Showing papers on "Virus published in 2000"

Dengue Viremia Titer, Antibody Response Pattern, and Virus Serotype Correlate with Disease Severity

Abstract: Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.

A DNA transfection system for generation of influenza A virus from eight plasmids

Abstract: We have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus from cloned cDNA. In this plasmid-based expression system, viral cDNA is inserted between the RNA polymerase I (pol I) promoter and terminator sequences. This entire pol I transcription unit is flanked by an RNA polymerase II (pol II) promoter and a polyadenylation site. The orientation of the two transcription units allows the synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template. This pol I–pol II system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters, presumably in different compartments of the nucleus. The interaction of all molecules derived from the cellular and viral transcription and translation machinery results in the generation of infectious influenza A virus. The utility of this system is proved by the recovery of the two influenza A viruses: A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1). Seventy-two hours after the transfection of eight expression plasmids into cocultured 293T and MDCK cells, the virus yield in the supernatant of the transfected cells was between 2 × 105 and 2 × 107 infectious viruses per milliliter. We also used this eight-plasmid system for the generation of single and quadruple reassortant viruses between A/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1). Because the pol I–pol II system facilitates the design and recovery of both recombinant and reassortant influenza A viruses, it may also be applicable to the recovery of other RNA viruses entirely from cloned cDNA.

Efficient initiation of HCV RNA replication in cell culture.

Abstract: Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-α rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.

Epstein-Barr Virus Infection

Abstract: The Epstein–Barr virus (EBV) was discovered 36 years ago by electron microscopy of cells cultured from Burkitt's lymphoma tissue by Epstein, Achong, and Barr.1 Four years later, in 1968, EBV was shown to be the etiologic agent of heterophile-positive infectious mononucleosis.2 EBV DNA was detected in tissues from patients with nasopharyngeal carcinoma in 1970.3 In the 1980s, EBV was found to be associated with non-Hodgkin's lymphoma and oral hairy leukoplakia in patients with the acquired immunodeficiency syndrome (AIDS).4,5 Since then, EBV DNA has been found in tissues from other cancers, including T-cell lymphomas and Hodgkin's disease.6,7 EBV is . . .

Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies.

Abstract: The development of the human immunodeficiency virus-1 (HIV-1)/simian immunodeficiency virus (SIV) chimeric virus macaque model (SHIV) permits the in vivo evaluation of anti-HIV-1 envelope glycoprotein immune responses. Using this model, others, and we have shown that passively infused antibody can protect against an intravenous challenge. However, HIV-1 is most often transmitted across mucosal surfaces and the intravenous challenge model may not accurately predict the role of antibody in protection against mucosal exposure. After controlling the macaque estrous cycle with progesterone, anti-HIV-1 neutralizing monoclonal antibodies 2F5 and 2G12, and HIV immune globulin were tested. Whereas all five control monkeys displayed high plasma viremia and rapid CD4 cell decline, 14 antibody-treated macaques were either completely protected against infection or against pathogenic manifestations of SHIV-infection. Infusion of all three antibodies together provided the greatest amount of protection, but a single monoclonal antibody, with modest virus neutralizing activity, was also protective. Compared with our previous intravenous challenge study with the same virus and antibodies, the data indicated that greater protection was achieved after vaginal challenge. This study demonstrates that antibodies can affect transmission and subsequent disease course after vaginal SHIV-challenge; the data begin to define the type of antibody response that could play a role in protection against mucosal transmission of HIV-1.

Nipah Virus: A Recently Emergent Deadly Paramyxovirus

Abstract: A paramyxovirus virus termed Nipah virus has been identified as the etiologic agent of an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore. The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 encephalitis cases, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. Electron microscopic, serologic, and genetic studies indicate that this virus belongs to the family Paramyxoviridae and is most closely related to the recently discovered Hendra virus. We suggest that these two viruses are representative of a new genus within the family Paramyxoviridae. Like Hendra virus, Nipah virus is unusual among the paramyxoviruses in its ability to infect and cause potentially fatal disease in a number of host species, including humans.

Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures.

Abstract: IP: 54.70.40.11 On: Sat, 15 Dec 2018 21:37:32 Journal of General Virology (2000), 81, 2341–2364. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors.

Abstract: A unique cohort of HIV-1-infected long term nonprogressors (LTNP) with normal CD4(+) T cell counts and <50 copies/ml of plasma were prospectively recruited for study. HLA typing revealed a dramatic association between the HLA B*5701 class I allele and nonprogressive infection [85% (11 of 13) vs. 9.5% (19 of 200) in progressors; P < 0. 001]. Antigen-specific CD8(+) T cells were enumerated by flow cytometric detection of intracellular IFN-gamma in response to HIV antigens and HLA B*57-gag tetramer staining. No quantitative differences in the total HIV-specific CD8(+) T cell responses were observed between B*57(+) LTNP and five B*57(+) progressors (P = 0.4). Although similar frequencies of peptide specific CD8(+) T cells were also found, the gag-specific CD8(+) T cell response in the LTNP group was highly focused on peptides previously shown to be B*57-restricted. These findings indicate that, within this phenotypically and genotypically distinct cohort, a host immune factor is highly associated with restriction of virus replication and nonprogressive disease. They also strongly suggest a mechanism of virus specific immunity that directly operates through the B*5701 molecule. Further characterization of qualitative differences in the virus-specific responses that distinguish HLA B*57(+) LTNP from progressors may ultimately define mechanisms of effective immune mediated restriction of virus replication.

Replication of the hepatitis C virus

Abstract: Infection with the hepatitis C virus (HCV) is a major cause of chronic liver disease. HCV is an enveloped plus-strand RNA virus closely related to flavi- and pestiviruses. The first cloning of the HCV genome, about 10 years ago, initiated research efforts leading to the elucidation of the genomic organization and the definition of the functions of most viral proteins. Despite this progress the lack of convenient animal models and appropriate in vitro propagation systems have hampered a full understanding of the way the virus multiplies. This review summarizes our current knowledge about HCV replication and describes attempts pursued in the last few years to establish efficient and reliable cell culture systems.

SLAM (CDw150) is a cellular receptor for measles virus.

Abstract: Measles virus continues to be a major killer of children, claiming roughly one million lives a year Measles virus infection causes profound immunosuppression, which makes measles patients susceptible to secondary infections accounting for high morbidity and mortality The Edmonston strain of measles virus, and vaccine strains derived from it, use as a cellular receptor human CD46 (refs 3, 4), which is expressed on all nucleated cells; however, most clinical isolates of measles virus cannot use CD46 as a receptor Here we show that human SLAM (signalling lymphocyte-activation molecule; also known as CDw150), a recently discovered membrane glycoprotein expressed on some T and B cells, is a cellular receptor for measles virus, including the Edmonston strain Transfection with a human SLAM complementary DNA enables non-susceptible cell lines to bind measles virus, support measles virus replication and develop cytopathic effects The distribution of SLAM on various cell lines is consistent with their susceptibility to clinical isolates of measles virus The identification of SLAM as a receptor for measles virus opens the way to a better understanding of the pathogenesis of measles virus infection, especially the immunosuppression induced by measles virus

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.

Abstract: Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.

The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen.

Abstract: The ribonucleoside analog ribavirin (1-beta-D-ribofuranosyl-1,2, 4-triazole-3-carboxamide) shows antiviral activity against a variety of RNA viruses and is used in combination with interferon-alpha to treat hepatitis C virus infection. Here we show in vitro use of ribavirin triphosphate by a model viral RNA polymerase, poliovirus 3Dpol. Ribavirin incorporation is mutagenic, as it templates incorporation of cytidine and uridine with equal efficiency. Ribavirin reduces infectious poliovirus production to as little as 0. 00001% in cell culture. The antiviral activity of ribavirin correlates directly with its mutagenic activity. These data indicate that ribavirin forces the virus into 'error catastrophe'. Thus, mutagenic ribonucleosides may represent an important class of anti-RNA virus agents.

The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.

Abstract: Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.

Neurologic complications of the reactivation of varicella-zoster virus.

Abstract: Varicella–zoster virus is an exclusively human herpesvirus that causes chickenpox (varicella), becomes latent in cranial-nerve and dorsal-root ganglia, and frequently reactivates decades later to produce shingles (zoster) and postherpetic neuralgia. In immunocompetent elderly persons or immunocompromised patients, varicella–zoster virus may produce disease of the central nervous system. Since the last major review of varicella–zoster virus in the Journal, 1,2 advances in molecular biology have provided important new insights into the pathogenesis of infection with varicella–zoster virus. The detection of varicella–zoster virus in blood vessels and other tissues by methods based on the polymerase chain reaction (PCR) has widened the recognized . . .

Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120

Abstract: T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC50) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log10 higher than the mean IC50 for CXCR4 (X4) isolates (P = 0.0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC50 for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log10 higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC50 for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log10 lower than the IC50 for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia.

Abstract: Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development

Human Immunodeficiency Virus Type 1 Spinoculation Enhances Infection through Virus Binding

Abstract: The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1(IIIB) virions at 1,200 x g for 2 h at 25 degrees C increases the number of particles that bind to CEM-SS T-cell targets by approximately 40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37 degrees C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, approximately 100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24(gag). Because the modest g forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.

A Novel Phenotypic Drug Susceptibility Assay for Human Immunodeficiency Virus Type 1

Abstract: Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Development of a preventive vaccine for Ebola virus infection in primates

Abstract: Outbreaks of haemorrhagic fever caused by the Ebola virus are associated with high mortality rates that are a distinguishing feature of this human pathogen. The highest lethality is associated with the Zaire subtype, one of four strains identified to date1,2. Its rapid progression allows little opportunity to develop natural immunity, and there is currently no effective anti-viral therapy. Therefore, vaccination offers a promising intervention to prevent infection and limit spread. Here we describe a highly effective vaccine strategy for Ebola virus infection in non-human primates. A combination of DNA immunization and boosting with adenoviral vectors that encode viral proteins generated cellular and humoral immunity in cynomolgus macaques. Challenge with a lethal dose of the highly pathogenic, wild-type, 1976 Mayinga strain of Ebola Zaire virus resulted in uniform infection in controls, who progressed to a moribund state and death in less than one week. In contrast, all vaccinated animals were asymptomatic for more than six months, with no detectable virus after the initial challenge. These findings demonstrate that it is possible to develop a preventive vaccine against Ebola virus infection in primates.

Hepatitis C Virus-Encoded Enzymatic Activities and Conserved RNA Elements in the 3′ Nontranslated Region Are Essential for Virus Replication In Vivo

Abstract: Hepatitis C virus (HCV) infection is a widespread major human health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A. A. Kolykhalov et al., Science 277:570-574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at the 3' terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3' terminal RNA element are essential for productive replication in vivo.

Avian-to-human transmission of H9N2 subtype influenza A viruses: Relationship between H9N2 and H5N1 human isolates

Abstract: In 1997, 18 cases of influenza in Hong Kong (bird flu) caused by a novel H5N1 (chicken) virus resulted in the deaths of six individuals and once again raised the specter of a potentially devastating influenza pandemic. Slaughter of the poultry in the live bird markets removed the source of infection and no further human cases of H5N1 infection have occurred. In March 1999, however, a new pandemic threat appeared when influenza A H9N2 viruses infected two children in Hong Kong. These two virus isolates are similar to an H9N2 virus isolated from a quail in Hong Kong in late 1997. Although differing in their surface hemagglutinin and neuraminidase components, a notable feature of these H9N2 viruses is that the six genes encoding the internal components of the virus are similar to those of the 1997 H5N1 human and avian isolates. This common feature emphasizes the apparent propensity of avian viruses with this genetic complement to infect humans and highlights the potential for the emergence of a novel human pathogen.

Activation of Interferon Regulatory Factor 3 Is Inhibited by the Influenza A Virus NS1 Protein

Abstract: We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-alpha/beta) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-alpha/beta gene expression. IRF-3 activation and, as a consequence, IFN-beta mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.

Influenza A Virus NS1 Protein Prevents Activation of NF-κB and Induction of Alpha/Beta Interferon

Abstract: The alpha/beta interferon (IFN-α/β) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-α/β-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-α/β genes, including IFN-β. It is known that transactivation of the IFN-β promoter depends on NF-κB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-κB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-κB pathway during delNS1 virus infection prevented the transactivation of the IFN-β promoter, demonstrating a functional link between NF-κB activation and IFN-α/β synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-κB pathway and of IFN-β synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-κB pathway may thus play a key role in the pathogenesis of influenza A virus.

Isolation of Hendra Virus From Pteropid Bats: A Natural Reservoir of Hendra Virus

Abstract: Since it was first described in Australia in 1994, Hendra virus (HeV) has caused two outbreaks of fatal disease in horses and humans, and an isolated fatal horse case. Our preliminary studies revealed a high prevalence of neutralizing antibodies to HeV in bats of the genus Pteropus, but it was unclear whether this was due to infection with HeV or a related virus. We developed the hypothesis that HeV excretion from bats might be related to the birthing process and we targeted the reproductive tract for virus isolation. Three virus isolates were obtained from the uterine fluid and a pool of foetal lung and liver from one grey-headed flying-fox (Pteropus poliocephalus), and from the foetal lung of one black flying-fox (P. alecto). Antigenically, these isolates appeared to be closely related to HeV, returning positive results on immunofluorescent antibody staining and constant-serum varying-virus neutralization tests. Using an HeV-specific oligonucleotide primer pair, genomic sequences of the isolates were amplified. Sequencing of 200 nucleotides in the matrix gene identified that these three isolates were identical to HeV. Isolations were confirmed after RNA extracted from original material was positive for HeV RNA when screened on an HeV Taqman assay. The isolation of HeV from pteropid bats corroborates our earlier serological and epidemiological evidence that they are a natural reservoir host of the virus.

B-1 and B-2 Cell–Derived Immunoglobulin M Antibodies Are Nonredundant Components of the Protective Response to Influenza Virus Infection

Abstract: We have studied the role of secreted immunoglobulin (Ig)M in protection from infection with influenza virus and delineated the relative contributions of B-1 versus B-2 cell–derived IgM in this process. Mice deficient in secreted IgM but capable of expressing surface IgM and secreting other Ig classes show significantly reduced virus clearance and survival rates compared with wild-type controls. Irradiation chimeras in which only either B-1 or B-2 cells lack the ability to secrete IgM show mortality rates similar to those of mice in which neither B-1 nor B-2 cells secrete IgM. Dependence on both sources of IgM for survival is partially explained by findings in allotype chimeras that broadly cross-reactive B-1 cell–derived natural IgM is present before infection, whereas virus strain–specific, B-2 cell–derived IgM appears only after infection. Furthermore, lack of IgM secreted from one or both sources significantly impairs the antiviral IgG response. Reconstitution of chimeras lacking B-1 cell–derived IgM only with IgM-containing serum from noninfected mice improved both survival rates and serum levels of virus-specific IgG. Thus, virus-induced IgM must be secreted in the presence of natural IgM for efficient induction of specific IgG and for immune protection, identifying B-1 and B-2 cell–derived IgM antibodies as nonredundant components of the antiviral response.

Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy

Abstract: The success of combination antiretroviral therapy for HIV-1 infection has generated interest in mechanisms by which the virus can persist in the body despite the presence of drugs that effectively inhibit key steps in the virus life cycle. It is becoming clear that viral reservoirs established early in the infection not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Mechanisms of viral persistence are best considered in the context of the dynamics of viral replication in vivo. Virus production in infected individuals is largely the result of a dynamic process involving continuous rounds of de novo infection of and replication in activated CD4(+) T cells with rapid turnover of both free virus and virus-producing cells. This process is largely, but not completely, interrupted by effective antiretroviral therapy. After a few months of therapy, plasma virus levels become undetectable in many patients. Analysis of viral decay rates initially suggested that eradication of the infection might be possible. However, there are several potential cellular and anatomical reservoirs for HIV-1 that may contribute to long-term persistence of HIV-1. These include infected cell in the central nervous system and the male urogenital tract. However, the most worrisome reservoir consists of latently infected resting memory CD4(+) T cells carrying integrated HIV-1 DNA. Definitive demonstration of the presence of this form of latency required development of methods for isolating extremely pure populations of resting CD4(+) T cells and for demonstrating that a small fraction of these cells contain integrated HIV-1 DNA that is competent for replication if the cells undergo antigen-driven activation. Most of the latent virus in resting CD4(+) T cells is found in cells of the memory phenotype. The half-life of this latent reservoir is extremely long (44 months). At this rate, eradication of this reservoir would require over 60 years of treatment. Thus, latently infected resting CD4(+) T cells provide a mechanism for life-long persistence of replication-competent forms of HIV-1, rendering unrealistic hopes of virus eradication with current antiretroviral regimens. The extraordinary stability of the reservoir may reflect gradual reseeding by a very low level of ongoing viral replication and/or mechanisms that contribute to the intrinsic stability of the memory T cell compartment. Given the substantial long-term toxicities of current combination therapy regimens, novel approaches to eradicating this latent reservoir are urgently needed.

Sialic Acid Species as a Determinant of the Host Range of Influenza A Viruses

Abstract: The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the α2,6(SAα2,6Gal) linkage, while equine viruses favor SAα2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcα2,6Gal but not NeuAcα2,3Gal or NeuGcα2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcα2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcα2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.