An Assessment of the Use of Chimpanzees in Hepatitis C Research Past, Present and Future: 2. Alternative Replacement Methods

The use of chimpanzees in hepatitis C virus (HCV) research was examined in the report associated with this paper (1: Validity of the Chimpanzee Model), in which it was concluded that claims of past necessity of chimpanzee use were exaggerated, and that claims of current and future indispensability were unjustifiable. Furthermore, given the serious scientific and ethical issues surrounding chimpanzee experimentation, it was proposed that it must now be considered redundant — particularly in light of the demonstrable contribution of alternative methods to past and current scientific progress, and the future promise that these methods hold. This paper builds on this evidence, by examining the development of alternative approaches to the investigation of HCV, and by reviewing examples of how these methods have contributed, and are continuing to contribute substantially, to progress in this field. It augments the argument against chimpanzee use by demonstrating the comprehensive nature of these methods and the valuable data they deliver. The entire life-cycle of HCV can now be investigated in a human (and much more relevant) context, without recourse to chimpanzee use. This also includes the testing of new therapies and vaccines. Consequently, there is no sound argument against the changes in public policy that propose a move away from chimpanzee use in US laboratories

An Assessment of the Use of Chimpanzees in Hepatitis C Research Past, Present and Future: 1. Validity of the Chimpanzee Model

The USA is the only significant user of chimpanzees in biomedical research in the world, since many countries have banned or limited the practice due to substantial ethical, economic and scientific concerns. Advocates of chimpanzee use cite hepatitis C research as a major reason for its necessity and continuation, in spite of supporting evidence that is scant and often anecdotal. This paper examines the scientific and ethical issues surrounding chimpanzee hepatitis C research, and concludes that claims of the necessity of chimpanzees in historical and future hepatitis C research are exaggerated and unjustifiable, respectively. The chimpanzee model has several major scientific, ethical, economic and practical caveats. It has made a relatively negligible contribution to knowledge of, and tangible progress against, the hepatitis C virus compared to non-chimpanzee research, and must be considered scientifically redundant, given the array of alternative methods of inquiry now available. The continuation of chimpanzee use in hepatitis C research adversely affects scientific progress, as well as chimpanzees and humans in need of treatment. Unfounded claims of its necessity should not discourage changes in public policy regarding the use of chimpanzees in US laboratories

Epstein-Barr virus: clinical and epidemiological revisits and genetic basis of oncogenesis

Epstein-Barr virus (EBV) is classified as a member in the order herpesvirales, family herpesviridae, subfamily gammaherpesvirinae and the genus lymphocytovirus. The virus is an exclusively human pathogen and thus also termed as human herpesvirus 4 (HHV4). It was the first oncogenic virus recognized and has been incriminated in the causation of tumors of both lymphatic and epithelial nature. It was reported in some previous studies that 95% of the population worldwide are serologically positive to the virus. Clinically, EBV primary infection is almost silent, persisting as a life-long asymptomatic latent infection in B cells although it may be responsible for a transient clinical syndrome called infectious mononucleosis. Following reactivation of the virus from latency due to immunocompromised status, EBV was found to be associated with several tumors. EBV linked to oncogenesis as detected in lymphoid tumors such as Burkitt's lymphoma (BL), Hodgkin's disease (HD), post-transplant lymphoproliferative disorders (PTLD) and T-cell lymphomas (e.g. Peripheral T-cell lymphomas; PTCL and Anaplastic large cell lymphomas; ALCL). It is also linked to epithelial tumors such as nasopharyngeal carcinoma (NPC), gastric carcinomas and oral hairy leukoplakia (OHL). In vitro, EBV many studies have demonstrated its ability to transform B cells into lymphoblastoid cell lines (LCLs). Despite these malignancies showing different clinical and epidemiological patterns when studied, genetic studies have suggested that these EBV- associated transformations were characterized generally by low level of virus gene expression with only the latent virus proteins (LVPs) upregulated in both tumors and LCLs. In this review, we summarize some clinical and epidemiological features of EBV- associated tumors. We also discuss how EBV latent genes may lead to oncogenesis in the different clinical malignancie

Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV

Development of a classical swine fever subunit marker vaccine and companion diagnostic test

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD50 of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R>1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population. (C) 2000 Elsevier Science B.V

Prevention of transplacental transmission of moderate-virulent classical swine fever virus after single or double vaccination with an E2 subunit vaccine

The use of a vaccine against classical swine fever virus (CSFV) during an outbreak of CSF should lead to a reduction in the horizontal or vertical transmission of CSFV. The reduction of vertical, i.e. transplacental, transmission of a moderate-virulent strain of CSFV from the sow to its offspring was studied in sows vaccinated once or twice with a CSFV E2 subunit vaccine. Two groups of nine sows were vaccinated with one PD95 dose of the E2 subunit vaccine, approximately four weeks before insemination. A third group of nine inseminated sows served as controls. One group of nine sows were vaccinated again at two weeks after insemination. At ten weeks after the primary vaccination, approximately six weeks after insemination, all 27 sows were challenged intranasally with 105 TCID50 of a moderate-virulent strain of CSFV, the Van Zoelen strain. The sows were euthanized at five weeks after challenge, and samples from the sows and fetuses were collected for detection of CSFV. All 27 sows were in gestation at the time of slaughter, CSFV was detected in the fetuses of all unvaccinated sows but it was not detected in any of the samples collected from fetuses of the double-vaccinated sows. Virus was however recovered from the fetuses of one out of nine sows vaccinated once. All the sows, except four double-vaccinated sows, developed CSFV Erns antibodies. Transplacental transmission of CSFV was reduced significantly (p <0.001) in all vaccinated sows. When the results from the experiment were extrapolated to a herd level, it could be concluded that, with 95% certainty, approximately 11% (single vaccination) or 0% (double vaccination), confidence intervals of 0.01-0.44 and 0.0-0.30 respectively, of the pregnant sows would still not be protected against vertical transmission of moderate-virulent CSFV. We conclude that vaccination with the CSFV E2 subunit vaccine can reduce the transmission of moderate-virulent strain of CSFV from the sow to its offspring significantly

Chimeric classical swine fever viruses containing envelope protein E(RNS) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response

Three chimeric classical swine fever virus (CSFV)/bovine viral diarrhoea virus (BVDV) full-length DNA copies were constructed, based on the infectious DNA copy of the CSFV vaccine strain C. The antigenic region of E2 and/or the complete ERNS gene were replaced by the analogous sequence of BVDV II strain 5250. Viable chimeric virus Flc11, in which ERNS was replaced, was directly recovered from supernatant of SK6.T7 cells transfected with full-length DNA. Viable chimeric virus Flc9, in which E2 was replaced, resulted in recovery of virus only when SK6.T7 transfected cells were maintained for several passages. However, no virus could be recovered after replacement of both ERNS and E2, even after 10 cell passages. Both Flc9 and Flc11 grow in swine kidney cells (SK6), stably maintain their heterologous BVDV sequences and, as assessed by monoclonal antibody typing and radio-immunoprecipitation assays, express their heterologous proteins. Flc9 showed a slower growth rate on SK6 cells than Flc11 and wild-type Flc2 virus. Replacement of ERNS or E2 of C-strain-based chimeric viruses did not alter cell tropism compared to wild-type C-strain virus for SK6 and FBE cells. Both Flc9 and Flc11 induced E2 or ERNS antibodies, which could be discriminated from those induced after wild-type virus infection, even after repeated vaccination. Furthermore, pigs were completely protected against a lethal CSFV challenge. These results indicate the feasibility of introduction of marker antigens in a live-attenuated marker C-strain vaccine for CSFV