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Introduction The human innate immune system provides early and effective responses against many invading pathogens and potential environmental threats. A critical component of innate immunity is pattern recognition of a [...], The Toll-like receptor 8 (TLR8) has an important role in innate immune responses to RNA viral infections, including respiratory syncytial virus (RSV). We previously reported that TLR8 expression was increased directly by the tumor suppressor and transcription factor p53 via a single nucleotide polymorphism (SNP) (rs3761624) in the TLR8 promoter, thereby placing TLR8 in the p53/immune axis. Because this SNP is in linkage disequilibrium with other SNPs associated with several infectious diseases, we addressed the combined influence of p53 and the SNP on downstream inflammatory signaling in response to a TLR8 cognate ssRNA ligand. Using human primary lymphocytes, p53 induction by chemotherapeutic agents such as ionizing radiation caused SNP-dependent synergistic increases in IL-6 following incubation with an ssRNA ligand, as well as TLR8 RNA and protein expression along with p53 binding at the TLR-p53 SNP site. Because TLR8 is X-linked, the increases were generally reduced in heterozygous females. We found a corresponding association of the p53-responsive allele with RSV disease severity in infants hospitalized with RSV infection. We conclude that p53 can strongly influence TLR8-mediated immune responses and that knowledge of the p53-responsive SNP can inform diagnosis and prognosis of RSV disease and other diseases that might have a TLR8 component, including cancer.

Author(s): Francesca Alfei [sup.1] , Kristiyan Kanev [sup.1] , Maike Hofmann [sup.2] , Ming Wu [sup.1] , Hazem E. Ghoneim [sup.3] [sup.12] , Patrick Roelli [sup.1] [sup.4] [sup.5] , Daniel [...], Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential.sup.1-4. This process, known as T cell exhaustion or dysfunction.sup.1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1).sup.5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood.sup.9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein.sup.13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1.sup.+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology. TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infections.

Introduction A paradigm of host immunity is that an individual's susceptibility to pathogens indicates a specific molecular defect. Yet, there are apparent exceptions to this paradigm: individuals who develop infections [...], Primary immunodeficiencies are often monogenic disorders characterized by vulnerability to specific infectious pathogens. Here, we performed whole-exome sequencing of a patient with disseminated Mycobacterium abscessus, Streptococcus viridians bacteremia, and cytomegalovirus (CMV) viremia and identified mutations in 2 genes that regulate distinct IFN pathways. The patient had a homozygous frameshift deletion in IFNGR2, which encodes the signal transducing chain of the IFN-[gamma] receptor, that resulted in minimal protein expression and abolished downstream signaling. The patient also harbored a homozygous deletion in IFNAR1 ([IFNAR1.sup.*557Gluext*46]), which encodes the IFN-[alpha] receptor signaling subunit. The [IFNAR1.sup.*557Gluext*46] resulted in replacement of the stop codon with 46 additional codons at the C-terminus. The level of [IFNAR1.sup.*557Gluext*46] mutant protein expressed in patient fibroblasts was comparable to levels of WT IFNAR1 in control fibroblasts. IFN-[alpha]-induced signaling was impaired in the patient fibroblasts, as evidenced by decreased STAT1/STAT2 phosphorylation, nuclear translocation of STAT1, and expression of IFN-[alpha]-stimulated genes critical for CMV immunity. Pretreatment with IFN-[alpha] failed to suppress CMV protein expression in patient fibroblasts, whereas expression of WT IFNAR1 restored IFN-[alpha]-mediated suppression of CMV. This study identifies a human IFNAR1 mutation and describes a digenic immunodeficiency specific to type I and type II IFNs.

CRISPR loci and Cas proteins provide adaptive immunity to bacteria and archaea against their viruses (1). To adapt to highly dynamic viral populations, CRISPR-Cas loci evolve rapidly, acquiring short phage [...], Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spaceracquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss [...]

Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti- and proviral properties which extend to a single-stranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual host-encoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses. RNAi | herpesvirus | RNA virus | RNA processing | phosphatidylinositol-3-kinase-Akt signalling doi/ 10.1073/pnas.1008861107

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.05.009 Byline: Ken Cadwell (1), Khushbu K. Patel (1), Nicole S. Maloney (1), Ta-Chiang Liu (1), Aylwin C.Y. Ng (3)(4), Chad E. Storer (5), Richard D. Head (5), Ramnik Xavier (3)(4), Thaddeus S. Stappenbeck (1), Herbert W. Virgin (1)(2)(6) Keywords: HUMDISEASE; MOLIMMUNO; MICROBIO Abstract: It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNF[alpha] and IFN[gamma] and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease. Author Affiliation: (1) Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA (2) Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA (3) Center for Computational and Integrative Biology and Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA (4) Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA (5) Inflammation and Immunology Research Unit, Pfizer Global Research and Development, St. Louis, MO 63017, USA (6) Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, St. Louis, MO 63110, USA Article History: Received 9 December 2009; Revised 26 February 2010; Accepted 19 April 2010 Article Note: (miscellaneous) Published: June 24, 2010

RNA viruses are the main agents of emerging and re-emerging diseases. It is therefore important to reveal the evolutionary processes that underpin their ability to jump species boundaries and establish themselves in new hosts. Here, I discuss how comparative genomics can contribute to this endeavor. Arguably the most important evolutionary process in RNA virus evolution, abundant mutation, may even open up avenues for their control through 'lethal mutagenesis.' Despite this remarkable mutational power, adaptation to diverse host species remains a major adaptive challenge, such that the most common outcome of host jumps are short-term 'spillover' infections. A powerful case study of the utility of genomic approaches to studies of viral evolution and emergence is provided by influenza virus and brought into sharp focus by the ongoing epidemic of swine-origin H1N1 influenza A virus (A/H1N1pdm). Research here reveals a marked lack of surveillance of influenza viruses in pigs, coupled with the possibility of cryptic transmission before the first reported human cases, such that the exact genesis of A/H1N1pdm (where, when, how) is uncertain. evolution | influenza | RNA virus | lethal mutagenesis | mutation rate doi/ 10.1073/pnas.0906193106

Cryphonectria parasitica, the chestnut blight fungus, can be infected by virulence-attenuating mycoviruses of the family Hypoviridae. Previous studies have led to the hypothesis that the hypovirus-infected phenotype is partly due to metabolic changes induced by the viral infection. To investigate this, we measured the metabolic rate and respiration of C. parasitica colonies grown on solid medium. These experiments supported historical observations of other fungal species done in liquid cultures that the metabolic rate steadily declines with age and differentiation of the mycelium. Hypovirus infection increased metabolic rate in the youngest mycelium, but a subsequent decline was also observed as the mycelium aged. By measuring both C[O.sub.2] production and [O.sub.2] consumption, we also observed that changes occur in carbohydrate metabolism as a result of ageing in both infected and uninfected mycelium. Mycelium on the periphery of the colony exploited fermentation pathways extensively, before transitioning to aerobic carbohydrate metabolism and finally lipid metabolism in the interior regions, despite abundant remaining glucose. However, the hypovirus affected the extent of these changes, with infected mycelium apparently unable to utilize lipid-related metabolic pathways, leading to an increased depletion of glucose. Finally, we used metabolic profiling to determine the changes in accumulation of primary metabolites in wild-type and hypovirus-infected mycelium and found that approximately one-third of the 164 detected metabolites were affected. These results are consistent with those expected from the physiological measurements, with significant alterations noted for compounds related to lipid and carbohydrate metabolism. Additionally, we observed an increase in the accumulation of the polyamine spermidine in the presence of hypovirus. Polyamines have been implicated in antiviral responses of mammalian systems; therefore this may suggest a novel antiviral response mechanism in fungi. DOI 10.1099/mic.0.029033-0

The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA. Here, we show that RIG-I ligands require base-paired structures in conjunction with a free 5'-triphosphate to trigger antiviral signaling. Hitherto unavailable chemically synthesized 5'-triphosphate RNA ligands do not trigger RIG-I-dependent IFN production in cells, and they are unable to trigger the ATPase activity of RIG-I without a base-paired stretch. Consistently, immunostimulatory RNA from cells infected with a virus recognized by RIG-I is sensitive to double-strand, but not single-strand, specific RNases. In vitro, base-paired stretches and the 5'-triphosphate bind to distinct sites of RIG-I and synergize to trigger the induction of signaling competent RIG-I multimers. Strengthening our model of a bipartite molecular pattern for RIG-I activation, we show that the activity of supposedly 'single-stranded' 5'-triphosphate RNAs generated by in vitro transcription depends on extended and base-paired by-products inadvertently, but commonly, produced by this method. Together, our findings accurately define a minimal molecular pattern sufficient to activate RIG-I that can be found in viral genomes or transcripts. immunostimulatory RNA | melanoma differentiation-associated protein 5 | retinoid acid-inducible gene-l-like helicases | virus infection interferon production

Human APOBEC3 (A3) proteins form part of the intrinsic immunity to retroviruses. Carrying 1 or 2 copies of a cytidine deaminase motif, A3s act by deamination of retroviral genomes during reverse transcription. HIV-1 overcomes this inhibition by the Vif protein, which prevents incorporation of A3 into virions. In this study we modeled and probed the structure of APOBEC3C (A3C), a single-domain A3 with strong antilentiviral activity. The 3-dimensional protein model was used to predict the effect of mutations on antiviral activity, which was tested in a [DELTA]vif simian immunodeficiency virus (SIV) reporter virus assay. We found that A3C activity requires protein dimerization for antiviral activity against SIV. Furthermore, by using a structure-based algorithm for automated pocket extraction, we detected a putative substrate binding pocket of A3C distal from the zinc-coordinating deaminase motif. Mutations in this region diminished antiviral activity by excluding A3C from virions. We found evidence that the small 5.8S RNA specifically binds to this locus and mediates incorporation of A3C into virus particles. Bioinformatics | immunodeficiency | protein structure | protein protein interaction | retrovirus

Coinfection in RNA virus populations results in two important phenomena, complementation and recombination. Of the two, complementation has a strong effect on selection against deleterious mutations, as has been confirmed in earlier studies. As complementadon delays the purging of less-fit mutations, coinfection may be detrimental to the evolution of a virus population. Here we employ both deterministic modeling and stochastic simulation to explore the mechanisms underlying the interactions between complementation and other evolutionary, factors, namely, mutation, selection, and epistasis. We find that strong complementation reduces slightly the overall fitness of a virus population but substantially enhances its diversity and robustness, especially when interacting with selection and epistasis. DOI: 10.1534/genetics.108.099796

Keywords: complementation, emergence, epistasis, evolution, fitness, mutation, natural selection, quasispecies, recombination, RNA secondary structure, robustness Abstract RNA viruses are the main agents of emerging disease. To understand how RNA viruses are able to jump species boundaries and spread in new hosts it is essential to determine the basic processes of evolutionary change in these infectious agents. RNA virus evolution is largely shaped by very high rates of mutation. This, coupled with potentially enormous intra- and interhost population sizes and continual replication, allows the rapid production of genetic diversity, including those mutations that facilitate host adaptation. However, high mutation rates also act to constrain aspects of RNA virus evolution, as the majority of mutations, including many at synonymous sites, are deleterious, which in turn places an upper limit on genome size. Ironically, although RNA viruses are characterized by their mutation rates, these rates may not be high enough to allow the onset of quasispecies dynamics, in which natural selection acts on the viral population as a whole.

During the last several decades corals have been in decline and at least one-third of all coral species are now threatened with extinction. Coral disease has been a major contributor to this threat, but little is known about the responsible pathogens. To date most research has focused on bacterial and fungal diseases; however, viruses may also be important for coral health. Using a combination of empirical viral metagenomics and real-time PCR, we show that Porites compressa corals contain a suite of eukaryotic viruses, many related to the Herpesviridae. This coral-associated viral consortium was found to shift in response to abiotic stressors. In particular, when exposed to reduced pH, elevated nutrients, and thermal stress, the abundance of herpes-like viral sequences rapidly increased in 2 separate experiments. Herpes-like viral sequences were rarely detected in apparently healthy corals, but were abundant in a majority of stressed samples. In addition, surveys of the Nematostella and Hydra genomic projects demonstrate that even distantly related Cnidarians contain numerous herpes-like viral genes, likely as a result of latent or endogenous viral infection. These data support the hypotheses that corals experience viral infections, which are exacerbated by stress, and that herpes-like viruses are common in Cnidarians. coral reefs | disease | Herpesviridae | viral-like particles | virome

Gene silencing by RNA interference (RNAi) has been shown to represent a recently discovered approach for the treatment of human diseases, including viral infection. A major limitation for the success of therapeutic strategies based on RNAi has been the delivery and shortlasting action of synthetic RNA. Multilayered polyelectrolyte films (MPFs), consisting of alternate layer-by-layer deposition of polycations and polyanions, have been shown to represent an original approach for the efficient delivery of DNA and proteins to target cells. Using hepatitis C virus infection (HCV) as a model, we demonstrate that siRNAs targeting the viral genome are efficiently delivered by MPFs. This delivery method resulted in a marked, dose-dependent, specific, and sustained inhibition of HCV replication and infection in hepatocyte-derived cells. Comparative analysis demonstrated that delivery of siRNAs by MPFs was more sustained and durable than siRNA delivery by standard methods, including electroporation or liposomes. The antiviral effect of siRNA-MPFs was reversed by a hyaluronidase inhibitor, suggesting that active degradation of MPFs by cellular enzymes is required for siRNA delivery. In conclusion, our results demonstrate that cell-degradable MPFs represent an efficient and simple approach for sustained siRNA delivery targeting viral infection. Moreover, this MPF-based delivery system may represent a promising previously undescribed perspective for the use of RNAi as a therapeutic strategy for human diseases. RNAi | therapy | hepatitis C virus | hepatocyte | liver

Human species C adenovirus serotype 5 (Ad5) is the most common viral vector used in clinical studies worldwide. Ad5 vectors infect liver cells in vivo with high efficiency via a poorly defined mechanism, which involves virus binding to vitamin K-dependent blood coagulation factors. Here, we report that the major Ad5 capsid protein, hexon, binds human coagulation factor X (FX) with an affinity of 229 pM. This affinity is 40-fold stronger than the reported affinity of Ad5 fiber for the cellular receptor coxsackievirus and adenovirus receptor, CAR. Cryoelectron microscopy and single-particle image reconstruction revealed that the FX attachment site is localized to the central depression at the top of the hexon trimer. Hexon-mutated virus bearing a large insertion in hexon showed markedly reduced FX binding in vitro and failed to deliver a transgene to hepatocytes in vivo. This study describes the mechanism of FX binding to Ad5 and demonstrates the critical role of hexon for virus infection of hepatocytes in vivo. gene transfer | virus targeting

Ref(2)P has been described as one of the Drosophila proteins that interacts with the sigma virus cycle. We generated alleles to identify critical residues involved in the restrictive (inhibiting viral multiplication) or permissive (allowing viral multiplication) character of Ref(2)P. We demonstrate that permissive alleles increase the ability of the sigma virus to infect Drosophila when compared to null alleles and we confirm that restrictive alleles decrease this capacity. Moreover, we have created alleles unfunctional in viral cycling while functional for Ref(2)P fly functions. This type of allele had never been observed before and shows that fly- and virus-related activities of Ref(2)P are separable. The viral status of Ref(2)P variants is determined by the amino-terminal PB1 domain polymorphism. In addition, an isolated PB1 domain mimics virus-related functions even if it is similar to a loss of function toward fly-related activities. The evolutionary tree of the Ref(2)P PB1 domain that we could build on the basis of the natural allele sequences is in agreement with an evolution of PB1 domain due to successive transient selection waves.

Some of the many molecular mechanisms that bacterial and viral and pathogens use to evade host immune defenses are highlighted. The success of each pathogen is directly dependant on its ability to mount effective anti-immune response within the infected host, which can ultimately result in acute disease, chronic infection or pathogen clearance.

The parasitic relationship of viruses with the host cytoskeleton provides an excellent basis for cell biologists to unlock the secrets of cytoplasmic transport and unravel mechanisms of disease. The way in which viruses use microtubule cytoskeleton to enhance their spread of infection is focused and the mechanism regulating cytoplasmic transport is highlighted.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by exogenous dsRNA. In plants an RNAi-like mechanism defends against viruses, but the hypothesis that animals possess a similar natural antiviral mechanism related to RNAi remains relatively untested. To test whether genes needed for RNAi defend animal cells against virus infection, we infected wild-type and RNAi-defective cells of the nematode C. elegans with vesicular stomatitis virus engineered to encode a GFP fusion protein. We show that upon infection, cells lacking components of the RNAi apparatus produce more GFP and infective particles than wild-type cells. Furthermore, we show that mutant cells with enhanced RNAi produce less GFP. Our observation that multiple genes required for RNAi are also required for resistance to vesicular stomatitis virus suggests that the RNAi machinery functions in resistance to viruses in nature. vesicular stomatitis virus | virus

A generalized method is developed for the rapid and sensitive detection of infectious viruses from cell culture by directly visualizing newly synthesized viral RNA with molecular beacons (MBs). This method, when combined with nuclease-resistant MBs, is useful not only for real-time detection of infectious viruses but also to assess the life cycle of viral processing in vitro.

Enterohemorrhagic Escherichia coli O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases. The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains. Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse XbaI-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning. This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity. In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage.

We have examined the progression of hepatitis C virus (HCV) infections by gene expression analysis of liver biopsies in acutely infected chimpanzees that developed persistent infection, transient viral clearance, or sustained clearance. Both common responses and outcome-specific changes in expression were observed. All chimpanzees showed gene expression patterns consistent with an IFN-[alpha] response that correlated with the magnitude and duration of infection. Transient and sustained viral clearance were uniquely associated with induction of IFN-[gamma]-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response. During the early stages of infection, host genes involved in lipid metabolism were also differentially regulated. We also show that drugs that affect these biosynthetic pathways can regulate HCV replication in HCV replicon systems. Our results reveal genome-wide transcriptional changes that reflect the establishment, spread, and control of infection, and they reveal potentially unique antiviral programs associated with clearance of HCV infection.

The NS1 protein of influenza A virus contributes to viral pathogenesis, primarily by enabling the virus to disarm the host cell type IFN defense system. We examined the downstream effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels by measuring expression of over 13,000 cellular genes in response to infection with wild-type and mutant viruses in human lung epithelial cells. Influenza A/PR/8/34 virus infection resulted in a significant induction of genes involved in the IFN pathway. Deletion of the viral NS1 gene increased the number and magnitude of expression of cellular genes implicated in the IFN, NF-KB, and other antiviral pathways. Interestingly, different IFN-induced genes showed different sensitivities to NS1-mediated inhibition of their expression. A recombinant virus with a C-terminal deletion in its NS1 gene induced an intermediate cellular mRNA expression pattern between wild-type and NS1 knockout viruses. Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.

Theiler's virus persistently infects the white matter of the spinal cord in susceptible strains of mice. This infection is associated with inflammation and primary demyelination and is studied as a model of multiple sclerosis. The H-2D gene is the major gene controlling viral persistence. However, the SJL/J strain is more susceptible than predicted by its [H-2.sup.s] haplotype. An (SJL/J x B10.S)[F.sub.1] x B10.S backcross was analyzed, and one quantitative trait locus (QTL) was located in the telomeric region of chromosome 10 close to the Ifng locus. Another one was tentatively mapped to the telomeric region of chromosome 18, close to the Mbp locus. We now report the study of 14 congenic lines that carry different segments of these two chromosomes. Although the presence of a QTL on chromosome 18 was not confirmed, two loci controlling viral persistence were identified on chromosome 10 and named Tmevp2 and Tmevp3. Furthermore, the Ifng gene was excluded from the regions containing Tmevp2 and Tmevp3. Analysis of the mode of inheritance of Tmevp2 and Tmevp3 identified an effect of sex, with males being more infected than females.

Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene N because infection of susceptible tobacco not carrying the N gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and de novo synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an N gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.

The role that subtle and widespread human gene polymorphisms play in virus-disease resistance is not well understood. The ways in which mouse-virus systems are used to identify candidate virus-resistance genes in humans have been studied. Various benefits can be found in such a study, which provides, among other things, probes to find functional homologues of resistance genes shares by lineages of rodents and primates. Not all of the aspects of virus-restriction are conserved, but functional similarities may be sufficient to be helpful to genetic studies of human resistance to viruses.

Interferon (IFN)-[Alpha]/[Beta]-mediated negative regulation of interleukin 12 (IL-12) and IFN-[Gamma] proteins is reported here. Both IFN-[Alpha] and IFN-[Beta] inhibited fixed Staphylococcus aureus Cowan strain induction of IL-12 and IFN-[Gamma] production by mouse splenic leukocytes in culture. Extended studies with IFN-[Alpha] demonstrated that inhibition was at the level of biologically active IL-12 p70. Effects were selective, as induction of tumor necrosis factor was unaffected and induction of IL-6 was enhanced. Neutralization of IFN-[Alpha]/[Beta] expressed endogenously during infections with murine cytomegalovirus (MCMV) enhanced early IL-12 and IFN-[Gamma] protein production. Furthermore, during infections of mice with lymphocytic choriomeningitis virus (LCMV), this treatment revealed a previously undetected early IL-12 and IFN-[Gamma] protein expression, and mice deficient in IFN-[Alpha]/[Beta] receptor function, but not control mice, also expressed endogenous LCMV-induced IL-12. The effects of IFN-[Alpha]/[Beta] neutralization on production of IL-12 and IFN-[Gamma] during the viral infections were detected in both serum samples and medium conditioned with splenic leukocytes isolated from infected animals. In vitro studies demonstrated that splenic leukocytes isolated from LCMV-infected mice were primed to produce IL-12 in response to stimulation with Staphylococcus aureus Cowan strain, but that this responsiveness was sensitive to added IFN-[Alpha]. Moreover, endogenous IFN-[Alpha]/[Beta] induced by LCMV inhibited in vivo lipopolysaccharide stimulation of IL-12 production. These results demonstrate a new pathway for regulating cytokine responses, and suggest a mechanism for inhibition of IL-12-dependent immune responses during viral infections.

Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.

An essential step in the initiation of a virus infection is the release of the viral genome from the other constituents of the virus particle, a process referred to as uncoating. We have used reverse transcription and polymerase chain reaction amplification procedures to determine the rate and direction of in vivo uncoating of the rod-shaped tobacco mosaic virus. The virus particles contain a single 6.4-kb RNA molecule that lies between successive turns of a helical arrangement of coat protein subunits. When the particles are introduced into plant cells, the subunits are removed via a bidirectional uncoating mechanism. Within 2-3 min, the part of the viral RNA from the 5' end to a position >70% toward the 3' end has been freed of coat protein subunits. This is followed by removal of subunits from the 3' end of the RNA and sequential uncoating of the RNA in a 3'-to-5' direction. An internal region of the viral RNA is the final part to be uncoated. Progeny virus particles are detected in the cells 35-40 min after inoculation.

Cells infected with orthopox virus strains including the Vaccinia virus contain a soluble type I interferon receptor that is encoded by gene B18R. The 60-65 kDa receptor belongs to the immunoglobulin superfamily and show high affinity for human IFNalpha and a variety of mammalian IFNs, which renders protection against poxvirus infections. A murine intranasal model explaining the mechanism used by poxviruses for counteracting IFN is discussed.

Neurovirulent Alphavirus Sindbis strains cause apoptotic death of cultured cells transfected with bcl-2 genes that cause viral infection. Apoptosis-induction is mainly influenced by E2 glycoprotein of Sindbis virus, resulting in neurovirulence killing of bcl-2 expressing cells.

For RNA viruses, rapid viral evolution and the biological similarity of closely related host species have been proposed as key determinants of the occurrence and long-term outcome of cross-species transmission. Using a data set of hundreds of rabies viruses sampled from 23 North American bat species, we present a general framework to quantify per capita rates of cross-species transmission and reconstruct historical patterns of viral establishment in new host species using molecular sequence data. These estimates demonstrate diminishing frequencies of both cross-species transmission and host shifts with increasing phylogenetic distance between bat species. Evolutionary constraints on viral host range indicate that host species barriers may trump the intrinsic mutability of RNA viruses in determining the fate of emerging host-virus interactions. 10.1126/science.1188836

Virus infection in mammals elicits a variety of defense responses that are initiated by signals from virus-sensing receptors expressed by the host. These receptors include the ubiquitously expressed RIG-I--like receptor (RLR) family of RNA helicases. RLRs are cytoplasmic proteins that act in cell-intrinsic antiviral defense by recognizing RNAs indicative of virus presence. Here, we highlight recent progress in understanding how RLRs discriminate between the RNA content of healthy versus virus-infected ceils, functioning as accurate sensors of virus invasion. 10.1126/science.1185068

The viral contamination by the four main viruses responsible for acute gastroenteritis in the hydrographic network that surrounds Manaus, Amazonas, Brazil, is assessed. The presence of viral genomes in areas where fecal contamination is not shown by bacterial indicators has suggested prolonged virus persistence in aquatic environments and has described the enteric group to be very reliable for environmental monitoring.

The genetic architecture of virus transmission is studied by crossing a vector and a non-vector genotype of the aphid Schizaphis graminum. The results have shown that in Schizaphis graminum, the transmission efficiency of both Cereal yellow dwarf virus (CYDV)-RPV and Barley yellow dwarf virus (BYDV)-SGV is regulated by a major gene and the transmission efficiency of each virus is affected by a unique set of minor genes.

The general concepts in virus entry are reviewed which reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication. At the level of cells an virus particles, development in morphological techniques from high-end light microscopy to electron microscopy are allowing events involved in virus to be analyzed with increased spatial and temporal resolution.