Avian influenza h6 viruses productively infect and cause illness in mice and ferrets

Influenza pandemic preparedness has focused on influenza virus H5 and H7 subtypes. However, it is not possible to predict with certainty which subtype of avian influenza virus will cause the next pandemic, and it is prudent to include other avian influenza virus subtypes in pandemic preparedness efforts. An H6 influenza virus was identified as a potential progenitor of the H5N1 viruses that emerged in Hong Kong in 1997. This virus continues to circulate in the bird population in Asia, and other H6 viruses are prevalent in birds in North America and Asia. The high rate of reassortment observed in influenza viruses and the prevalence of H6 viruses in birds suggest that this subtype may pose a pandemic risk. Very little is known about the replicative capacity, immunogenicity, and correlates of protective immunity for low-pathogenicity H6 influenza viruses in mammals. We evaluated the antigenic and genetic relatedness of 14 H6 influenza viruses and their abilities to replicate and induce a cross-reactive immune response in two animal models: mice and ferrets. The different H6 viruses replicated to different levels in the respiratory tracts of mice and ferrets, causing varied degrees of morbidity and mortality in these two models. H6 virus infection induced similar patterns of neutralizing antibody responses in mice and ferrets; however, species-specific differences in the cross-reactivity of the antibody responses were observed. Overall, cross-reactivity of neutralizing antibodies in H6 virus-infected mice did not correlate well with protection against heterologous wild-type H6 viruses. However, we have identified an H6 virus that induces protective immunity against viruses in the North American and Eurasian lineages.     

Protective Efficacy of Passive Immunization with Monoclonal Antibodies in Animal Models of H5N1 Highly Pathogenic Avian Influenza Virus Infection

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.     

Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on the ability of HIV to completely evade neutralizing antibody responses

Acute human immunodeficiency virus (HIV) infection is associated with the rapid development of neutralization escape mutations. The degree to which viral evolution persists in chronic infection has not been well characterized, nor is it clear if all patients develop high-level neutralization antibody escape. We therefore measured neutralizing antibody responses against autologous and heterologous viruses in a cohort of acutely and chronically infected subjects (n = 65). Neutralizing antibody responses against both autologous virus and heterologous viruses were lower among individuals with acute infection than among those with chronic infection. Among chronically infected individuals, there was a negative correlation between the level of neutralizing antibodies against autologous virus and the level of viremia. In contrast, there was a positive correlation between the level of neutralizing antibodies against a panel of heterologous viruses and the level of viremia. Viral evolution, as defined by the presence of higher neutralizing titers directed against earlier viruses than against contemporaneous viruses, was evident for subjects with recent infection but absent for those with chronic infection. In summary, neutralizing antibody responses against contemporaneous autologous viruses are absent in early HIV infection but can be detected at low levels in chronic infection, particularly among those controlling HIV in the absence of therapy. HIV replication either directly or indirectly drives the production of increasing levels of antibodies that cross-neutralize heterologous primary isolates. Collectively, these observations indicate that although HIV continuously drives the production of neutralizing antibodies, there may be limits to the capacity of the virus to evolve continuously in response to these antibodies. These observations also suggest that the neutralizing antibody response may contribute to the long-term control of HIV in some patients while protecting against HIV superinfection in most patients.     

Antibody-mediated protection against cytotoxic T-cell escape in coronavirus-induced demyelination

C57BL/6 (B6) mice infected with mouse hepatitis virus (MHV) strain JHM develop a clinically evident, demyelinating encephalomyelitis. Infectious virus can be isolated from the spinal cords of these mice and is invariably mutated in the immunodominant CD8 T-cell epitope recognized in this strain. We showed previously that these persistently infected mice did not mount a measurable serum anti-MHV neutralizing antibody response. Here we show that cytotoxic T-lymphocyte (CTL) escape was not detected in MHV-infected BALB/b mice (H-2(b) haplotype), even though the same CD8 T-cell epitopes were recognized as in B6 mice. BALB/b mice had 25-fold more MHV-specific antibody-secreting cells in the central nervous system, the site of infection, than B6 mice, suggesting that local production of anti-MHV antibody contributed to this absence of CTL escape. Additionally, administration of anti-MHV neutralizing antibody to infected B6 mice suppressed the development of CTL escape mutants. These findings indicate a key role for the anti-MHV antibody response in suppressing virus replication, thereby minimizing the emergence and competitive advantage of CTL escape mutants.     

Perforin and Fas cytolytic pathways coordinately shape the selection and diversity of CD8+-T-cell escape variants of influenza virus

Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+ cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.     

Suppression of virus-specific antibody production by CD8+ class I-restricted antiviral cytotoxic T cells in vivo.

The question of whether virus-induced immunosuppression includes the antibody response against the infecting virus itself was evaluated in a model situation. Transgenic mice expressing the T-cell receptor (TCR) specific for peptide 32-42 of lymphocytic choriomeningitis virus (LCMV) glycoprotein 1 presented by Db reacted with a strong transgenic cytotoxic T-lymphocyte (CTL) response starting on day 3 after infection with a high dose (10(6) PFU intravenously [i.v.]) of the WE strain of LCMV (LCMV-WE); LCMV-specific antibody production in the spleen was suppressed in these mice. Low-dose (10(2) PFU i.v.) infection resulted in an antiviral antibody response comparable to that of the transgene-negative littermates. The induction of suppression of LCMV-specific antibody responses was specifically mediated by CD8+ TCR transgenic CTLs, since the LCMV-8.7 variant virus (which is not recognized by transgenic TCR-expressing CTLs because of a point mutation) did not induce suppression. In addition, treatment with CD8 monoclonal antibody in vivo abrogated suppression. Once suppression had been established, it was found to be nonspecific. The abrogation of antibody responses depended on the relative kinetics of the antibody response involved and the kinetics of the anti-LCMV CTL response. Analysis of T- and B-cell subpopulations showed no significant changes, but immunohistochemical analysis of spleens revealed extensive destruction of follicular organization in lymphoid tissue by day 4 in transgenic mice infected with LCMV-WE but not in those infected with the CTL escape mutant LCMV-8.7. Impairment of antigen presentation rather than of T or B cells was also suggested by adoptive transfer experiments, showing that transferred infected macrophages may improve the anti-LCMV antibody response in LCMV-immunosuppressed transgenic recipients; also, T and B cells from suppressed transgenic mice did respond in irradiated and virus-infected nontransgenic mice with antibody formation to LCMV. Such virus-triggered, T-cell-mediated immunopathology causing the suppression of B cells and of protective antibody responses, including those against the infecting virus itself, may permit certain viruses to establish persistent infections.     

Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine.

We have mapped a linear epitope recognized by the virus-neutralizing monoclonal antibody 6-15C4 within the primary sequence of the G protein from the Evelyn-Rokitnicki-Abelseth strain of rabies virus. This was accomplished by using fragments of the rabies virus G protein and deduced amino acid sequences of neutralization-resistant variant rabies viruses. The monoclonal antibody 6-15C4 specifically recognized a synthetic peptide (peptide G5-24) which resembles the 6-15C4 epitope in structure. In addition, a tandem peptide constructed from the G5-24 peptide and a dominant TH cell epitope of the rabies virus N protein induced protective immunity against lethal rabies virus challenge infection in mice.     

Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses.

We developed an in vitro assay for antibodies to hepatitis C virus (HCV) that bind to virions and prevent initiation of the replication cycle in susceptible cells in vitro. These antibodies therefore appear to be capable of neutralizing the virus. Using this assay and a standard inoculum of HCV of known infectivity, we have measured the antibody in serial serum samples obtained from the same chronically infected patient over 14 years following onset of his hepatitis. Such antibody was found in sera collected within 5 years of onset of hepatitis but not in later sera. In double immunoprecipitation experiments with anti-human immunoglobulin, the same sera that contained neutralizing antibody were found to contain antibody that bound to HCV to form antigen-antibody complexes immunoprecipitable with anti-human globulin. Similarly, plasma collected from this patient in 1990, 13 years after onset of hepatitis, and which contained HCV that had diverged genetically from the 1977 strain, did not contain antibody capable of neutralizing either the 1977 or the 1990 strain of HCV. However, plasma collected a year later (1991, 14 years after onset of hepatitis) contained neutralizing antibody to the 1990, but not the 1977, strain of HCV. These results suggest that HCV does induce antivirion antibody, as measured by blocking of initiation of the replication cycle of virus in cells and by the formation of immunoprecipitable antigen-antibody complexes but that these antibodies are isolate specific and change over time. Thus, these antivirion antibodies function as neutralizing antibodies and are probably in vitro correlates of the attempt of the host to contain the emergence of neutralization-resistant variants of HCV over time.     

Small rodent models of hepatitis B and C virus replication and pathogenesis

The narrow host range of infection supporting the long-term propagation of hepatitis B and C viruses is a major limitation that has prevented a more thorough understanding of persistent infection and t...     

Pre-Existing Immunity with High Neutralizing Activity to 2009 Pandemic H1N1 Influenza Virus in Shanghai Population

Pre-existing immunity is an important factor countering the pandemic potential of an emerging influenza virus strain. Thus, studying of pre-existing immunity to the 2009 pandemic H1N1 virus (2009 H1N1) will advance our understanding of the pathogenesis and epidemiology of this emerging pathogen. In the present study, sera were collected from 486 individuals in a hospital in Shanghai, China, before the 2009 H1N1 influenza pandemic. The serum anti-hemagglutinins (HA) antibody, hemagglutination inhibition (HI) antibody and neutralizing antibody against the 2009 H1N1 were assayed. Among this population, 84.2%, 14.61% and 26.5% subjects possessed anti-HA antibody, HI antibody and neutralizing antibody, respectively. Although neutralizing antibody only existed in those sera with detectable anti-HA antibody, there was no obvious correlation between the titers of anti-HA and neutralizing antibody. However, the titers of anti-HA and neutralizing antibody against seasonal H1N1 virus were highly correlated. In the same population, there was no correlation between titers of neutralizing antibody against 2009 H1N1 and seasonal H1N1. DNA immunization performed on mice demonstrated that antibodies to the HA of 2009 pandemic and seasonal H1N1 influenza viruses were strain-specific and had no cross-neutralizing activity. In addition, the predicted conserved epitope in the HA of 2009 H1N1 and recently circulating seasonal H1N1 virus, GLFGAIAGFIE, was not an immunologically valid B-cell epitope. The data in this report are valuable for advancing our understanding of 2009 H1N1 influenza virus infection.     

Stoichiometry of antibody neutralization of human immunodeficiency virus type 1

The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single antibody molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine.     

Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus

Background

The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti et al. and by Gioia et al. demonstrate that heterosubtypic neutralizing antibodies against the highly pathogenic H5N1 virus can be elicited with a seasonal influenza vaccine in humans. However, whether such response offers immune protection against highly pathogenic H5N1 virus remained to be determined.

Methodology/Principal Findings

In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge.

Conclusions/Significance

Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.     

Persistent infection of chimpanzees with human immunodeficiency virus: serological responses and properties of reisolated viruses.

Persistent infection by human immunodeficiency virus (HIV-1) in the chimpanzee may be valuable for immunopathologic and potential vaccine evaluation. Two HIV strains, the tissue culture-derived human T-cell lymphotropic virus type IIIB (HTLV-IIIB) and in vivo serially passaged lymphadenopathy-associated virus type 1 (LAV-1), were injected intravenously into chimpanzees. Two animals received HTLV-IIIB as either virus-infected H9 cells or cell-free virus. A third animal received chimpanzee-passaged LAV-1. Evaluation of their sera for virus-specific serologic changes, including neutralizations, was done during a 2-year period. During this period all animals had persistently high titers of antibodies to viral core and envelope antigens. All three animals developed a progressively increasing type-specific neutralizing LAV-1 versus HTLV-IIIB antibody titer during the 2-year observation period which broadened in specificity to include HTLV-HIRF, HTLV-IIIMN, and HTLV-IIICC after 6 to 12 months. The antibody titers against both viruses were still increasing by 2 years after experimental virus inoculation. Sera from all animals were capable of neutralizing both homologously and heterologously reisolated virus from chimpanzees. A slightly more rapid type-specific neutralizing response was noted for the animal receiving HTLV-IIIB-infected cells compared with that for cell-free HTLV-IIIB. Sera from all persistently infected chimpanzees were capable of mediating group-specific antibody-mediated complement-dependent cytolysis of HIV-infected cells derived from all isolates tested. Viruses reisolated from all three animals at 20 months after inoculation revealed very similar peptide maps of their respective envelope gp120s, as determined by two-dimensional chymotrypsin oligopeptide analysis. One peptide, however, from the original HTLV-IIIB-inoculated virus was deleted in viruses from all three animals, and in addition, we noted the appearance of a new or modified peptide which was common to LAV-1 as well as to HTLV-IIIB reisolated from infected chimpanzees. It thus appears that a group-specific neutralizing antibody response as well as a group-specific cytotoxic response can develop in chimpanzees after an inoculation of a single HIV variant. This finding suggests that a common, less immunodominant determinant(s) is present on a single HIV strain which could induce group-specific antibodies during viral infection and replication. The identification of this group-specific epitope and the induction of analogous immunity may be relevant to vaccine development against human acquired immunodeficiency syndrome.     

Persistent infection with mouse hepatitis virus of low virulence in nude mice.

A persisting type of infection with wasting syndrome was established in congenitally athymic nude mice after intraperitoneal inoculation with a mouse hepatitis virus which was not fully pathogenic for heterozygous haired littermates. From the liver, spleen, lymph nodes, and brain of most infected nude mice, the virus was detected at high titers during aperiod from 6 to 35 days postinfection, occurrence of degenerative and necrotic lesions being correlated with virus titers in these organs. The titer of serum neutralizing antibody remained undetectable or very low in most diseases nude mice, whereas some animals resisting the infection could produce antibody at a later stage. In heterozygous haired mice, some lesions were detectable at a very early stage of infection in the spleen and liver, but they seemed to disappear with a marked elevation of the neutralizing antibody titer. Nude mice were able to resist the virus infection when they had previously received transfer of thymocytes from weanling heterozygous littermates.     

Anti-influenza A virus characteristics of a fucoidan from sporophyll of Undaria pinnatifida in mice with normal and compromised immunity

Undaria pinnatifida, an edible brown alga, contains fucoidan (FuC), a sulfated polysaccharide, that inhibited the in vitro replication of influenza A virus, and stimulated both innate and adaptive immune defense functions in virus-infected mice. In the present study, the effects of oral administration of FuC were evaluated on influenza virus infection in immunocompetent and immunocompromised mice, where the efficacy of FuC was demonstrated in reducing viral replication, decreasing weight loss and mortality, and prolonging survival. Oral FuC resulted in increased neutralizing antibody production in the mucosa and blood. In contrast, while suppressing virus yields in mice more markedly than FuC, oseltamivir significantly reduced the neutralizing antibody titers in both the mucosa and blood. In immunocompromised mice, drug-resistant viruses frequently recovered after oseltamivir treatment; no resistant viruses were isolated from FuC-treated mice. FuC could be a candidate for the development of new therapeutic options including its combination with neuraminidase inhibitors such as oseltamivir.     

Generation,Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

Background

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.

Methodology/Principal Findings

We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.

Conclusions/Significance

Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.     

Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys

Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.     

Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.     

Chimeric Mouse Model for the Infection of Hepatitis B and C Viruses

While the chimpanzee remains the only animal that closely models human hepatitis C virus (HCV) infection, transgenic and immunodeficient mice in which human liver can be engrafted serve as a partial solution to the need for a small animal model for HCV infection. The established system that was based on mice carrying a transgene for urokinase-type plasminogen activator (uPA) gene under the control of the human albumin promoter has proved to be useful for studies of virus infectivity and for testing antiviral drug agents. However, the current Alb-uPA transgenic model with a humanized liver has practical limitations due to the inability to maintain non-engrafted mice as dizygotes for the transgene, poor engraftment of hemizygotes, high neonatal and experimental death rates of dizygous mice and a very short time window for hepatocyte engraftment. To improve the model, we crossed transgenic mice carrying the uPA gene driven by the major urinary protein promoter onto a SCID/Beige background (MUP-uPA SCID/Bg). These transgenic mice are healthy relative to Alb-uPA mice and provide a long window from about age 4 to 12 months for engraftment with human hepatocytes and infection with hepatitis C or hepatitis B (HBV) viruses. We have demonstrated engraftment of human hepatocytes by immunohistochemistry staining for human albumin (30-80% engraftment) and observed a correlation between the number of human hepatocytes inoculated and the level of the concentration of human albumin in the serum. We have shown that these mice support the replication of both HBV and all six major HCV genotypes. Using HBV and HCV inocula that had been previously tittered in chimpanzees, we showed that the mice had approximately the same sensitivity for infection as chimpanzees. These mice should be useful for isolating non-cell culture adapted viruses as well as testing of antiviral drugs, antibody neutralization studies and examination of phenotypic changes in viral mutants.