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The fertile mind of John Hunter
GB agent
The development of specific diagnostic reagents and the distribution of experimental reagents to various laboratories are essential to answer many questions on the epidemiology of the GB viruses, their pathogenesis in man, their clinical and public health significance, and whether these are indeed common human hepatotropic viruses.
presented at a briefing meeting held in Washington April 7. The original plasma pool containing the GB agent passage 11in marmosets was transmitted serially to tamarins (Saguinus labiatus). The subtractive polymerase chain reaction method known as representational difference analysis (RDA)3 was then used to clone specific nucleotide sequences present in infectious plasma from a GB-infected tamarin. 7 of 11 unique clones that were identified were examined in detail. Sequences of the RDA-derived clones were exogenous to the genomic DNAs of tamarins, human beings, yeast (Saccharomyces cerevisiae), and Escherichia coli, were not detected in preinoculation tamarin plasma, but were found in the infectious plasma of tamarins inoculated with GB. Extension of these sequences and subsequent sequence comparisons indicate that the GB plasma contains two were on
distinct flavivirus-like RNA genomes. Further studies showed that the two RNA species present in the GB inoculum can be filtered, diluted, and passaged separately in tamarins, thereby confirming the existence of two independent viruses, GBV-A and GBV-B.4 GBV-A does not replicate in the liver of tamarins whereas GBV-B causes hepatitis. Cross-challenge experiments showed that infection with the original infectious tamarin inoculum conferred protection from reinfection with GBV-B but not GBV-A.5 A third virus, GBV-C, was isolated subsequently from a human specimen which was immunoreactive with a GBV-B protein. GBV-C RNA was found in several patients with clinical hepatitis, and shown to have substantial sequence identity to GBV-A. A series of studies including phylogenetic analysis of genomic sequences showed that GBV-A, B, and C are not genotypes of hepatitis C virus (HCV), and that GBV-A and GBV-C are closely related. GBV-A/C and GBV-B and the hepatitis C viruses are members of distinct viral groups. The organisation of the genes of the GBV-A, B, and C genomes shows that they are related to other positive-strand RNA viruses with local regions of sequence identity with various flaviviruses. The three GB viruses and HCV share only limited overall aminoacid
identity. Reagents were developed with recombinant antigens for use by ELISA, and limited testing was carried out in human populations with a high incidence of viral hepatitis-patients with non-A, B, C, D, E hepatitis, intravenous drug abusers, and multitransfused patients. The presence of antibody to each of the GB viruses was remarkable. For example, antibody was found in 14% of 101 multitransfused patients, with anti-GBV-A in 3%, anti-GBV-B in 11 %, and anti-GBV-C in 7% (there was some overlap). In 112 intravenous drug abusers, positive reactions were found overall in 11-6%, and in 8%, 4-4%, and 1-8% respectively; and the seroprevalence in 1300 samples from West Africa was 26-9% overall, and 7-7%, 13-9%, and 14-6% to the respective GBV groups. A survey of a small sample of 200 blood donors from one region in the USA indicated positive anti-GBV reaction with these viruses in 4 (2%) donors. A reverse-transcription PCR was designed for monitoring viraemia associated with a GBV infection, and this will help to monitor viraemia and establish whether chronic viraemia develops. sequence
1454
Arie J Zuckerman WHO Collaborating Centre for
Reference and Research on Viral
Diseases, Royal Free Hospital School of Medicine, London, UK 1
2 3 4
5
Deinhardt F, Holmes AW, Capps RB, et al. Studies on the transmission of human viral hepatitis to marmoset monkeys. I Transmission of disease, serial passages, and description of liver lesions. J Exp Med 1967; 125: 673-88. Parks WP, Melnick JL. Attempted isolation of hepatitis viruses in marmosets. J Infect Dis 1969; 120: 539-47. Lisitsyn N, Lisitsyn N, Wigler M. Cloning the differences between two complex genomes. Science 1993; 259: 946-51. Simons JN, Pilot-Matias TJ, Leary TP, et al. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci USA (in press). Schlauder GC, Dawson GJ, Simons JN, et al. Molecular and serologic analysis in the transmission of the GB hepatitis agents. J Med Virol 1995; 46: 81-90.
The fertile mind of John Hunter The Hunterian Museum of the Royal College of Surgeons at Lincoln’s Inn Fields in London is a feast for inquiring minds. It is a source of both inspiration and humiliationeven the briefest of visits makes one realise how the myopia of present-day specialisation has restricted our breadth of vision of the natural world. Not so John Hunter, that indefatigable Scot, whose curiosity seemingly knew no bounds and who was one of the first to try by experiment to solve some of Nature’s mysteries. There are few people alive today who share his breadth of interest, and hence can begin to appreciate the full significance of his magnificent museum. Sir Cyril Clarke, physician and lepidopterist, is one of those few, and in an article’ published by the Royal Society, of which John Hunter was also a Fellow, he recounts how, in 1792, Hunter attempted in-vitro fertilisation in the silkworm moth, Bombyx mon, and apparently succeeded. Hunter’s technique was to take a female moth immediately after emergence from the pupa, and keep it in isolation until it started to lay eggs. He then cut open a male moth, collected semen from the seminal ducts with the aid of a fine camel-hair brush, and painted it onto the newly laid eggs, which went on to hatch normally. The empty egg cases are there in the museum to prove it. Hunter merely claimed that he had demonstrated that "the eggs could be impregnated by art". The irony is that nobody, not even Sir Cyril himself, has been able to repeat this success with in-vitro fertilisation, either in silkworms or indeed in any of the Lepidoptera. Since John Hunter had an uncanny knack of being right, perhaps there was more to his art than meets the eye? R V Short Department of Physiology, Monash University, Victoria, Australia 1
Clarke CA. In vitro fertilization in Bombyx mori, the silkworm: was or wrong in 1792? Notes Rec R Soc Lond 1995; 49: 85-92.
John Hunter right
The development of specific diagnostic reagents and the distribution of experimental reagents to various laboratories are essential to answer many questions on the epidemiology of the GB viruses, their pathogenesis in man, their clinical and public health significance, and whether these are indeed common human hepatotropic viruses.
presented at a briefing meeting held in Washington April 7. The original plasma pool containing the GB agent passage 11in marmosets was transmitted serially to tamarins (Saguinus labiatus). The subtractive polymerase chain reaction method known as representational difference analysis (RDA)3 was then used to clone specific nucleotide sequences present in infectious plasma from a GB-infected tamarin. 7 of 11 unique clones that were identified were examined in detail. Sequences of the RDA-derived clones were exogenous to the genomic DNAs of tamarins, human beings, yeast (Saccharomyces cerevisiae), and Escherichia coli, were not detected in preinoculation tamarin plasma, but were found in the infectious plasma of tamarins inoculated with GB. Extension of these sequences and subsequent sequence comparisons indicate that the GB plasma contains two were on
distinct flavivirus-like RNA genomes. Further studies showed that the two RNA species present in the GB inoculum can be filtered, diluted, and passaged separately in tamarins, thereby confirming the existence of two independent viruses, GBV-A and GBV-B.4 GBV-A does not replicate in the liver of tamarins whereas GBV-B causes hepatitis. Cross-challenge experiments showed that infection with the original infectious tamarin inoculum conferred protection from reinfection with GBV-B but not GBV-A.5 A third virus, GBV-C, was isolated subsequently from a human specimen which was immunoreactive with a GBV-B protein. GBV-C RNA was found in several patients with clinical hepatitis, and shown to have substantial sequence identity to GBV-A. A series of studies including phylogenetic analysis of genomic sequences showed that GBV-A, B, and C are not genotypes of hepatitis C virus (HCV), and that GBV-A and GBV-C are closely related. GBV-A/C and GBV-B and the hepatitis C viruses are members of distinct viral groups. The organisation of the genes of the GBV-A, B, and C genomes shows that they are related to other positive-strand RNA viruses with local regions of sequence identity with various flaviviruses. The three GB viruses and HCV share only limited overall aminoacid
identity. Reagents were developed with recombinant antigens for use by ELISA, and limited testing was carried out in human populations with a high incidence of viral hepatitis-patients with non-A, B, C, D, E hepatitis, intravenous drug abusers, and multitransfused patients. The presence of antibody to each of the GB viruses was remarkable. For example, antibody was found in 14% of 101 multitransfused patients, with anti-GBV-A in 3%, anti-GBV-B in 11 %, and anti-GBV-C in 7% (there was some overlap). In 112 intravenous drug abusers, positive reactions were found overall in 11-6%, and in 8%, 4-4%, and 1-8% respectively; and the seroprevalence in 1300 samples from West Africa was 26-9% overall, and 7-7%, 13-9%, and 14-6% to the respective GBV groups. A survey of a small sample of 200 blood donors from one region in the USA indicated positive anti-GBV reaction with these viruses in 4 (2%) donors. A reverse-transcription PCR was designed for monitoring viraemia associated with a GBV infection, and this will help to monitor viraemia and establish whether chronic viraemia develops. sequence
1454
Arie J Zuckerman WHO Collaborating Centre for
Reference and Research on Viral
Diseases, Royal Free Hospital School of Medicine, London, UK 1
2 3 4
5
Deinhardt F, Holmes AW, Capps RB, et al. Studies on the transmission of human viral hepatitis to marmoset monkeys. I Transmission of disease, serial passages, and description of liver lesions. J Exp Med 1967; 125: 673-88. Parks WP, Melnick JL. Attempted isolation of hepatitis viruses in marmosets. J Infect Dis 1969; 120: 539-47. Lisitsyn N, Lisitsyn N, Wigler M. Cloning the differences between two complex genomes. Science 1993; 259: 946-51. Simons JN, Pilot-Matias TJ, Leary TP, et al. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci USA (in press). Schlauder GC, Dawson GJ, Simons JN, et al. Molecular and serologic analysis in the transmission of the GB hepatitis agents. J Med Virol 1995; 46: 81-90.
The fertile mind of John Hunter The Hunterian Museum of the Royal College of Surgeons at Lincoln’s Inn Fields in London is a feast for inquiring minds. It is a source of both inspiration and humiliationeven the briefest of visits makes one realise how the myopia of present-day specialisation has restricted our breadth of vision of the natural world. Not so John Hunter, that indefatigable Scot, whose curiosity seemingly knew no bounds and who was one of the first to try by experiment to solve some of Nature’s mysteries. There are few people alive today who share his breadth of interest, and hence can begin to appreciate the full significance of his magnificent museum. Sir Cyril Clarke, physician and lepidopterist, is one of those few, and in an article’ published by the Royal Society, of which John Hunter was also a Fellow, he recounts how, in 1792, Hunter attempted in-vitro fertilisation in the silkworm moth, Bombyx mon, and apparently succeeded. Hunter’s technique was to take a female moth immediately after emergence from the pupa, and keep it in isolation until it started to lay eggs. He then cut open a male moth, collected semen from the seminal ducts with the aid of a fine camel-hair brush, and painted it onto the newly laid eggs, which went on to hatch normally. The empty egg cases are there in the museum to prove it. Hunter merely claimed that he had demonstrated that "the eggs could be impregnated by art". The irony is that nobody, not even Sir Cyril himself, has been able to repeat this success with in-vitro fertilisation, either in silkworms or indeed in any of the Lepidoptera. Since John Hunter had an uncanny knack of being right, perhaps there was more to his art than meets the eye? R V Short Department of Physiology, Monash University, Victoria, Australia 1
Clarke CA. In vitro fertilization in Bombyx mori, the silkworm: was or wrong in 1792? Notes Rec R Soc Lond 1995; 49: 85-92.
John Hunter right