Detection and genotyping of GBV-C/HGV variants in China.

We detected GBV-C/HGV sequences in the sera from 64 out of a total of 324 subjects in the south of China. In agreement with findings of others, we noted an especially high rate of infection among intravenous drug addicts and patients with chronic hepatitis C virus infection. The detection was achieved by nested PCR to amplify the 5' noncoding region (5'NCR) of the viral genome. Sequence analysis of the resulting 234 bp product revealed a total of 26 different sequences of which 25 were found to belong to the genotype G3, which is the most prevalent genotypes among Asian isolates, and one belonged to genotype G1, common among African isolates. The sequence divergence between the genotypes was largely clustered in a short variable region (V2) within the 5'NCR, and we showed that genotyping may be achieved equally well by analysis of this variable region as by the more detail analysis of the entire 5'NCR or of the entire viral genome.

Phenylalanine biosynthesis in Escherichia coli K-12: mutants derepressed for chorismate mutase P-prephenate dehydratase.

Mutants were isolated which are derepressed for the synthesis of chorismate mutase P-prephenate dehydratase. No other enzymes involved in the synthesis of phenylalanine are derepressed in these strains. These mutants are able to grow in concentrations of o- and p-fluorophenylalanine that inhibit the growth of AB3259, the strain from which they were derived. They also excrete phenylalanine. Genetic analysis shows that the mutations causing this derepression are closely linked to the structural gene for this enzyme (cotransduction frequency of 95% or more with pheA). The gene in which they occur has been designated pheO since this gene has all of the properties predicted for an operator gene controlling the pheA structural gene. Finally, the pheO mutant alleles have been shown to be dominant in diploids.

Tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: complementation between different tyrR alleles.

Dominance tests in diploids have confirmed that the product of the tyrR gene is involved in a negative control system affecting the synthesis of both 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and DAHP synthetase (phe). Some tyrR mutants are derepressed for the synthesis of both DAHP synthetase (tyr) and (phe), whereas others are derepressed for the synthesis of DAHP synthetase (tyr) but overrepressed for the synthesis of DAHP synthetase (phe). Complementation tests between these alleles confirm that they are in the same cistron. The allele causing overrepression of enzyme synthesis is dominant over both the wild type and the derepressing allele in diploids.

Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A.

Mutant strains of Escherichia coli have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (phe) is derepressed, in addition to those enzymes of tyrosine biosynthesis previously shown to be controlled by the gene tyrR. The major enzyme of the terminal pathway of phenylalanine biosynthesis chorismate mutase-prephenate dehydratase is not derepressed in these strains. Genetic analysis of the mutants shows that the mutation or mutations causing derepression map close to previously reported tyrR mutations. A study of one of the mutations has shown it to be recessive to the wild-type allele in a diploid strain. It is proposed that the tyrR gene product is involved in the regulation of the synthesis of DAHP synthetase (phe) as well as the synthesis of DAHP synthetase (tyr), chorismate mutase-prephenate dehydrogenase, and transaminase A.

Evaluation of three serological tests for detection of antibody to Pseudomonas aeruginosa in human sera.

Four hundred and ninety-five sera from 325 patients from whom Pseudomonas aeruginosa had been isolated and 86 control sera were tested for antibody by indirect haemagglutination tests (HAT) and complement fixation tests (CFT) using a polyvalent pseudomonas serotype-specific vaccine antigen, PEV-02. Sera were also tested by countercurrent immunoelectrophoresis (CIE) for precipitins to a species-specific protein antigen. Control sera gave titres of 160 or less by HAT and 20 or less by CFT. 2-Mercaptoethanol resistant antibody titres (immunoglobulin G) were below 40 for all control sera and none of the latter contained precipitins to common antigen. Of 325 patients, 156 (48%) gave titres of 320 or greater by HAT and of these, 114 (73%) showed elevated immunoglobulin G titres. Less patients with positive blood cultures than expected were positive by HAT and more patients with bone infections gave raised immunoglobulin G titres than expected. Cystic fibrosis patients were invariably seropositive by all tests. There was a correlation between positive CIE and CFT tests, especially in patients who were positive by HAT. Approximately half of 83 patients tested gave a serotype-specific antibody response. The tests were of little value in confirming clinically evident acute infections, but in cases of doubtful infection they did provide confirmatory evidence of an antibody response in approximately one-third of patients culture-positive for Pseudomonas aeruginosa.

Acute sporadic hepatitis E virus infection in southern China.

BACKGROUND/AIMS: The hepatitis E virus is responsible for epidemic and sporadic hepatitis in northwestern China, but its role as a cause of acute sporadic hepatitis in southern China has not been reported. METHODS: We applied the most practical current methods for diagnosis of hepatitis E virus infection, IgM and IgG anti-HEV detection by enzyme linked immunosorbent assay, to investigate the prevalence of hepatitis E virus infection among acute sporadic hepatitis. RESULTS: Anti-HEV IgM was found in 1 of 26 (3.8%), 4 of 20 (20.0%), 4 of 19 (21.1%), and 51 of 142 (35.9%), with acute hepatitis A, B, C and non-ABC, respectively. Anti-HEV IgM was not detectable in healthy subjects, while IgG anti-HEV was found in 14 of 77 healthy subjects (18.2%) and was long-lasting. Ninety-one cases without any evidence of hepatitis A, B or C infections and anti-HEV IgM were tentatively classified as non-A, B, C, D, E (non-ABCDE) hepatitis. By comparison with non-ABCDE, cases with hepatitis E were more frequently icteric and exhibited higher alanine aminotransferase levels (92.2% vs. 45.1%, 770 iu/l vs 377 iu/l, respectively, p < 0.005). Chronic cases were not observed in hepatitis E virus infections. However, 14 of 91 (15.4%) cases with non-ABCDE developed to chronicity (p < 0.005). CONCLUSIONS: Hepatitis E virus infection is sporadic as well as endemic in southern China. Only IgM anti-HEV but not IgG anti-HEV can be used as an appropriate marker of acute hepatitis E virus infection. Superinfection of hepatitis E virus with other types of hepatitis viruses is frequent in this area. While the disease was associated with more severe clinical manifestations, patients usually recovered completely.

Follow-up study of acute hepatitis C.

An extended follow-up study of hepatitis C virus (HVC) infection was conducted in Guangzhou and its nearby regions on patients hospitalized with acute hepatitis. Routine screening of blood donors for HCV was not yet instituted at the time of this study. HCV was found to be a common cause of the disease, and the infection had a close association with recent histories of blood transfusion. Sequential sera obtained from patients during hospitalization and after discharge were tested for the presence of HCV antibodies by the first and the second generation of commercial test kits, for levels of alanine aminotransferase (ALT), and for the presence of HCV-RNA. The development of HCV antibodies in some of the patients may be delayed for protracted period following clinical onset. HCV-RNA was only intermittently detectable both before and after seroconversion. Six of 33 patients studied showed seroreversion and 5 of them were accompanied by loss of HCV-RNA and serum ALT returned to normal levels. The disease persisted in 24 of 27 patients without seroreversion, accompanied by intermittent detection of HCV-RNA throughout the protracted course of the infection. Our results indicate that both EIA for detection of HCV antibodies and PCR for serum HCV-RNA should be used in combination for reliable diagnosis of HCV infection and screening of blood for transfusion.

Genetic instability of R plasmids in relation to the shift of drug resistance patterns in Salmonella johannesburg.

Observation of the resistance of Salmonella johannesburg to the six drugs ampicillin (A), streptomycin (S), tetracycline (T), chloramphenicol (C), kanamycin(K) and sulphadiazine (Su) was made over the 7 years from 1973 to 1979. Strains with ASTCKSu- and ASCKSu- resistance patterns predominated in the years 1973-1975 and 1976-1979, respectively. These resistances were found to be mediated by autotransferring plasmids belonging to the incompatibility group FIme. The ASTCKSu-resistance plasmids were unstable, giving rise to deletion variants at a much higher frequency than ASCKSu-resistance plasmids either of natural origin or derived in vitro from the ASTCKSu-resistance plasmids. Thus, the ASCKSu-resistance plasmid might be a deletion variant of the ASTCKSu-resistance plasmid. This is supported by the extensive similarity of their cleavage patterns produced by specific restriction endonucleases.

Two murine monoclonal antibodies against serogroup E Salmonellae.

A monoclonal antibody (MAb), MO15, was raised against the lipopolysaccharide antigen of an epsilon15-lysogenized serogroup E(1) Salmonella strain. The O factor 15-specific MAb MO15, together with another serogroup E-specific MAb, can differentiate among phage lysogenization variants in serogroup E salmonellae. Their epitope specificities in relation to conventional O-antigenic structures are discussed.

Conformational antigenic determinants generated by interactions between a bacterially expressed recombinant peptide of the hepatitis E virus structural protein.

A 23 kDa peptide locating to amino acid residues 394 to 604 of the major Hepatitis E Virus (HEV) structural protein was expressed in E. coli. This peptide was found to interact naturally with one another to form homodimers and it was recognized strongly and commonly in its dimeric form by HEV reactive human sera. The antigenic activity associated with the dimeric form was abrogated when the dimer was dissociated into monomer and the activity was reconstituted after the monomer was re-associated into dimer again. The dimeric form of the peptide elicited a vigorous antibody response in experimental animals and the resulting antisera were found to cross-react against HEV, effecting an efficient immune capture of the virus. These results attributed the antigenic activity associated with the dimeric form of the peptide to conformational antigenic determinants generated as a result of interaction between the peptide molecules. It is suggested that some of these antigenic determinants may be expressed by the HEV capsid and raised the possibility of this bacterially expressed peptide as an HEV vaccine candidate.

A bacterially expressed peptide prevents experimental infection of primates by the hepatitis E virus.

A 23 kDa peptide of the major structural protein of the hepatitis E virus (HEV) expressed in E. coli was found to naturally interact with one another to form homodimers and the peptide was recognized strongly in its dimeric form by HEV reactive human sera. To determine if the peptide may confer protection against HEV infection, three monkeys were immunized with the purified peptide and three were given placebo. Both groups of animals were challenged with 10(5) genome equivalent dose of the homologous strain of HEV. All control animals excreted the virus for 10-12 days beginning 5 days after the infection. The viral genome was also present in the peripheral blood monocyte (PBMC) samples from two animals, but it was not detected in the plasma samples from any of the animals. The infection in two control animals was accompanied by HEV seroconversion. Immunization was found to abrogate HEV stool excretion in two animals and reduced the viral excretion to one day in the third. None of the immunized animals showed detectable HEV in plasma or PBMC samples nor did the animals showed evidence of HEV seroconversion. These results suggested that immunization with the bacterially expressed peptide may prevent experimental infection of primates with the homologous strain of HEV.

Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma.

Tumor cells from NPC patients are regularly and latently infected with EBV. To examine whether the virus serves as target for immune intervention of the cancer, we determined levels of EBV-specific CTLp in peripheral blood from NPC patients, long-term survivors of the cancer and healthy subjects. CTLp levels of test subjects varied between 3- 3,000/10(6) PBMCs. The plasma EBV burden increased when the CTLp level fell below 150, whereas the EBV burden of PBMCs was not correlated with CTLp level. Compared with healthy carriers, CTLp levels of patients were lower and varied over a wider range, between 3-1,500/10(6) PBMCs. The quantitative immune deficit was probably attributed to the tumor because, first, CTLp in survivors was restored to levels similar to those in healthy carriers after the tumor had been successfully treated. Second, the CTLp level changed as disease progressed, being lower in local disease, increased in locoregional disease and decreased again when the tumor metastasized. Based on these findings, 4 patients with advanced disease were infused with 5 x 10(7)-3 x 10(8) autologous EBV CTLs. The treatment was safe and unaccompanied by inflammatory or other complications, but whether it improved tumor control could not be discerned from the large tumor bulk. Nevertheless, the treatment regularly increased CTLp levels of patients, maintained it at higher levels for protracted periods and, in 3 patients, restored host surveillance of EBV replication, reducing the plasma EBV burden. Taken together, these results provided a rationale to further explore EBV as a target of immune intervention of NPC.