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Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection. All four immunized animals resolved HCV infection. The difference in the rate of chronicity between the immunized and the control animals was close to statistical significance ( P = 0.067). Immunized animals developed vigorous gamma interferon enzyme-linked immunospot responses and moderate proliferative responses. To investigate cross-genotype protection, the immunized recovered chimpanzees were challenged with a pool of six major HCV genotypes. During the acute phase after the multigenotype challenge, all animals had high-titer viremia in which genotype 4 dominated (87%), followed by genotype 5 (13%). However, after fluctuating low-level viremia, the viremia finally turned negative or persisted at very low levels. This study suggests the potential efficacy of replicating recombinant vaccinia virus-based immunization against chronic HCV infection.

Background— We previously reported successful therapeutic immunization in a chimpanzee having a relatively low viral load, which was immunized with recombinant plasmid hepatitis B surface antigen (HBsAg) DNA and boosted with recombinant HBsAg encoding canarypox virus. In the present study, we attempted to confirm these findings in an animal with a high virus load. Methods and Results— We tested three immunization strategies successively over a 3-year period. In the first of these, we administered four monthly injections of DNA encoding HBsAg + PreS2 + hepatitis B core antigen (HBcAg) + DNA encoding interleukin (IL)-12, (given 3 days later), and boosted with canarypox expressing all of the above HBV genes 6 months after initial immunization. No reduction in viral load was observed. In the second trial, we administered lamivudine for 8 weeks, and then began monthly DNA-based immunization with plasmids expressing the above viral genes; however, viral loads rebounded 1 week after termination of lamivudine therapy. In a third trial, we continued lamivudine therapy for 30 weeks and immunized with vaccinia virus expressing the above viral genes 18 and 23 weeks after the start of lamivudine therapy. Again viral loads rebounded shortly after cessation of lamivudine treatment. Analysis of cell-mediated immune responses, and their avidity, revealed that DNA-based immunization produced the strongest enhancement of high avidity T-cell responses, while recombinant vaccinia immunization during lamivudine therapy enhanced low avidity responses only. The strongest low and high avidity responses were directed to the middle surface antigen. Conclusions— Three strategies for therapeutic immunization failed to control HBV viremia in a chronically infected chimpanzee with a high viral load.

An open question for hepatitis C virus (HCV) vaccine development is whether the various genotypes of this virus protect against the development of chronic infection after heterologous infection with different genotypes. We approached this question by challenging chimpanzees that had recovered from HCV genotype 1a or 1b infection with 6 heterologous genotypes as well as with a homologous genotype (for chimpanzees originally infected with genotype 1a). All 9 chimpanzees rechallenged with a homologous genotype developed self-limited infections. Of 11 chimpanzees challenged with 100 chimpanzee infectious doses of heterologous genotypes, 6 developed self-limited infections, with peak viral loads in acute-phase serum that were ~5-fold lower than those seen during primary infections. One chimpanzee (which had recovered from genotype 1b infection and was rechallenged with genotype 6a) did not develop viremia but did show an anamnestic cell-mediated immune response after rechallenge. Four of the 11 chimpanzees rechallenged with heterologous genotypes developed chronic infections with the genotypes used for rechallenge. These findings suggest that a universally protective HCV vaccine may need to incorporate epitopes from multiple genotypes.

The availability of molecular beacon-based, real time polymerase chain reaction (PCR) and a semi-automated sample extraction procedure have made it possible for us to retrospectively examine HCV replication kinetics in HCV naive chimpanzees infected during the past 20 years. We compared these in 17 animals that developed chronic infection, and in 21 that developed self-limited infection. No differences were found in infecting dose, or replication kinetics in the acute phase between these two types of infection. An unanticipated finding was the fact that 10 of 17 animals developing chronic infection partially controlled virus replication for 48 +/- 48 weeks after typical acute phase viraemia, and prior to development of chronic infection. Twenty-nine out of 30 (29/30) sera, which were negative by quantitative PCR during the downregulated period, were, however, positive by the more sensitive Genprobe isothermal transcription-mediated amplification (TMA) assay. Thus, downregulation was not complete. Ten animals showing self-limited infection showed complete resolution of viraemia by TMA assay. Quasispecies analysis revealed that in all, except one case, the virus reappearing after downregulation was essentially identical to that of the originally infecting virus.

In hepatitis C virus (HCV) infection, there is accumulating data suggesting the presence of cellular immune responses to HCV in exposed but seemingly uninfected populations. Some studies have suggested cross-reactive antigens rather than prior HCV exposure as the main reason for the immune responses. In this study we address this question by analyzing the immune response of chimpanzees that have been sequentially exposed to increasing doses of HCV virions. The level of viremia, as well as the immune responses to HCV at different times after virus inoculation, were examined. Our data indicate that HCV infective doses as low as 1–10 RNA (+) virions induce detectable cellular immune responses in chimpanzees without consistently detectable viremia or persistent seroconversion. However, increasing the infective doses of HCV to 100 RNA (+) virions overcame the low-inoculum-induced immune response and produced high-level viremia followed by seroconversion.

BACKGROUND: Numerous reports have noted the existence of sera, particularly from resolving cases of HBV infection, that are positive for HBV DNA by PCR, despite being negative for HBsAg and IgM anti-HBc. If such blood is infective and detectable by HBV NAT screening, it seems desirable to introduce such screening for transfused blood. STUDY DESIGN AND METHODS: Three chimpanzees were inoculated with serum and lymphocytes from three patients who were HBV DNA PCR positive, but HBsAg negative. The animals were tested over a period of 15 months for HBsAg, anti-HBs, anti-HBc, and HBV DNA by PCR. RESULTS: All animals remained uninfected. CONCLUSION: Small amounts of plasma and MNCs from HBV DNA-positive HBsAg-negative blood do not appear to be infectious; however, further studies with larger volumes of inoculum should be conducted.

To determine whether there was a correlation between the kinetics or frequency of antibody to mammalian-derived hepatitis C virus (HCV) second envelope protein (E2) and development of chronicity or self-limitation of HCV infections, serial sera were examined for anti-E2, anti-HCV with confirmation with Matrix 2.0 (Abbott Laboratories, Abbott Park, IL), and reverse transcriptase-polymerase chain reaction (RT-PCR) from 6 cases of self-limited infection and 6 cases of chronic infection in chimpanzees, and from 5 cases of self-limited infection and 3 cases of chronic infection in patients. Anti-E2 developed earlier, more frequently, and to higher titer in chimpanzees and patients who were developing chronic infection than in those with self-limited infections. Thus anti-E2 is unlikely to play a role in self-limitation of the infection. However, long-term persistence of anti-E2 correlates with chronic infection. There was little or no correlation between the timing of development of anti-E2 and anti-HCV.

Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of filarial parasites and other nematode species. We have investigated the possible involvement of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro. The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine, and N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-al anine benzylamide. None of the inhibitors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100 to 200 microM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylcadaverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide epsilon-(gamma-glutamyl)lysine. These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed reactions.

Improved serologic and polymerase chain reaction (PCR)-based tests for hepatitis C virus (HCV) infection provided an opportunity to reexamine a posttransfusion follow-up study done from 1969 to 1972. A total of 213 cardiac surgery patients was prospectively followed after receiving an average of 18 units of blood, 24% of which was from paid donors. Serial sera were tested for antibody to recombinant DNA-derived C100-3 and capsid polypeptides; selected cases were also tested against synthetic peptides derived from different regions of the HCV sequence. PCR and RIBA II immunoblot assays were done on selected sera. Each of 55 probable and 5 of 11 possible hepatitis cases who were seronegative before transfusion seroconverted. Anti-HCV seroconversion also occurred in 6 (4%) of 148 subjects without hepatitis. Among subjects followed > 1 year, PCR positivity persisted in 14 (82%) of 17. If the results of this study can be generalized, all bloodborne non-A, non-B hepatitis may be due to HCV.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.

Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by- product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-naive chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.

The treatment of plasma with organic solvent/detergent mixtures at the time of plasma collection or pooling could reduce the exposure of technical staff to infectious viruses and enhance the viral safety of the final product. Treatment of plasma for 4 hours with 2-percent tri(n-butyl)phosphate (TNBP) at 37 degrees C, with 1-percent TNBP and 1-percent polyoxyethylensorbitan monooleate (Tween 80) at 30 degrees C, or with 1-percent TNBP and 1-percent polyoxyethylene ethers, (Triton X-45) at 30 degrees C resulted in the rapid and complete inactivation of greater than or equal to 10(4) tissue culture-infectious doses (TCID50) of vesicular stomatitis and Sindbis viruses, which are used as surrogates. Treatment of plasma with TNBP and TNBP and Tween-80 was shown to inactivate greater than or equal to 10(4) TCID50 of human immunodeficiency virus. TNBP treatment of plasma contaminated with 10(6) chimpanzee-infectious doses (CID50) of hepatitis B virus and 10(5) CID50 of non-A,non-B hepatitis virus prevented the transmission of hepatitis to chimpanzees. Immediately after treatment of plasma with 2-percent TNBP, the recovery of factors VIII, IX, and V and antithrombin III was 80, 90, 40, and 100 percent, respectively. Recovery of all factors was greater than or equal to 90 percent after treatment with TNBP and detergent mixtures. Treated plasma was fractionated by standard techniques into antihemophilic factor and prothrombin complex concentrates, immune globulin, and albumin. Prior treatment with TNBP or TNBP and detergent did not affect the separations of desired proteins. Therefore, it appears possible to inactivate viruses in plasma before the execution of standard fractionation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Chimpanzee is a unique animal model for HCV infection, in which about 50% of infections resolve spontaneously. It has been reported that the magnitude of T cell responses to HCV core in recovered chimpanzees is greater than that in chronically infected ones. However, the mechanism(s) by which the chimpanzees with resolved infection overcome core-mediated immunosuppression remains unknown. In this study, we examined the effect of HCV core on T cell responsiveness in chimpanzees with resolved and chronic HCV infection. We found that core protein strongly inhibited T cell activation and proliferation in chimpanzees with chronic infection, while this inhibition was limited in chimpanzees with resolved infection. Notably, the level of gC1qR, as well as the binding of core protein, on the surface of T cells was lower in recovered chimpanzees when compared to chimpanzees with chronic HCV infection. Intriguingly, the observed differences in gC1qR expression levels and susceptibility to core-induced suppression amongst HCV-chronically infected and recovered chimpanzees were observed prior to HCV challenge, suggesting a possible genetic determination of the outcome of infection. These findings suggest that gC1qR expression on the surface of T cells is crucial for HCV core-mediated T cell suppression and viral clearance, and that represents a novel mechanism by which a virus usurps host machinery for persistence.

Chimpanzees, because of their near-human nature, have special needs that must be met by those who carry out medical research with them. Perhaps the most important of these is the need for companionship. Chimpanzees kept alone become obviously depressed, and manifest stereotypic behavior such as compulsive rocking. We have followed a policy of keeping chimpanzees in groups of two or more, whenever possible. This has virtually prevented overt depressive symptomatology. This policy has not significantly affected studies on hepatitis B and C (1).

summary. The immune response to hepatitis C virus (HCV) is believed to be critical in determining the outcome of the disease. In this study we have analysed epitope recognition, cytokine profile, and anti-HCV antibody responses in chronically HCV-infected and recovered chimpanzees. Quantitative measurement of anti-HCV antibody in HCV-infected chimpanzees revealed that the response in HCV- recovered chimpanzees peaked within 4–20 weeks. In contrast, the anti-HCV antibody responses in chronically HCV infected chimpanzees did not peak until 100–200 weeks after infection, and decreased gradually thereafter. T cell proliferation assays measuring responses to pooled HCV proteins revealed significant increases in the 3H-uptake during the early stages of infection in recovered chimpanzees in comparison to the chronically infected ones. Class I-restricted epitopes of the core, and NS3 proteins of HCV were analysed using 9–10 mer overlapping peptides covering the core and NS3 proteins, and IFN-γ ELISPOT technique. Our data indicated early and broad class-I restricted core, and NS3 protein epitope recognitions in HCV-recovered chimpanzees but not in chimpanzees that had been chronically infected. Additionally, dominant epitopes recognized early in infection (8 weeks) were no longer recognized later in infection (followed up to 64 weeks). Cytokines profiling revealed a 50-fold increase in TNF-α secretion in the supernatant of core-specific CD8 memory cells of the chronically infected chimpanzees in comparison to the recovered ones. In summary, multiple parameters correlate with HCV recovery in chimpanzees.

Serum from 387 chimpanzees (Pan troglodytes verus), caught in the wild or bred in captivity, was tested for antibody to HIV-1 and HIV-2, using second- and third-generation enzyme immunoassays. Six samples were repeatedly positive; however, only one of these was Western blot positive. Serial sera drawn before and after the Western blot-positive samples were seronegative, and thus we conclude that this sample represented specimen contamination, or mislabeling. Thus, none of the 387 Pan troglodytes verus from West Africa were spontaneously infected with SIVcpz. Chimpanzees are known to be exquisitely susceptible to infection with HIV-1 when experimentally inoculated, and thus our findings suggest that HIV-1-related viruses do not exist in Pan troglodytes verus in the wild. As it has been convincingly shown that SIVcpz exists in wild Pan troglodytes troglodytes in Central Africa, this suggests that HIV-1 arose in Central Africa, but not in West Africa.

Chimpanzees have been shown to be exquisitely susceptible to human hepatitis viruses, without themselves developing clinical illness, thus providing an important model for studies on these agents. Chimpanzees have contributed substantially to human welfare by making possible the development of hepatitis B vaccines, which now prevent development of cirrhosis and hepatocellular carcinoma in millions of people. They have provided a means to evaluate the efficacy of virus inactivation strategies, which have made blood derivatives formerly contaminated with blood-borne viruses (hepatitis B, C, and human immunodeficiency viruses) safe with respect to their transmission. In exchange for these contributions, humans owe chimpanzees lifelong retirement in sanctuaries that offer socialization and environmental enrichment.

There are about 200 million chronic hepatitis B virus (HBV) carriers at high risk of development of cirrhosis and hepatocellular carcinoma. Termination of the carrier state may avert these risks. We have investigated immunotherapy for chronic HBV infection in a chimpanzee HBV carrier using recombinant DNA-based immunization followed by a recombinant canarypox booster. One week after the booster, HBV DNA declined greater than 400-fold and remained undetectable by the quantitative polymerase chain reaction (PCR) assay for 186 weeks. Plasma levels of hepatitis B surface antigen (HBsAg) declined for only a short time. The decline in HBV DNA correlated with a boost in gamma interferon production without a corresponding boost in cytotoxic T lymphocyte levels, and decline in the transcriptional template or covalently closed circular DNA level. Confirmation of these findings requires further studies in chimpanzees and/or in humans.

The aim of this study was to evaluate HCV-cytotoxic T lymphocyte response from PBMC in bulk CTL assays and in CTL precursor analyses using in vitro stimulation with canarypox virus (ALVAC) expressing HCV-capsid/E1/E2/NS2/NS3 antigens. Canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology on mammalian cells. PBMC were obtained from four chimpanzees with chronic hepatitis C infection and one uninfected chimpanzee. CTL from bulk culture of PBMC and CTL precursor frequencies were found in three of the four chronically infected chimpanzees using ALVAC in vitro stimulation. No CTL response was detected in PBMC from the uninfected chimpanzee. The precursor frequencies of CTL specific for capsid, NS2 and NS3 proteins ranged between 1/2663 and 1/27202. No correlation was observed between percent cytolysis in bulk culture and CTL precursor frequencies. This method may prove useful in assessing the correlation between HCV–CTL response and virological or histological status.

We have previously reported that chimpanzees chronically infected with hepatitis C virus (HCV) could be reinfected, even with the original infecting strain. In this study we tested the hypothesis that this might reflect the presence of minor quasispecies to which there was little or no immunity. To evaluate this hypothesis, we sequenced multiple clones taken at intervals after primary infection and rechallenge from four chronically infected chimpanzees. The inoculum used in these studies (HCV-H, genotype 1a) revealed 17 separate variants among 46 clones sequenced. Following challenge, each of the four challenged animals showed marked alterations of their quasispecies distribution. The new variants, which appeared 1 to 6 weeks after challenge, were either identical to or closely resembled variants present in the challenge inoculum. These results, paralleled by an increase in viremia in some of the challenged animals, suggest that quasispecies in the challenge inoculum were responsible for signs of reinfection and that there was little immunity. However, the newly emerged quasispecies completely took over infection in only one animal. In the remaining three chimpanzees the prechallenge quasispecies were able to persist. The natural evolution of infection within chimpanzees resulted in variants able to compete with the inoculum variants. Whether through reexposure or the natural progression of infection, newly emerged quasispecies are likely to play a role in the pathogenesis of chronic HCV infection.

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 μM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Hepatitis C virus (HCV)-infected humans and chimpanzees were studied for reactivity with linear epitopes in HCV H strain structural proteins. In 10 HCV-infected patients, epitopes were mostly mapped to the capsid and E1 proteins but not to E2. However, serum from 1 HCV-infected blood donor with a high anti-capsid titer reacted with multiple epitopes including E2. By contrast, antibody to capsid epitopes was seen in sera from HCV-rechallenged chimpanzees but not from chronically infected animals. No reactivity was observed to GOR epitope in chimpanzees, while 6 of 11 human subjects reacted with this host-coded antigen. Reactivity to rare epitopes in E2 was seen in chimpanzees with chronic and self-limited infections. Reactivity to one peptide of E1 (aa 316-329) was observed in 10 of 11 sera from HCV-infected humans and 11 of 15 chimpanzee sera. However, reactivity to this epitope was also seen in normal chimpanzees and in 6 (7.1%) of 84 uninfected human subjects.

Polymerase chain reaction (PCR) and newer serologic assays for hepatitis C virus (HCV) were used to investigate 19 HCV cross-challenge episodes in chimpanzees. In these cross-challenges, 59% showed seroconversion after challenge, 33% showed reappearance of HCV-associated hepatocellular ultrastructural changes, 5 animals not PCR-positive at the time of challenge showed return of PCR positivity, and 26% developed hepatitis after rechallenge. A total of 74% showed at least one of these signs of reinfection. The frequency of development of serologic and ultrastructural responses was, however, reduced in secondary compared with primary infections (P less than .01). In 10 animals, the cross-challenge was done with heterologous strains, and in 9 with the originally infecting virus. There was no significant difference in the responses to homologous and heterologous challenges. The data suggest relatively weak immunity in HCV infections.

The identification and characterization of a recombinant cDNA clone (OV103) expressing a microfilarial surface-associated antigen of Onchocerca volvulus is described. OV103 was identified and isolated from a λgt11 cDNA expression library derived from adult O. volvulus mRNA using a chimpanzee antiserum, taken 2 years after infection with third-stage larvae of O. volvulus . The cDNA clone encodes a 12.5-kDa protein that corresponds to a 15-kDa parasite protein present in microfilariae and adult female worms. The antigen encoded by this clone is located in the basal layer of the cuticle and the hypodermis of the female adult worm, and on the surface of microfilariae. OV103 fusion polypeptide is recognized only by some sera from onchocerciasis infected subjects (57%), but more significantly (89%) by sera from individuals that have low levels of patent infection. In addition, the antibody response to this protein developed before appearance of microfilariae in the skin of chimpanzees that had developed non-patent or low level patent infections, while the antibody response in chimpanzees with high levels of microfilariae appeared later at the time of appearance of microfilariae. Preliminary experiments indicated that affinity purified antibodies directed against OV103 fusion polypeptide mediated killing of nodular microfilariae in vitro in the presence of normal peripheral blood granulocytes.

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.

We would like to comment on the controversy concerning the use of a virulent strain of human immunodeficiency virus (HIV) to assess the protective efficacy of candidate HIV vaccines (A. M. Prince and L. Andrus, Letters, Science 's Compass, 18 Dec. p. [2195][1]; N. L. Letvin, ibid. ). We believe that this must be viewed from both scientific and ethical perspectives. The controversy stems from the report of a chimpanzee that developed an AIDS-like syndrome 10 years after infection with various laboratory isolates of HIV and which was euthanized in 1996 ([1][2]). Inoculation of 40 milliliters of blood from this chimpanzee to a second animal resulted in an extremely high acute viremia (>107 HIV RNA molecules per milliliter of plasma) and a rapid depletion of CD4+ cells within 14 weeks after infection. The virulence of the primary infection in this animal is not representative of most primary infections in humans. Acute viremia in humans is characteristically 10- to 100-fold lower, and CD4+ cell depletion does not occur until several years after infection. Use of a challenge virus having unusual virulence could seriously jeopardize the HIV vaccine effort, because protection against such viruses could be missed with vaccines that effectively protect against less virulent wild-type HIV. Suitable challenge viruses for vaccine evaluation should have virulence characteristics similar to those of wild-type viruses that infect humans. They should also ideally be primary isolates grown only on peripheral blood lymphocytes because of the relative resistance of such viruses to antibody-mediated neutralization. An expanded stock of the HIV-1Han2 isolate was recently developed by Program EVA (European Vaccine Against AIDS) to fulfill these requirements ([2][3]). This clade-B primary isolate exhibits growth characteristics in chimpanzees similar to those seen in humans. It reliably infects chimpanzees using small challenge inocula (10 to 100-tissue-culture infectious doses) and maintains a detectable chronic viremia similar to that obtained with the laboratory isolate HIV-1Lai. It does not cause AIDS rapidly, if at all. From an ethical perspective, our concerns about the use of virulent HIV strains stem to a large extent from precedents set in the field of simian immunodeficiency virus (SIV)/SHIV (a genetically engineered hybrid virus with an HIV envelope and an SIV core) research in monkeys. Pathogenic SIV strains have emerged that have subsequently been passaged through monkeys to develop isolates with increased virulence for that species and which cause death within weeks after infection. We point out that euthanasia of chimpanzees (our nearest relative) is universally condemned. The development of virulent HIV strains that cause AIDS in a short time would necessitate euthanasia and should be opposed on that ground alone. We urge those who carry out vaccine research in the chimpanzee model to seriously question the use of virulent HIV challenge inocula from both a scientific and an ethical standpoint. 1. [↵][4]1. F. J. Novembre 2. et al. , J. Virol 71, 4086 (1997). [OpenUrl][5][Abstract/FREE Full Text][6] 2. [↵][7]1. W. J. Bogers 2. et al. , J. Gen. Virol. 79, 2895 (1998). [OpenUrl][8][Abstract/FREE Full Text][9] [1]: /lookup/doi/10.1126/science.282.5397.2194d [2]: #ref-1 [3]: #ref-2 [4]: #xref-ref-1-1 "View reference 1 in text" [5]: {openurl}?query=rft.jtitle%253DJournal%2Bof%2BVirology%26rft.stitle%253DJ.%2BVirol.%26rft.aulast%253DNovembre%26rft.auinit1%253DF.%2BJ.%26rft.volume%253D71%26rft.issue%253D5%26rft.spage%253D4086%26rft.epage%253D4091%26rft.atitle%253DDevelopment%2Bof%2BAIDS%2Bin%2Ba%2Bchimpanzee%2Binfected%2Bwith%2Bhuman%2Bimmunodeficiency%2Bvirus%2Btype%2B1%26rft_id%253Dinfo%253Apmid%252F9094687%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [6]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoianZpIjtzOjU6InJlc2lkIjtzOjk6IjcxLzUvNDA4NiI7czo0OiJhdG9tIjtzOjI1OiIvc2NpLzI4My81NDA1LzExMTUuNS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30= [7]: #xref-ref-2-1 "View reference 2 in text" [8]: {openurl}?query=rft.jtitle%253DJournal%2Bof%2BGeneral%2BVirology%26rft.stitle%253DJ.%2BGen.%2BVirol.%26rft.aulast%253DBogers%26rft.auinit1%253DW.%2BM.%26rft.volume%253D79%26rft.issue%253D12%26rft.spage%253D2895%26rft.epage%253D2903%26rft.atitle%253DCharacteristics%2Bof%2Bprimary%2Binfection%2Bof%2Ba%2BEuropean%2Bhuman%2Bimmunodeficiency%2Bvirus%2Btype%2B1%2Bclade%2BB%2Bisolate%2Bin%2Bchimpanzees%26rft_id%253Dinfo%253Apmid%252F9880002%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [9]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoidmlyIjtzOjU6InJlc2lkIjtzOjEwOiI3OS8xMi8yODk1IjtzOjQ6ImF0b20iO3M6MjU6Ii9zY2kvMjgzLzU0MDUvMTExNS41LmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==

Simian immunodeficiency virus (SIV) is a Lentivirus that exhibits both morphological and genomic similarities to HIV. SIV has been isolated from sooty mangabeys in several primate colonies, and has been shown to be about 80% related to HTV-2. Since HIV-2 and sooty mangabeys are both indigenous to West Africa, isolation of a virus from sooty mangabeys in West Africa would be of great interest in determining the origins of these two viruses. We report here thin section transmission electron microscopy of a lentivirus from a pet sooty mangabey living in Liberia, West Africa.Peripheral blood mononuclear cells (PEMC) from a healthy pet sooty mangabey in Liberia were frozen in liquid nitrogen and shipped to the California Primate Research Center. The cells were thawed and stimulated for 72h with 0.5μg/ml Staphylococcal enterotoxin A in RPMI 1640 containing 10% fetal calf serum. 2×106PEMC were then co-cultivated with 2×106CEMxl74 cells and processed for EM.CEMxl74 cells co-cultivated with infected PBMC exhibited syncytial giant cells with patchy foamy cytoplasm (Fig 1). large numbers of virus particles were observed within these vacuoles (Fig 2).

A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to replicate in cell cultures derived from chimpanzee liver after inoculation of serum containing infective non-A, non-B (NANB) hepatitis viruses from two independent sources. Replication of this agent was not observed when the same cells were inoculated with a chloroform-extracted inoculum or were left uninoculated. Replication involves assembly of virus cores on tubular structures similar to those seen in liver cells of chimpanzees infected with most isolates of NANB virus.

Repeated administration of an intravenous immunoglobulin containing antibody to hepatitis B e antigen (anti-HBe) and antibody to hepatitis B core antigen (anti-HBc) but free of antibody to hepatitis B surface antigen (anti-HBs) before and after the inoculation of 10(4.9) 50% chimpanzee infective doses of hepatitis B virus (HBV) markedly prolonged the incubation period of HBV in experimentally infected chimpanzees. Similar administration of an immunoglobulin preparation containing anti-HBc but free of anti-HBe and anti-HBs or intramuscular administration of a single dose of immunoglobulin containing anti-HBe and anti-HBc 3 days before or after inoculation with HBV did not appear to modulate HBV infection. These observations suggested that anti-HBe, or an unidentified antibody associated with it, may have biological activity in the modulation of HBV replication.

Fourteen chimpanzees were challenged with the Hutchinson strain inoculum that has been shown by many workers to produce non-A, non-B (NANB) hepatitis associated with characteristic cytoplasmic ultrastructural changes observable by electron microscopy. Nine of these animals had a history of definite NANB hepatitis induced by seven different human viral isolates; all of these animals resisted rechallenge. The five animals without a history of NANB hepatitis all developed definite histological changes associated with NANB hepatitis after challenge. Homologous rechallenge with a 100-fold higher infectivity titer was carried out in five of the nine chimpanzees. Cytoplasmic ultrastructural changes developed after challenge in two of these animals; the remaining three had evidence of possible mild reinfection on the basis of liver histopathology or mild elevations of transaminase or both. We conclude that most, if not all, blood-borne NANB isolates belong to a single class of agents and that this virus produces immunity to rechallenge, but this immunity may be overwhelmed by high-dose inocula.

Blood product sterilisation with 0.3% tri(n-butyl)phosphate (TNBP)/0.2% sodium cholate (CA), a combination known to permit high recovery of factor VIII and factor IX, was evaluated for its effect on hepatitis B (HBV), non-A, non-B (NANB), and human T-lymphotropic type III (HTLV-III) viruses. 2 chimpanzees received factor VIII preparations contaminated with 10(4) chimpanzee infectious doses (CID50) of HBV and treated with TNBP/CA; neither had evidence of HBV infection during 9 months follow-up, but hepatitis B surface antigen (HBsAg) developed 5 and 6 weeks, respectively, after challenge with untreated inoculum. 2 chimpanzees were similarly exposed to 10(4) CID50 of Hutchinson NANB inoculum treated with TNBP/CA; neither became infected during 26 weeks of follow-up but both had characteristic NANB-associated ultrastructural changes 3-5 weeks after exposure to untreated inoculum. 2 chimpanzees inoculated with 80 ml of TNBP/CA-treated factor VIII derived from a pool of thirteen lots obtained from five US manufacturers remained free of any evidence of NANB infection during 32 weeks of follow-up. Subsequently, NANB infection developed in both animals 3-4 weeks after exposure to untreated inoculum. Exposure of HTLV-III diluted into a factor VIII preparation to TNBP/CA inactivated greater than or equal to 10(4.2) tissue culture infective doses within 20 min at 24 degrees C.Blood product sterilization with 0.3% tri(n-butyl)phosphate (TNBP)/0.2% sodium cholate (CA), a combination known to permit high recovery of factor VIII and factor IX, was evaluated for its effect on hepatitis B (HBV), non-A, non-B (NANB), and human T-lymphotropic type III (HTLV-III) viruses. 2 chimpanzees received factor VIII preparations contaminated with 100,000 chimpanzee infectious doses of HBV and treated with TNBP/CA. Neither had evidence of HBV during the 9 month follow-up, but hepatitis B surface antigen (HBsAG) developed 5 and 6 weeks, respectively, after challenge with untreated inoculum. 2 chimpanzees were similarly exposed to NAMB inoculum treated with TNBP/CA. Neither became infected during 26 weeks of follow-up, but both had characteristic NANB-associated ultrastructural changes 3-5 weeks after exposure to untreated inoculum. 2 chimpanzees inoculated with TNBP/CA-treated factor VIII derived from a pool of 13 lots obtained from 5 US manufacturers remained free of any evidence of NANB infection during 32 weeks of follow-up. Subsequently, NANB infection developed in both animals 3-4 weeks after exposure to untreated inoculum. Exposure of HTLV-III diluted into a factor VIII preparation to TNBP/CA inactivated tissue culture infected doses within 20 minutes. These results demonstrate that exposure of labile blood derivatives to TNBP/CA effectively inactivates HBV, NANB, and HTLV-III viruses, although additional experiments with more potent inocula are needed to establish limits of inactivation efficacy against these agents. To provide absolute safety, a process efficacy of = or 5-6 log 10 is preferable.

Titrated stocks of hepatitis B virus and Hutchinson strain non-A, non-B hepatitis virus were diluted in normal serum to contain, respectively, greater than or equal to 10(6) and greater than or equal to 10(4) chimpanzee infectious doses (CID50) per milliliter and exposed to 1% Tween 80 and 20% ether at 4 degrees C for 18 h. After evaporation of the ether, the treated sera were each inoculated into two chimpanzees. The animals remained free of serologic and biochemical evidence of hepatitis during a 6-month follow-up period, and were then shown to be susceptible to infection by challenge with the original untreated inocula. To assess the effect of exposure to Tween 80/ether on coagulation factors, four lots of antihemophilic factor (AHF) concentrate and 2 lots of commercial factor IX concentrate were treated as above. For the AHF concentrate there was an average of 70% recovery of factor VIII procoagulant activity, 93% recovery of factor VIII-related antigen, and 73% recovery of fibronectin opsonin activity and no detectable change in ristocetin cofactor activity or in fibronectin antigen. Crossed immunoelectrophoresis revealed no change in migration rate of fibrinogen, fibronectin, and von Willebrand factor (vWF), although the quantity of fibrinogen was reduced. Factor VIII procoagulant activity and vWF activity remained associated during chromatography on BioGel A15.

Inoculation of chimpanzees revealed that starting material for the preparation of a pooled stabilized human serum preparation was contaminated with non-A, non-B hepatitis virus(es). This material was subjected to beta-propiolactone/ultraviolet irradiation treatment and adsorption with an insolubilized silicic acid under conditions employed for sterilization of the final product (Biseko, Biotest). The treated material did not transmit hepatitis to 2 inoculated chimpanzees. These animals were subsequently found to be susceptible to non-A, non-B infection by challenge with the untreated material. These findings demonstrate a non-A, non-B type hepatitis virus to be susceptible to inactivation by the methods employed for sterilization of this stabilized human serum preparation.

The thrombogenicity of beta-PL/UV-treated PPSB (factor IX concentrate) was evaluated in chimpanzees. PPSB isolated from beta-propiolactone-treated and UV-irradiated plasma was injected into chimpanzees at a dose of approximately 100 units/kg body weight. An FDA licensed PPSB preparation served as the negative control, and a preparation containing activated as well as precursor clotting factors served as the positive control. 15 minutes, 1 h, 4 h, and 24 h after the PPSB application the following parameters were determined in the chimpanzee blood: factors II, VII, IX, X, VIII, fibrinogen, AT III, thrombin coagulase, Quick value, APTT and platelet count. Neither the untreated control preparation, nor the PPSB isolated from beta-propiolactone-treated and UV-irradiated plasma, showed signs of thrombogenicity in the chimpanzee model. The positive control indicated that the chimpanzee is a suitable model for the thrombogenicity testing of activated clotting factors.

The bivalent form of an aqueous formalin-inactivated hepatitis B vaccine was evaluated for safety and immunogenicity in chimpanzees. To evaluate safety five animals were inoculated intravenously with vaccine containing 500 micrograms HBsAg and two animals with 50 micrograms. None of these animals developed hepatitis or any serologic marker indicative of the presence of residual live virus in the vaccine. Twenty-four animals were used to evaluate immunogenicity and protective efficacy. Seven of these immunized animals produced weak or no anti-HBs responses. Two doses of 50 micrograms HBsAg given subcutaneously 1 month apart protected each of four animals that were challenged with 10(3.5) CID50 HBV at 6 and 12 months after immunization and protected three of four animals challenged at 24 months against development of hepatitis or HBsAg. Three of 4 animals in each group immunized with two doses of 20, 10, or 5 micrograms HBsAg were similarly protected when challenged 6 months after immunization. Thirteen of 20 immunized animals that did not develop HBsAg after challenge with HBV developed anamnestic anti-HBs or anti-HBc responses between 2 and 18 months after challenge, indicating minimal replication of challenge virus. The time of onset and frequency of occurrence of these delayed responses was related to the titer of anti-HBs at the time of challenge. False positive Ausab test results were observed in quarantined chimpanzees. These were neither preceded by appearance of HBsAg nor accompanied by development of anti-HBc. In most cases these reactions were due to a reactant having a sedimentation coefficient and an electrophoretic mobility resembling that of IgM. This reactant generally did not appear to confer resistance to challenge with HBV. The humoral immune response was characterized as being entirely of the IgM class 2 weeks after immunization and switched entirely into the IgG class by 10-12 weeks after vaccine administration. At the time of challenge all animals with antibody had anti-HBs of subtype a.

The efficacy of combined beta-propiolactone/ultraviolet irradiation (betaPL/UV) for inactivation of hepatitis B virus in labile blood derivatives has been reviewed. The initial evaluations of these procedures were hampered by inadequate process control that resulted in excessive protein denaturation; furthermore, adequate evaluation of process efficacy for virus inactivation was prevented by the absence of titered hepatitis virus stocks, the lack of an animal model, and the failure to carry out controlled trials. Finally, it was not appreciated that the power of these procedures lay especially in their use in combination. These deficits have now been remedied. To permit quantitation of process efficacy, a regression analysis of the relation between virus dose and incubation period in chimpanzees has been carried out. This has provided a means of estimating virus titer and determining the accuracy of such estimates. The most recent data suggest that betaPL/UV can reduce the titer of hepatitis B virus about 10 million fold (10(-7)). The process efficacy for betaPL/UV followed by the special adsorption procedures used in preparation of a stabilized human serum containing most human serum proteins except for factor VIII, the factor IX complex, fibrinogen, and the lipoproteins was estimated as a 10(8)-fold reduction in virus titer. This degree of virus inactivation should be more than sufficient to sterilize the amounts of hepatitis B virus that could be expected in pooled human plasma that has been screened for hepatitis B surface antigen. Preliminary data also suggest that the betaPL/UV procedure effectively inactivates non-A, non-B hepatitis virus(es).

beta-propiolactone (beta-PL) treatment has been evaluated for its ability to inactivate 10(3.5) chimpanzee infectious doses (CID50) of the Hutchinson strain of hepatitis non-A, non-B virus (HNANBV). Two chimpanzees were inoculated with a beta-PL-treated immunoglobulin solution to which this dose of the titrated virus had been added prior to beta-PL treatment. beta-PL treatment was performed in accordance with the production procedure used for a licensed intravenous immunoglobulin preparation. Neither animal developed hepatitis. When subsequently challenged with the same spiked immunoglobulin solution that had not been beta-PL treated, both animals developed clear-cut hepatitis non-A, non-B. The results of this experiment demonstrate that beta-PL treatment is effective for the inactivation of hepatitis non-A, non-B virus in intravenous immunoglobulin.

Alanine aminotransferase (ALT) is currently widely used as a surrogate marker for detection of blood having a higher risk of transmission of non-A, non-B hepatitis (NANBH). Studies in the chimpanzee model, in which the NANBH carrier state is well defined, have revealed gamma-glutamyltransferase to be considerably more sensitive for the detection of NANBH carrier blood. Further study is required to determine whether this marker is also more sensitive for detection of the chronic NANBH carrier state in man.

A beta-propiolactone/ultraviolet irradiation procedure (beta PL/UV) has been evaluated for its ability to inactivate 30,000 chimpanzee infectious doses of the Hutchinson strain of non-A, non-B (NANB) virus. The chimpanzees were inoculated with plasma to which this dose of the titrated virus had been added prior to application of the beta PL/UV process in accordance with a procedure used for licensed blood derivatives in Germany. Neither animal developed hepatitis. When subsequently challenged with the same contaminated plasma, which had not been sterilized, both animals promptly developed typical NANB hepatitis. This study extends the high (approximately 10(7)-fold) process efficiency of the beta PL/UV procedure previously reported for hepatitis B virus to a blood-borne NANB virus.

SummaryRecent experiments have shown that a preparation of PPSB (factor IX concentrate) derived from βPL/UV treated plasma was not infectious in chimpanzees with respect to hepatitis B and non-A, non-B. To answer the question whether the βPL/UV treatment influences the tolerance and efficacy of the PPSB concentrate, long-term application of PPSB-Biotest was carried out in chimpanzees. After 10 applications of 25 U factor IX/kg at weekly intervals, no signs of intolerance were observed by measurement of blood pressure during i. v. application and by means of skin-testing. Determination of coagulation factor activity during the application period revealed the same factor IX recovery at the beginning and at the end of the study.

Hepatitis-B antigen (HBAg) was detected by radioimmunoassay in 17% of engorged mosquitoes and in 1 out of 18 pools of engorged bed bugs caught in the Ivory Coast. Antigen was not found in any pools of gravid or unfed mosquitoes or bed bugs or in any pools of phlebotomine sand flies, black flies, or tsetse flies tested. 6·5% of Ivoiriens living in villages where insect collections were made had HBAg detectable by radioimmunoassay. Detection of antigen in engorged insects but not in gravid or unfed insects suggests that replication of the virus does not occur in these arthropods but that mechanical transmission to the human host may occur. Although it is questionable whether control of blood-sucking insects would significantly affect the high rate of exposure to hepatitis-B infections among indigenous populations, blood-sucking insects may serve as important vectors of infection among visitors to tropical regions.

Routine screening by immunoelectroosmophoresis has revealed that 1.5 volunteer blood donors per 1,000 from the New York metropolitan area are carriers of the hepatitis B antigen (HB Ag). Almost all (94%) of the donors with HB Ag appear to be chronic carriers. A significant excess of HB Ag carriers was observed among males, among donors aged 20-29 years, and among blacks. Jews were found to be carriers of HB Ag far less frequently than other whites. Only 3% of HB Ag carriers gave a past history of overt viral hepatitis. The prevalence of antibody to HB Ag (HB Ab) was found to be three times as high among paid blood donors in comparison to volunteer donors (20% vs. 6.7%). In both volunteer and paid donors the prevalence of HB Ab was significantly higher among blacks than among nonblacks. No differences in the prevalence of HB Ab were observed between males and females, or between Jews and other whites. The prevalence of HB Ab in contrast to that of HB Ag was found to increase with advancing age.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus of the family Hepadnaviridae. Having been found in various animals (duck, heron, woodchuck, ground squirrel, and primates), hepadnaviruses must have undergone a long history of evolution and may comprise more members than currently recognized. Chimpanzees may also have their own hepadnavirus, even if it might be very close to HBV. We analyzed HBV-like sequences from three chimpanzees ( Pan troglodytes ) that were most likely infected during their life in Africa in the wild. Two chimpanzees (Ch256 and Ch258) possessed a viral genome of 3182 nt in length with a 33-nt deletion in the preS1 region, which could not be classified into any of the six genotypes (A–F) of human HBV but was very homologous to a previously reported isolate from a London Zoo chimpanzee. Phylogenetically distinct from the HBV-like sequences from gibbons, orangutans, and a gorilla so far reported, the Ch256 and Ch258 isolates would represent an indigenous chimpanzee HBV (tentatively ChHBV). A third chimpanzee (Ch195) had a 3212-nt genome, classifiable into the genotype E of HBV. Because HBV-E has been found mostly in Africans, Ch195 may have been infected from a human source in Africa. However, an inverse scenario is also possible: a spread of HBV-E might have occurred from chimpanzees to humans a long time ago in Africa. Analysis of the arginine-rich C-terminal region of the core protein, which is well conserved among mammalian hepadnaviruses, indicated that HBV-E/F and nonhuman primate hepadnaviruses are much closer than HBV-A/B/C/D to the hepadnaviruses of woodchuck and ground squirrel. Our results support an “ex-nonhuman primate” hypothesis for the origin of HBV.

SummaryTo assess the sterilization efficacy of a combined Tween 80®, β-propiolactone and ultraviolet irradiation procedure applied to a FVIII preparation to which an estimated 105.9 chimpanzee infectious doses (CID50) of hepatitis B virus had been added per ml, two chimpanzees were inoculated with 10 ml each of treated and untreated preparations. The untreated preparation, which was obtained from donors with normal alanine aminotransferase (ALT) levels, induced non-A, non-B hepatitis in both recipient animals, and delayed hepatitis B infection in one of these. Neither animal receiving the treated preparation developed either type of hepatitis. When subsequently challenged with the untreated material, both of the latter animals developed non-A, non-B and hepatitis B infection, proving their susceptibility to both types of infection.It was concluded that the combined procedure inactivated an estimated 106.9 CID50 of hepatitis B virus and an unknown quantity of a non-A, non-B virus.The finding of non-A, non-B virus infectivity in a pooled FVIII preparation despite careful ALT screening of plasma donors emphasizes the necessity of subjecting such preparations to sterilization procedures.