Larry G. Birkenmeyer

Prevalence of TT virus infection in US blood donors and populations at risk for acquiring parenterally transmitted viruses.

Two overlapping sets of TT virus (TTV)-specific polymerase chain reaction primers were used to test for presence of TTV, which was found in approximately 10% of US volunteer blood donors, 13% of commercial blood donors, and 17% of intravenous drug abusers. The rate of TTV infection among US non-A, non-B, non-C, non-D, non-E hepatitis patients was only 2%. Among commercial blood donors and intravenous drug abusers, only 1%-3% of the TTV-positive individuals were coinfected with GB virus C (GBV-C), a parenterally transmitted virus. This suggests that GBV-C and TTV may have different routes of transmission. Comparison of the sensitivities of 2 TTV polymerase chain reaction (PCR) primer sets showed that the majority of samples were detected with only 1 of the 2 sets. Therefore, previous studies in which only a single PCR primer pair was used may have significantly underestimated the true prevalence of TTV.

Isolation of a GB virus-related genome from a chimpanzee

Recently, two new flaviviruses, GB virus A (GBV‐A) and GB virus B (GBV‐B), were identified in the plasma of a tamarin infected with the hepatitis GB agent. A third virus, GB virus C (GBV‐C), was subsequently identified in humans. In the current study, representational difference analysis (RDA) was used to search for a new virus in the serum of a chimpanzee that developed acute resolving hepatitis following inoculation with a pool of chimpanzee plasma. The plasma pool originated from serial passages of a human sample containing virus‐like particles. Numerous cDNA clones were obtained that exhibited 62–80% identity with GBV‐C. With the exception of the extreme 5′ and 3′ ends, the complete viral genome was sequenced, revealing a single large open reading frame encoding a 2833 amino acid polyprotein that contains two envelope proteins, two proteases, a helicase, and an RNA‐dependent RNA polymerase. Phylogenetic analysis of the new virus indicates that it is closely related to GBV‐C, yet still sufficiently divergent as to be placed in a separate group, tentatively labeled GB virus Ctroglodytes (GBV‐Ctro). Numerous human samples were screened by reverse transcriptase‐polymerase chain reaction (RT‐PCR), but GBV‐Ctro sequence was not detected. However, a second chimpanzee inoculated with the same plasma pool was shown to develop a GBV‐Ctro infection. Although isolated from an Old World primate with hepatitis, the primary host of GBV‐Ctro and any association with disease remains to be determined. J. Med. Virol. 56:44–51, 1998.

Pathogenesis of chronic liver disease in patients with chronic hepatitis B virus infection without serum HBeAg

Chronic hepatitis B in patients lacking hepatitis B e antigen has been attributed to a hepatitis B virus variant (G-to-A mutation at nucleotide 1896 in the precore region of the genome). We therefore assessed the frequency and significance of this variant among 43 United States patients (10 with chronic hepatitis B seropositive for e antigen, 19 seronegative for e antigen, and 14 healthy carriers). Sera were tested for HBV DNA by polymerase chain reaction and branched DNA assay. The A1896 variant was detected by direct sequencing and ligase chain reaction. Serum HBV DNA was more frequently found among patients with e antigenpositive than e antigen-negative chronic hepatitis B. Viral titers were generally higher in those with e antigen. None of the e antigen-positive and only 24% of e antigen-negative patients harbored the A1896 variant. Patients infected with the variant were more often Asian, had had hepatitis B for longer and had higher levels of viral DNA than HBeAg-negative patients with the wild-type virus. The A1896 variant was found exclusively in patients infected with HBV genotypes C and D. Thus, the A1896 variant is uncommon in the United States. The activity of liver disease appears to be more closely related to the level of HBV replication than the presence of mutations at nucleotide 1896 in the genome.

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.

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

ABSTRACT 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.

Engineering of the Hepatitis C Virus Helicase for Enhanced Seroreactivity

Hepatitis C Virus c33, a recombinant protein comprising residues 1192-1457 of NS3 helicase, has been a mainstay of HCV serology for decades. With seven unpaired cysteines, seroreactivity of E. coli expressed c33 is dependant on reductants. While engineering a c33 replacement for new anti-HCV serological tests, we sought to reduce oxidation sensitivity, a liability for immunodiagnostic reagent stability. A series of cysteine-to-serine substituted variants of a c33-like antigen was constructed and evaluated for reactivity against a panel of HCV-positive sera. Several variants were essentially nonreactive while others exhibited reactivity similar to or better than the wild-type construct. One demonstrated equivalent potency to wild-type but also diminished DTT dependence. To explore enhanced anti-NS3 reactivity, we constructed and examined an expanded series of antigens comprising individual helicase domains, the full-length helicase, additional cysteine-to-serine variants, and variants at positions critical to catalytic activity. Immunoassays using these latter NS3 helicase recombinants demonstrated that domain 1 possessed significantly more seroreactivity than previously believed, that the use of soluble full-length helicase protein enhanced sensitivity by several-fold over c33, and that anti-NS3 helicase seroreactivity was further enhanced by the introduction of point mutations which altered the catalytic activity or oxidation sensitivity of the antigen.