GB Agent Research Articles

Highly sensitive retrospective determination of organophosphorous nerve agent biomarkers in human urine implemented in vivo in rabbit.

Highly toxic organophosphorous nerve agents (OPAs) have been used in several armed conflicts and terror attacks in the last few decades. A new method for retrospective determination of alkyl methylphosphonic acid (AMPA) metabolites in urine after exposure to VX, GB and GF nerve agents was developed. This method enables a rapid, sensitive and selective determination of trace levels of the nerve agent biomarkers ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) in urine. The new technique involves a unique combination of two solid phase extraction (SPE) cartridges: a Ba/Ag/H cartridge for urine interference removal, and a ZrO2 cartridge for selective reconstitution and enrichment of the AMPAs. Extraction of AMPAs from the ZrO2 cartridge was accomplished with a 1% ammonium hydroxide (NH4OH) solution and was followed by analysis via liquid chromatography-mass spectrometry (LC-MS). The limits of quantitation (LOQs) were in the range of 10-100pg/mL with recoveries of 64-71% (± 5-19%) after fast sample preparation and a total LC-MS analysis cycle time of 15min and 13min, respectively. This method was successfully applied in vivo in a rabbit that was exposed to 0.5 LD50 (7.5µg/kg, i.v.) sarin for retrospective monitoring of the IMPA metabolite in urine. For the first time, IMPA was determined in rabbit urine samples for 15days post-exposure, which is longer than any reported post-exposure method for AMPAs. To the best of our knowledge, this new method is the most sensitive and rapid for AMPA determination in urine by LC-MS/MS analysis.

Catalytic Degradation of the Nerve Agent VX by Water-Swelled Polystyrene-Supported Ammonium Fluorides

The catalytic degradation of the nerve agent VX (O-ethyl S-2-(diisopropylamino)ethyl methylphosphonothioate) by water-swelled polymer-supported ammonium fluorides is described. VX (0.06-0.53 mol/mol F(-)) is rapidly degraded (t(1/2) ∼ 10-30 min) to form the "G-analogue" (O-ethyl methylphosphonofluoridate), which hydrolyzes (t(1/2) ∼ 1-1.5 h) to the nontoxic EMPA (ethyl methylphosphonic acid). The toxic desethyl-VX is not formed. The catalytic effect of fluoride is maintained even when 6 equiv of VX are loaded. GB (O-isopropyl methylphosphonofluoridate) and desethyl-VX agents are also degraded under these conditions.

RIDDING THE NATION OF CHEMICAL WEAPONS

THE ARMY'S 18-YEAR-OLD PROGRAM to rid the nation of its 31,000-ton arsenal of chemical weapons has been bedeviled by technical glitches, equipment malfunctions, and cost overruns. At its Anniston, Ala., incinerator facility, the Army is again facing one of its thorniest problems: aging rockets filled with the nerve agent GB. A chemical stabilizer was added to some, but not all, of the GB agent that the Army produced during the Cold War era. Over time, the stabilizer—which the Army calls di-c-di for diisopropylcarbodiimide—has reacted with GB to form crystals or a gel, Anniston facility spokesman Mike Abrams explains. Army estimates that up to 30% of the nearly 43,000 GB rockets in the Anniston inventory have undergone this chemical reaction. So far, Anniston has only found—and incinerated—crystal-containing GB rockets. The crystals appear to look like the rock salt used to make ice cream, Abrams says. Army labs are now analyzing the crystals to determine ...

GB virus B as a model for hepatitis C virus.

GB viruses A and B (GBV-A and GBV-B) are members of the Flaviviridae family and are isolated from tamarins injected with serum from a human hepatitis patient. Along with a related human virus, GB virus C, or alternatively, hepatitis G virus (GBV-C/HGV), the three viruses represent the GB agents. Of the three viruses, GBV-B has been proposed as a potential surrogate model for the study of hepatitis C virus (HCV) infections of humans. GBV-B is phylogenetically most closely related to HCV and causes an acute, self-resolving hepatitis in tamarins as indicated by an increase in alanine aminotransferase and changes in liver histology. Similarities between GBV-B and HCV are found at the nucleotide sequence level with the two viruses sharing 28% amino acid homology over the lengths of their open reading frames. Short regions have even higher levels of homology that are functionally significant as shown by the ability of the GBV-B NS3 protease to cleave recombinant HCV polyprotein substrates. The shared protease substrate specificities suggest that GBV-B may be useful in testing antiviral compounds for activity against HCV. Although there are numerous similarities between GBV-B and HCV, there are important differences in that HCV frequently causes chronic infections in people, whereas GBV-B appears to cause only acute infections. The acute versus chronic course of infection may point to important differences between the two viruses that, along with the numerous similarities, will make GBV-B in tamarins a good surrogate model for HCV.

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.

Prevalence and infectivity of hepatitis G virus and its strain variant, the GB agent, in volunteer blood donors in Taiwan.

The prevalence of hepatitis G virus (HGV) and its strain variant, the GB agent (GBV-C) is high in non-virus-inactivated plasma products, but, persistent infection in recipients is relatively low. Stored samples from transfusion donors and recipients in a prospective study of posttransfusion hepatitis were tested for HGV RNA and antibody to the E2 protein (anti-E2). Thirty-two (2.1%) of the 1500 qualified donors were positive for HGV RNA. Twenty-four persons had received a transfusion of blood from one of these 32 viremic donors. Of these 24 recipients, 3 were positive for HGV RNA before transfusion. Of the remaining 21 recipients, 8 became viremic after transfusion, while the other 13 were not infected. Four of the eight infected recipients were persistently positive for HGV RNA, while four became negative in 1 to 3 years. Three of the four patients with HGV clearance seroconverted to anti-E2 positivity. Comparison of the viral titer, viral sequences at E2, storage period of blood donations, and clinical data in the infected and noninfected recipients revealed no significant differences. However, the noninfected recipients seemed to have a higher prevalence of anti-E2 before transfusion. The prevalence of HGV viremia in volunteer blood donors in Taiwan is 2.1 percent, and blood from 0.6 percent of them actually causes HGV infection in the recipients. In half of infected recipients, clearance of HGV occurs. Anti-E2 appears in most recipients whose viremia is cleared.

Genomic analysis of two GB virus A variants isolated from captive monkeys.

The recent isolation of GB viruses A and B from GB agent infected tamarins and their lack of involvement in human hepatitis has sparked interest in the origin of these viruses. Several healthy non-human primate species have been shown to harbour sequences 52-79% identical to the GBV-A 5' nontranslated region. In this paper we report the near genome length sequence of GBV-Amx 70047 and GBV-Atri 1122. These sequences support previous observations about the genomic organization of GBV-A and provide insight into the genomic variability within this virus genus. Although the GBV-A variant polyproteins possess many motifs conserved between other members of the Flaviviridae, they do not encode a basic core-like protein. Amino acid sequence comparisons and phylogenetic analysis demonstrate variability within the GBV-A genus similar to that observed between hepatitis C virus (HCV) types. However, genomic organization and disease association demonstrate a closer evolutionary relationship to GBV-C than to HCV.

Evaluation of combined toxic effects of GB/GF and efficacy of Jielin Injection against combined poisoning in mice

A computer program (Q-test) was used to evaluate the combined toxic effects of nerve agent GF and its combined form with sarin (GB/GF) in mice. Efficacy of Jielin Injection, the 2-PAM-containing antidote used successfully in China for the treatment of organophosphate pesticide poisoning, was also evaluated and compared with HI-6 against single and combined poisonings. The two agents were basically additive in toxicity when combined. However, toxic signs (convulsions) appeared later in combined poisoning than after exposure to each agent alone. The protective ratio of Jielin Injection against GF poisoning was low but significantly higher when against poisoning by GB or combined agent. When HI-6 was substituted for 2-PAM, the antidote was more effective against poisoning by both single and combined agents. Results of in vitro reactivation of GF-inhibited human erythrocyte acetylcholinesterase by these oximes agreed with the in vivo antidotal efficacy.

Infection with hepatitis G virus and its strain variant, the GB agent (GBV-C), among blood donors in Japan.

The purpose of the study was to survey the epidemiology of recently reported non-A through -E hepatitis virus designated hepatitis G virus (HGV) and its strain variant, the GB agent (GBV-C). Pilot samples from 2461 blood donors in Japan, randomly selected to form cohorts with different levels of alanine aminotransferase (ALT) and markers of hepatitis B virus or hepatitis C virus (HCV) infection, were tested for RNA of HGV/GBV-C by reverse transcription-polymerase chain reaction with nested primers deduced from the 5'-noncoding region. HGV/GBV-C RNA was detected in 23 (7.4%) of the 361 donors with anti-HCV and HCV RNA. This detection is more frequent than that in donors without elevated ALT levels (< or = 45 U/L) or markers of HCV or hepatitis B virus infection (15/1303; 1.2%) (p < 0.001), donors with ALT values between 46 and 99 U per L (0/108) (p < 0.002), donors with ALT values > or = 100 U per L (5/361; 1.4%), and donors with anti-HCV but without detectable HCV RNA (1/93; 1.1%) (p < 0.05). More than 1 percent of Japanese blood donors were infected with HGV/GBV-C, and the prevalence was much higher in those with HCV RNA. Should persistent infection with HGV/GBV-C induce any hepatotoxic sequelae, either alone or in concert with the other hepatitis viruses, screening of blood units for HGV/GBV-C would deserve consideration.

Prevalence of hepatitis G virus and its strain variant, the GB agent, in blood donations and their transmission to recipients.

Hepatitis G virus (HGV) and its strain variant, the GB agent (GBV-C) are independent isolates of a recently identified non-A through -E hepatitis virus. Prevalence in United States volunteer blood donors is 1.5 to 1.9 percent, but no data on European blood donors are available. Epidemiologic data suggest a preferred parenteral transmission route. The prevalence of HGV/GBV-C in European blood donors and the efficiency of transmission to transfusion recipients were investigated. Plasma samples from unpaid volunteer German blood donors were tested for HGV/GBV-C by in-house reverse transcription-polymerase chain reaction. Positive donors were independently retested and interviewed for parenteral transmission risks. Amplification products were sequenced and subjected to phylogenetic analysis. Recipients of reverse transcription-polymerase chain reaction-positive donations were traced and tested for HGV/GBV-C infection. A total of 14 (1.34%) of 1048 donors (alanine aminotransferase < 45 IU/L) were repeatedly positive for HGV/GBV-C with 9 (2.18%) of 413 urban and 5 (0.78%) of 635 rural donors (chi 2-test; p = 0.04). Isolates differed in nucleotide sequence homology over a range of 12.5 to 19.6 percent. All but one positive donor reported parenteral transmission risks. Transmission of HGV/GBV-C was detected in 4 of 9 transfusion recipients. The prevalence of HGV/GBV-C in donors with an alanine aminotransferase level > 45 IU per L was 3 percent (3/100). Two mother/child pairs were identified with highly homologous isolates. A significantly greater prevalence of HGV/GBV-C was detected in urban volunteer blood donors than in rural donors. The high prevalence in urban donors (2.18%) suggests specific transmission risks for this group. The less than 50-percent efficiency of HGV/GBV-C transmission via blood components may indicate the presence of defective viruses with reduced infectivity. There is evidence for vertical transmission.

SEROLOGIC DIAGNOSIS OF VIRAL HEPATITIS

Viral hepatitis has become a difficult field in which clinical and laboratory skills are needed to establish the correct diagnosis and plan for the appropriate therapy. For example, it is no longer enough to diagnose chronic hepatitis B or C. Now, the viral titer or viral genotype must be known. The laboratory test then must be understood in the context of the clinical presentation. This article helps the clinician to acquire such working knowledge. It summarizes available data for hepatitis A, B, C, D, and E. It also includes the recently discovered viral agents, hepatitis G and the hepatitis GB agents.

Immunodiagnosis of viral hepatitides A to E and non-A to -E

Primary viral infection of liver cells is caused by several distinct hepatotropic viruses transmitted by oral and parenteral routes. While the clinical, epidemiological, pathological, and immunological aspects of viral hepatitides have many common attributes and subtle differences (39), the etiologies of liver disorders cannot be established on the basis of clinical signs and symptoms or abnormal liver function tests (e.g., elevated alanine aminotransferase [ALT]). Following the discovery of Australia antigen (8), which was subsequently termed hepatitis B surface antigen (HBsAg), the identification and characterization of hepatitis B virus (HBV), hepatitis A virus (HAV), hepatitis D virus (HDV), hepatitis C virus (HCV), and hepatitis E virus (HEV) occurred successively between 1972 and 1992 (46, 60, 61, 63). The virological and immunological properties of these five well-established agents of viral hepatitis are summarized in Table 1. The rapid evolution of the field of viral hepatitis is reviewed periodically at the Triennial International Symposia on Viral Hepatitis, which originated at the University of California, San Francisco, in 1972 (83). The proceedings of the last symposium, which was held in Tokyo in 1993, provide recent and exhaustive information about the hepatitis viruses (56). More than 25,000 papers and 40 books and monographs published during the past 2 decades reflect the pace of advances in the field of viral hepatitis, and yet the progress continues, with recently published reports of non-A, -B, -C, -D, and -E viruses, i.e., hepatitis F virus (HFV), hepatitis G virus (HGV), and three novel hepatitis agents termed GB-A, GB-B, and GB-C viruses (21, 65, 69). HGV and the GB agents may be closely related to one another. GB agents are representatives of two new genera of the family Flaviviridae and are distantly related to HCV (54). The recognition of a variety of immunological and virological markers in patients’ sera enables the establishment of sensitive and specific diagnostic assays for discriminating between various forms of viral hepatitides. While infections with HAV, HEV, and HFV resolve in less than 3 months, HBV, HCV, HDV, and HGV tend to establish persistent infections, which are defined by the presence of serological markers for periods exceeding 6 months. The direct detection of viral genomes by hybridization with cloned DNA or RNA probes has been superseded by the recent advent of PCR for amplification of specific viral gene sequences. However, enzyme immunoassays (EIA) for viral antigens and antibodies formed during the natural history of the host response to hepatotropic viral infection serve as the most practical approach to establishing an immunodiagnosis of viral hepatitis. The purpose of this brief review is to present succinctly a composite picture of hepatitis virology and serology relevant to the laboratory diagnosis of viral hepatitis and liver diseases.

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.

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.

Supercritical water oxidation of chemical agents, and solid propellants

Abstract Supercritical water oxidation (SCWO), also referred to as hydrothermal oxidation (HTO), is a developing technology for the destruction of hazardous and nonhazardous wastes. SCWO destroys combustible materials using an oxidant in water at temperatures in the range of 350°C (662°F) to 600°C (1112°F) and pressures of 17 MPa (2500 psi) or greater. General Atomics and its subcontractors are currently conducting two comprehensive research and demonstration programs geared toward the destruction of Department of Defense (DoD) wastes utilizing SCWO technology. Wastes of primary interest include chemical agents and solid propellants. Technical challenges, particularly corrosion and solids handling, were overcome, and the destruction of chemical agents and solid propellants was demonstrated on a bench scale. SCWO results for chemical agents show destruction and removal efficiencies for GB, VX, and mustard agents to be in excess of 99.9999%, limited only by detection capability. SCWO results for hydrolyzed Class 1.1 solid propellant show destruction and removal efficiencies for total organic carbon (TOC) of >99.9%. Design and fabrication of a transportable SCWO pilot plant for chemical warfare agents, propellants and other DoD hazardous wastes and a prototype HTO system for solid rocket propellant disposal are complete and demonstration testing is underway.

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.

The UV Absorption Spectra of Chemical Agents and Simulants

The uv spectra of chemical agents GA, GB, GD, VX, HD, and L and common agent simulants have been measured in cyclohexane solution from 350 to 200 nm. The purity of the agents, measured by gas chromatographic analysis, was in general &gt;96%. At 230 nm, the strength of the uv absorption followed the general trend L &gt; VX &gt; GA &gt; HD &gt; GD &gt; GB &gt; DMMP. Except for HD and L, agent spectra lacked characteristic features that will be useful for remote detection by uv sensors.

Transmission of human non-A, non-B hepatitis to chimpanzees following failure to transmit GB agent hepatitis.

Two colony-born infant chimpanzees were inoculated with documented infectious serum containing the GB agent. Serum aminotransferase levels remained within normal limits in weekly serum samples, and no abnormalities were detected in weekly liver biopsy specimens. Each of these chimpanzees was subsequently inoculated with serum containing an agent of human non-A, non-B hepatitis. Each developed non-A, non-B hepatitis characterized by elevation of serum aminotransferase levels and histopathologic changes in liver biopsy specimens. Thus, the chimpanzee appears not to be susceptible to the GB agent, and prior inoculation with this agent does not appear to confer immunity to subsequent infection with human non-A, non-B hepatitis.

Lack of Susceptibility of Marmosets to Human Non-A, Non-B Hepatitis

Infectious sera from three humans with chronic non-A, non-B hepatitis, whose blood or serum had transmitted non-A, non-B hepatitis both to other humans and to experimentally inoculated chimpanzees, were inoculated into five marmosets. A sixth uninoculated marmoset served as a control. No elevations in levels of serum alanine aminotransferase or isocitric dehydrogenase occurred in serum samples obtained weekly from any of the marmosets during three months following inoculation. This study indicates that certain species of marmoset, which are susceptible to and provide well-documented animal models for hepatitis A and GB-agent hepatitis, do not appear to be susceptible to the agent(s) of human non-A, non-B hepatitis. In addition, this study suggests that the agent(s) of human non-A, non-B hepatitis and the GB agent are probably different.

Virus-like particles and GB agent hepatitis.

ALTHOUGH the MS-11 and CR3262 strains of human hepatitis A virus have been well characterised morphologically and shown to be indistinguishable, controversy has plagued characterisation of the GB agent of hepatitis3. Presumably derived from a surgeon with acute hepatitis, the GB agent has been transmitted serially in marmoset monkeys3 but has been shown to be unrelated serologically to any known human hepatitis virus4. Recently, Almeida et al.5 reported the detection after prolonged search of 20–22-nm particles aggregated by endogenous antibody in a pool of marmoset sera (pool H205, GB pass 11) known to contain the GB agent. These antibody-coated particles resembled parvoviruses morphologically, appeared in “empty”, “full”, and fragmented crescent-shaped forms, and were undetectable in normal marmoset serum. On the basis of previous filtration data from Deinhardt et al.6 which suggest that the GB agent is approximately 20 nm in diameter and the previous finding by others that anti-complementary activity—thought to represent the presence of circulating antigen–antibody complexes—occurs during acute viral hepatitis, Almeida et al. suggested that the virus-like particle they have detected is the GB agent5.