any DT-unique bands through a side-by-side comparison of DT and DC subtraction products. In comparison to conventional RDA, SPAD-RDA achieved greater enrichment of viral sequences from an HCV infected chimpanzee, resulting in isolation of 13.7% of the viral genome, and an overall enrichment for HCV sequences of 239-fold. Virus fragments were also obtained from an HCV-infected human sample subtracted against non-paired human driver sequences. J. Med. Virol. 71:150-159, 2003.

GB virus C genotype determination in GB virus-C/HIV co-infected individuals.

Several recent studies have indicated that patients infected with human immunodeficiency virus (HIV) exhibit a beneficial effect of co-infection with GB virus C (GBV-C). The benefit is demonstrated by slower progression to acquired immunodeficiency syndrome (AIDS) and prolonged survival time after the development of AIDS. In some but not all studies, a significant association between GBV-C/HIV co-infection and increased CD4(+) cell counts has been reported. To understand further the possible role that GBV-C might play in the reduced morbidity and mortality among HIV-infected patients, we sought to examine the presence of different GBV-C genotypes in a cohort of co-infected patients. PCR products derived from the 5'-untranslated region (5'-UTR) and the second envelope gene (E2) were sequenced directly and genotyped by phylogenetic analysis. While 5'-UTR analysis delineated the major type, analysis of the complete E2 gene was required for identification of group 2 subtypes, designated 2a and 2b. Among 35 patients tested, GBV-C genotype was determined for 33: two patients were infected with genotype 1, 12 with type 2a, and 19 with type 2b. Clinical data were available for 25 genotyped patients: one infected with genotype 1, nine with genotype 2a, and 15 with type 2b. CD4 cell counts tended to be lower in patients infected with genotype 2a compared with those with genotype 2b (310 +/- 136 vs 430 +/- 199, P = 0.054). Additional studies with larger cohorts from separate geographical regions are needed to determine whether a particular GBV-C genotype is associated with reduced morbidity or mortality among HIV co-infected patients.

Detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral L1 genome segment.

A rapid, reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of reovirus RNA in cell culture is described. Total nucleic acids are extracted from a small volume of cell culture supernatant and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR. The PCR primers correspond to sequences conserved between prototype reovirus strains type 1 Lang, type 2 Jones, and type 3 Dearing, as well as those of several reovirus field-isolate strains. Reactions are analyzed by agarose gel electrophoresis, and samples showing a band of the appropriate size in the first and second amplification, or in the second amplification alone, are designated as positive. This protocol allows for the rapid and sensitive detection of reovirus in cell culture. The RT-PCR methods described below can easily be adapted to the amplification of reovirus from other media, including preserved tissues, clinical specimens, and water.

Detection of mammalian reovirus RNA by using reverse transcription-PCR: sequence diversity within the lambda3-encoding L1 gene.

Reoviruses infect virtually all mammalian species, and infection of humans is associated with mild gastrointestinal or upper respiratory illnesses. To improve reovirus detection strategies, we developed a reverse transcription-PCR technique to amplify a fragment of the reovirus L1 gene segment. This assay was capable of detecting 44 of 44 reovirus field isolate strains and was sufficiently sensitive to detect nearly a single viral particle (1.16 +/- 0.13) per PCR of prototype strain type 3 Dearing. Pairwise comparisons of the 44 partial L1 gene sequences revealed that nucleotide variability ranged from 0 to 24.7%, with most of the nucleotide polymorphism occurring at synonymous positions. Phylogenetic trees generated from amplified L1 gene sequences suggest that multiple alleles of the L1 gene cocirculate in nature and that genetic diversity of the L1 gene is largely independent of the host species, geographic locale, or date of isolation. Phylogenetic trees constructed from the L1 gene sequences are distinct from those constructed from the four reovirus S-class gene segments, which supports the hypothesis that reovirus gene segments reassort in nature. This study establishes a new sensitive and specific technique for the identification of mammalian reoviruses and enhances our understanding of reovirus evolution.

Improved detection systems for TT virus reveal high prevalence in humans, non-human primates and farm animals.

TT virus is a newly described agent infecting humans. Initially isolated from a patient (initials T.T.) with unexplained hepatitis, the virus has since been found in both normal and diseased individuals. In the present study, we utilized genomic-length sequences from distinct genotypes of TT virus to design PCR-based assays using conserved oligonucleotide primers from three independent regions of the virus genome. Each of the three assays was found to be superior to the PCR-based assays previously published. The most sensitive of the new assays was utilized to demonstrate the prevalence of TT virus to be at least 34.1% in volunteer blood donors, 39.6% in commercial blood donors, 59.6% in non-A-GB hepatitis cases, 81.7% in injectable drug users and 95.9% in haemophiliacs. In an attempt to identify a possible source of human infection, we found TT virus sequences to be present in 19% of chickens, 20% of pigs, 25% of cows and 30% of sheep. Sequence determination and phylogenetic analyses demonstrated that isolates from farm animals were not genetically distinct from those found in humans. This study clearly demonstrates that previously reported PCR assays dramatically underestimate the true prevalence of TT virus within the human population. Due to the high rate of infection in both blood donors and those with non-A-GB hepatitis, these results question the causal role of TT virus in cases of unexplained hepatitis. Further, it is possible that domesticated farm animals serve as a source of human infection.