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In the context of an A/H1N1 influenza pandemic situation, this study demonstrates that heterologous vaccination with an AS03-adjuvanted 2008/2009 seasonal trivalent and pandemic H5N1 monovalent split vaccine conferred partial protection in influenza-naive ferrets after challenge with the influenza pandemic H1N1 A/The Netherlands/602/09 virus. Further, unlike saline control and non-adjuvanted vaccine, it was shown that immunization of naive ferrets with an AS03-adjuvanted pandemic H1N1 A/California/7/09 influenza split vaccine induced increased antibody response and enhanced protection against the challenge strain, including significant reduction in viral shedding in the upper respiratory tract and reduced lung pathology post-challenge. These results show the need for vaccination with the adjuvanted vaccine to fully protect against viral replication and influenza disease in unprimed ferrets. (C) 2011 Elsevier Ltd. All rights reserved.

We investigated the development of pulmonary lesions in ferrets by means of computed tomography (CT) following infection with the 2009 pandemic A/H1N1 influenza virus and compared the scans with gross pathology, histopathology and immunohistochemistry. Ground-glass opacities observed by CT scanning in all infected lungs corresponded to areas of alveolar oedema at necropsy. These areas were most pronounced on day 3 and gradually decreased from days 4 to 7 post-infection. This pilot study shows that the non-invasive imaging procedure allows quantification and characterization of influenza-induced pulmonary lesions in living animals under biosafety level 3 conditions and can thus be used in pre-clinical pharmaceutical efficacy studies.

Several felid species have been shown to be susceptible to infection with highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype. Infection of felids by H5N1 HPAI virus is often fatal, and cat-to-cat transmission has been documented. Domestic cats may then be involved in the transmission of infection to other animals but also to humans. A particular concern is the hypothetical role of the cat in the adaptation of the virus to mammalian species, thus increasing the pandemic risk. Therefore, the development of a HPAI vaccine for domestic cats should be considered a veterinary and also a public health priority. Here we show that vaccination of cats with a recombinant canarypox (ALVAC)(1)) virus, expressing the hemagglutinin (HA) of influenza virus A/chicken/Indonesia/03 (H5N1) confers protection against challenge infection with two antigenically distinct H5N1 virus isolates from humans. Despite low hemagglutination inhibiting (HI) antibody titers at the time of challenge, all vaccinated cats were protected against mortality and had reduced histopathological changes in the lungs. Importantly, viral shedding was reduced in vaccinated cats as compared to controls, suggesting that vaccination of cats could reduce the risk of viral transmission. In conclusion this study showed that the recombinant canarypox virus protected cats against homologous and heterologous H5N1 HPAI virus challenges.

Background. The newly emerged influenza A(H1N1) virus (new H1N1 virus) is causing the first influenza pandemic of this century. Three influenza pandemics of the previous century caused variable mortality, which largely depended on the development of severe pneumonia. However, the ability of the new H1N1 virus to cause pneumonia is poorly understood. Methods. The new H1N1 virus was inoculated intratracheally into ferrets. Its ability to cause pneumonia was compared with that of seasonal influenza H1N1 virus and highly pathogenic avian influenza (HPAI) H5N1 virus by using clinical, virological, and pathological analyses. Results. Our results showed that the new H1N1 virus causes pneumonia in ferrets intermediate in severity between that caused by seasonal H1N1 virus and by HPAI H5N1 virus. The new H1N1 virus replicated well throughout the lower respiratory tract and more extensively than did both seasonal H1N1 virus (which replicated mainly in the bronchi) and HPAI H5N1 virus (which replicated mainly in the alveoli). High loads of new H1N1 virus in lung tissue were associated with diffuse alveolar damage and mortality. Conclusions. The new H1N1 virus may be intrinsically more pathogenic for humans than is seasonal H1N1 virus.

The etiology of acute respiratory tract illnesses is sometimes unclear due to limitations of diagnostic tests or the existence of as-yet-unidentified pathogens. Here we describe the identification and characterization of a not previously recognized coronavirus obtained from an 8-mo-old boy suffering from pneumonia. This coronavirus replicated efficiently in tertiary monkey kidney cells and Vero cells, in contrast to human coronaviruses (HCoV) 229E and OC43. The entire cDNA genome sequence of the previously undescribed coronavirus was determined, revealing that it is most closely related to porcine epidemic diarrhea virus and HCoV 229E. The maximum amino acid sequence identity between ORFs of the newly discovered coronavirus and related group 1 coronaviruses ranged from 43% to 67%. Real-time RT-PCR assays were designed to test for the prevalence of the previously undescribed coronavirus in humans. Using these tests, the virus was detected in four of 139 individuals (3%) who were suffering from respiratory illness with unknown etiology. All four patients suffered from fever, runny nose, and dry cough, and all four had underlying or additional morbidity. Our data will enable the development of diagnostic tests to study the prevalence and clinical impact of this virus in humans in more detail. Moreover, it will be important to discriminate this previously undescribed coronavirus from HCoV 229E and OC43 and the severe acute respiratory syndrome coronavirus.

Sequence-independent amplification and specific reverse transcription PCRs identified genogroup I and II picobirnaviruses in respiratory tracts of pigs. These data expand knowledge of picobirnavirus diversity and tropism. Genetic relationships between porcine respiratory and human enteric picobirnaviruses suggest cross-species transmission of picobirnaviruses between pigs and humans., link_to_OA_fulltext

Animal cells have developed many ways to suppress viral replication, and viruses have evolved diverse strategies to resist these. Here we provide evidence that the virion infectivity factor protein of human immunodeficiency virus type 1 (HIV-1) functions to counteract a newly discovered activity in human cells that otherwise inhibits virus replication. This anti-viral phenotype is shown by human T cells, the principal in vivo targets for HIV-1, and, based on our present understanding of virion infectivity factor action, is presumed to act by interfering with a late step(s) in the virus life cycle. These observations indicate that the inhibition of virion infectivity factor function in vivo may prevent HIV-1 replication by 'unmasking' an innate anti-viral phenotype.

The viral infectivity factor (Vif) of human immunodeficiency virus type-1 (HIV-1) functions at a late stage of the viral life cycle to confer infectivity on progeny virions. Although Vif is present in HIV-1 particles, both the relevance of incorporation for function and the mechanism that underlies incorporation remain unresolved. Using matched T cell systems that express high or low levels of Vif, we demonstrate that the extent of Vif incorporation into virions varies in relation to cellular expression levels. Because viral infectivity is not affected by these variations, we suggest that the packaging of Vif is neither specific nor necessary for function.

The primate immunodeficiency virus Vif proteins are essential for replication in appropriate cultured cell systems and, presumably, for the establishment of productive infections in vivo. We describe experiments that define patterns of complementation between human and simian immunodeficiency virus (HIV and SIV) Vif proteins and address the determinants that underlie functional specificity. Using human cells as virus producers, it was found that the HIV-1 Vif protein could modulate the infectivity of HIV-1 itself, HIV-2 and SIV isolated from African green monkeys (SIVAGM). In contrast, the Vif proteins of SIVAGM and SIV isolated from Sykes' monkeys (SIVSYK) were inactive for all HIV and SIV substrates in human cells even though, at least for the SIVAGM protein, robust activity could be demonstrated in cognate African green monkey cells. These observations suggest that species-specific interactions between Vif and virus-producing cells, as opposed to between Vif and virus components, may govern the functional consequences of Vif expression in terms of inducing virion infectivity. The finding that the replication of murine leukemia virus could also be stimulated by HIV-1 Vif expression in human cells further supported this notion. We speculate that species restrictions to Vif function may have contributed to primate immunodeficiency virus zoonosis.

CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus.

The vif gene of human immunodeficiency virus type 1 is absolutely required for productive infection of primary cells derived from human blood and certain immortalized T lymphoid cells, for example, H9. Cells with this restrictive phenotype are termed nonpermissive, whereas cell lines in which vif-deficient virus can replicate efficiently are known as permissive. In this paper, we describe experiments in which virus stocks derived from single-cycle infections of strictly nonpermissive H9 cells were used to determine the fate of vif-deficient infections. By PCR-based approaches, it was found that Vif has no significant impact on the biosynthetic capability of the virion reverse transcriptase in infected C8166 T cells. Specifically, the initial appearance of all DNA species up to and including initiated second (plus) strands as well as the early accumulation of these replicative intermediates is equivalent for wild-type and vif-deficient infections. However, whereas these viral DNAs are stably maintained in wild-type infections and can proceed to establish proviruses, they are largely degraded by the later time points of vif-deficient infections and, as a result, are prevented from forming proviruses. Subcellular fractionation analyses indicated that the majority of viral DNA is localized to the nucleus within 2 h of infection and that the turnover of reverse transcripts that occurs in these vif-deficient infections presumably takes place in the nucleus. Given that the ultimate infection phenotype of the virions is determined during virus production, we propose that Vif is required for an aspect of virus assembly and/or maturation that endows penetrating viral nucleoprotein cores with the ability to mature into functional preintegration complexes that can proceed to provirus establishment. In contrast, viruses that are produced in the absence of Vif give rise to nucleoprotein complexes that disassemble prematurely in challenged cells and fail to protect their RNA/DNA contents from nucleolytic destruction.

The primary complication of seasonal influenza in humans is viral pneumonia. A conventional animal model—intranasal inoculation of ferrets with 106 median tissue culture infectious dose of virus—results in disease that is neither consistent nor comparable with severe viral pneumonia in humans. Therefore, the authors modified the experimental procedures by increasing the median tissue culture infectious dose to 109 and by inoculating via the intratracheal route, testing these procedures with H1N1 strains (A/Bilthoven/3075/1978 and A/Netherlands/26/2007) and H3N2 strains (A/Bilthoven/16190/1968 and A/Netherlands/177/2008) of seasonal influenza virus. The ferrets of all groups ( n = 3 per virus strain) had clinical signs, increased body temperature, virus excretion from day 1, loss of body weight, and increased relative lung weight at 4 days postinoculation. All ferrets had severe pulmonary consolidation, and histologic examination revealed moderate to severe necrotizing bronchointerstitial pneumonia with severe edema, necrosis of alveolar epithelium, inflammatory infiltrates in alveolar septa and lumina, epithelial regeneration, and perivascular and peribronchiolar inflammatory infiltrates. The lesions were associated with the presence of influenza virus antigen in respiratory epithelium by immunohistochemistry. Although all 4 virus strains caused pulmonary lesions of comparable severity, virus isolation in the lungs, trachea, nasal concha, and tonsils showed higher mean virus titers in the H1/07 and H3/68 groups than in the H1/78 and H3/08 groups. In conclusion, the above H1N1 and H3N2 strains cause severe pneumonia in ferrets by use of the modified experimental procedures and provide a good model for pneumonia caused by seasonal influenza A virus infection in humans.

The use of chimeras of rat and human CD4 to probe the HIV-1 gpl20 and antibody binding properties of CD4 is reviewed. Short segments of human CD4 sequence were substituted for the equivalent regions of rat CD4 which does not bind gpl20, and analysis of the properties of these chimeras established: (i) that residues 33-58 of the NH2-terminal domain of human CD4 encompass the high-affinity gpl20 binding site; and (ii) that chimeras containing residues 33-62 mediate HIV-1 infection. The chimerabinding specificities of gpl20 and a large panel of anti-CD4 antibodies were also determined. This allowed a critical test of the popular notion that receptor mimics appear at high frequency among antibodies elicited by immunization with receptor ligands and that anti-idiotypic antibodies can be used to identify novel receptors. The data suggest that such mimics appear infrequently, if at all, a result which is consistent with the failure of the anti-idiotype approach to identify new genes encoding receptors with prescribed functions.

Mass vaccination against the 2009 A/H1N1 influenza virus, with different vaccine formulations, is being implemented globally. Because of the urgency with which the vaccine has been prepared, little information has been gathered on variables that influence the effectiveness of the vaccine. Specifically, it is not clear whether priming by previous infection with or vaccination against seasonal influenza affects the response to the vaccine. Similarly, the role of vaccine adjuvants in vaccine response is not known. To address these questions, we injected ferrets with seasonal influenza vaccine and then vaccinated with 2009 A/H1N1 vaccine with or without the oil-in-water adjuvant MF59. The results show that the seasonal flu vaccine, although it did not induce functional antibodies against the 2009 A/H1N1 virus, provided immunological priming and allowed production of protective antibodies to 2009 A/H1N1 after one dose of 2009 A/H1N1 vaccine. The vaccine given without adjuvant significantly reduced viral load in the lungs but did not protect from infection. Only the vaccine with adjuvant completely prevented both pulmonary and nasal infection.

We investigated the prophylactic and therapeutic efficacy of an intravenous (IV) formulation of zanamivir in a macaque infection model for highly pathogenic influenza A (H5N1) virus. Antiviral efficacy was dose-dependent, with no reduction in viral load observed at 2 mg/kg, but a significant reduction observed at 10 mg/kg (p = 0.039) and at 20 mg/kg in the combined prophylactic and therapeutic groups (p = 0.049) with both prophylaxis (commencing 12 h before infection) and therapy (commencing 4 h after infection) showing similar reductions in viral load. Combined gross pathology and microscopic pneumonia scores in the treated animals relative to untreated controls were significantly reduced at 10 mg/kg (p = 0.02) and at 20 mg/kg in the prophylaxis group (p, = 0.02), but were not significant in the treatment group (p = 0.145). in this new animal model for evaluation of influenza antivirals, despite variability observed between individual animals, IV zanamivir showed evidence of efficacy against highly pathogenic H5N1 virus. (C) 2008 Elsevier B.V. All rights reserved.

We investigated the ability of adjuvanted, inactivated split-virion influenza A virus (H5N1) vaccines to protect against infection and demonstrated that the disease exacerbation phenomenon seen with adjuvanted formaldehyde-inactivated respiratory syncytial virus and measles virus investigational vaccines did not occur with these H5N1 vaccines. Macaques were vaccinated twice with or without an aluminum hydroxide or oil-in-water emulsion adjuvanted vaccine. Three months later, animals were challenged with homologous wild-type H5N1. No signs of vaccine-induced disease exacerbation were seen. With either adjuvant, vaccination induced functional and cross-reactive antibodies and protected the lungs and upper respiratory tract. Without an adjuvant, the vaccine provided partial protection. Best results were obtained with the emulsion adjuvant.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259–5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55 Gag polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.

Lentivirus Vif proteins are potent regulators of virus infectivity. However, relatively little is known about the functional domains, peptide motifs, or residues of any Vif protein. In this report, we present the first extensive mutagenesis analysis of the 192-amino-acid human immunodeficiency virus type 1 (HIV-1) Vif protein. A large number of scanning missense (mostly alanine substitution) and deletion mutations were introduced into the HIV-1 HXB3 vif gene, and the resulting proteins were evaluated for the induction of virus infectivity as well as subcellular localization. The results show that amino acids dispersed throughout Vif’s linear sequence are important for function. However, because many of the inactive proteins also appear to be mislocalized, we suggest that many of them may actually be misfolded rather lacking an intracellular targeting signal. Interestingly, disruptions within an internal region spanning residues 114 to 146 give rise to mutant proteins that either retain function or are inactive but are not substantially mislocalized. We therefore speculate that this region, which harbors two essential cysteine residues and one essential serine residue, may contain aspects of a putative Vif effector domain.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) performs a number of functions that are associated with the nucleus. Vpr enhances the nuclear import of postentry viral nucleoprotein complexes, arrests proliferating cells in the G2 phase of the cell cycle, and acts as a modest transcriptional activator. For this paper, we have investigated the nuclear import of Vpr. Although Vpr does not encode a sequence that is recognizable as a nuclear localization signal (NLS), Vpr functions as a transferable NLS both in somatic cells and in Xenopus laevis oocytes. In certain contexts, Vpr also mediates substantial accumulation at the nuclear envelope and, in particular, at nuclear pore complexes (NPCs). Consistent with this, Vpr is shown to interact specifically with nucleoporin phenylalanine-glycine (FG)-repeat regions. These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr’s functions may take place at the NPC. The accessory gene vpr is present in all viruses that belong to the five phylogenetic lineages of primate lentiviruses. The human immunodeficiency virus type 1 (HIV-1) Vpr protein is well conserved in terms of primary sequence and is approximately 96 amino acids in length. To date, a number of functions have been ascribed to HIV-1 Vpr (18). First, Vpr has been shown to act early in viral infection as a facilitator of postentry nucleoprotein complex (often referred to as the preintegration complex [PIC]) nuclear import (30, 40). This activ

Human immunodeficiency virus type-1 (HIV-1) is able to infect non-dividing cells such as tissue macrophages productively because post-entry viral nucleoprotein complexes are specifically imported into the nucleus in the absence of mitosis. Although it has been proposed that an amino-terminal region of the viral matrix (MA, p17Gag) protein harbors a basic-type nuclear localization sequence (NLS) that contributes to this process, utilization of three distinct nuclear import assays failed to provide any direct supporting evidence. Instead, we found that disruption of this region (26KK-->TT) reduces the rate at which the viral Gag polyprotein (p55Gag) is post-translationally processed by the viral protease. Consistent with the fact that appropriate proteolytic processing is essential for efficient viral growth in all cell types, we also show that the 26KK-->TT MA mutation is equivalently deleterious to the replication of a primary macrophage-tropic viral isolate in cultures of non-dividing and dividing cells. Taken together, these observations suggest that proteins other than MA supply the NLS(s) that enable HIV-1 to infect non-dividing cells.

Soon after the identification of HIV as the causative agent of AIDS, it became clear that CD4 was its primary cellular receptor. The clinical observation that AIDS was characterized by a loss of the T helper/inducer subset of lymphocytes (Th) prompted the studies which showed that HIV replicated in vitro only in cells expressing the Th marker, CD4 (Klatzmann et al. 1984a), and that antibodies to CD4 specifically inhibited infection and syncytium formation (Dalgleish et al. 1984; Klatzmann et al. 1984b). The receptor function of CD4 was conclusively demonstrated when its expression was shown to confer HIV susceptibility on previously insusceptible cells (Maddon et al. 1986). Finally, when recombinant soluble CD4 was mixed with virus or virus-infected cells, infection and syncytium formation were inhibited, presumably because of competition with the cell surface receptor (Smith et al. 1987; Fisher et al. 1988; Hussey et al. 1988; Traunecker et al. 1988).

The productive infection of many susceptible human cells, including lymphocytes and macrophages derived from peripheral blood, by the pathogenic lentivirus human immunodeficiency virus type 1 requires expression of the virally encoded vif (for virion infectivity factor) gene. Interestingly, this gene appears to have been conserved among all of the lentiviruses of primates and almost all of the lentiviruses of nonprimates. Using T cells constitutively expressing vif genes derived from diverse sources and virus replication assays, we show that the vif gene of a second primate lentivirus, simian immunodeficiency virus from macaques, complements vif-defective human immunodeficiency virus type 1 but that those of three distinct nonprimate lentiviruses do not. Although the molecular basis for Vif function has yet to be defined, the potential implications of this noted restriction of vif complementarity are discussed.

Expression of CD4 is not sufficient for cellular susceptibility to HIV-1 entry. The majority of nonprimate cells appear to lack a factor that plays an accessory role to CD4 during virus-cell fusion. We have previously reported CD4-dependent entry in rat myeloma Y3 cells, and here we show that Y3 and CD4-expressing mouse L929 cells fuse spontaneously to produce heterokaryons susceptible to entry by HIV-1. The results suggest that a putative accessory factor necessary for entry of HIV-1 into cells is expressed by the nonprimate Y3 cell line.

Replication of human immunodeficiency virus type 1 (HIV-1) is restricted to CD4-expressing primate cells. This tropism may be due partly to the absence from nonprimate cells of a species-specific factor which has an accessory role to CD4 during virus penetration. In this study we describe a rat B lymphocyte cell line in which there is efficient CD4-dependent entry of HIV-1. However, this cell line has a block to productive infection of HIV-1 at a stage between reverse transcription and integration. Our results demonstrate that the putative accessory factor for HIV-1 penetration is not restricted to primate cells and that there is a novel, uncharacterized cell-virus interaction at a stage between penetration and integration.

CD4 is the primary receptor for the human immunodeficiency virus type 1 (HIV-1). Early mutational studies implicated a number of residues of CD4, centered in the region 41-59, in binding to gp120. However, further mutational analyses, together with studies using inhibitory antibodies or CD4-derived peptides, have suggested that other regions of CD4 are also involved in binding or postbinding events during infection. To resolve these ambiguities, we used rat CD4 mutants in which particular regions were replaced with the corresponding sequence of human CD4. We have previously shown that some of these are able to bind HIV-1 gp120, and here we test their ability to act as functional receptors. We find that the presence of human CD4 residues 33-62 is enough to confer efficient receptor function to rat CD4, and we conclude that it is unlikely that regions of CD4 outside this sequence are involved in specific interactions with HIV-1 during either infection or syncytium formation.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is required for efficient replication in primary cells and certain immortalized cell lines in vitro and, in all likelihood, for the establishment of pathogenic infections in vivo. Current hypotheses concerning Vif's mechanism of action posit that it operates in virus-expressing cells during virion assembly, budding, or maturation such that released virions are modified in a manner that enables them to undergo productive infection in subsequent viral challenges. To gain further insight into the mechanism of action of lentivirus Vif proteins, we have performed a variety of in situ localization and biochemical fractionation studies using cells in which Vif is essential for efficient replication. Double-label immunofluorescence analyses of cells productively infected with HIV-1 or feline immunodeficiency virus revealed dramatic patterns of colocalization between Vif and the virally encoded Gag proteins. Subcellular fractionations of human T cells expressing HIV-1 Vif performed in the absence of any detergent demonstrated that greater than 90% of Vif is associated with cellular membranes. Additional purification using a continuous density gradient indicated that the majority of the membrane-bound Vif copurifies with the plasma membrane. Taken together, these observations suggest that lentivirus Vif and Gag proteins colocalize at the plasma membrane as virion assembly and budding take place. As a result, Vif is able to exert its modulatory effect(s) on these late steps of the virus life cycle.