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Both monogenic diseases and viral infections can manifest in a broad spectrum of clinical phenotypes that range from asymptomatic to lethal, suggesting that other factors modulate disease severity. Here, we examine the interplay between the genetic neuronopathic Gaucher's disease (nGD), and neuroinvasive Sindbis virus (SVNI) infection. Infection of nGD mice with SVNI had no influence on nGD severity. However, nGD mice were more resistant to SVNI infection. Significantly different inflammatory responses were seen in nGD brains when compared with SVNI brains: the inflammatory response in the nGD brains consisted of reactive astrocytes and microglia with no infiltrating macrophages, but the inflammatory response in the brains of SVNI-infected mice was characterized by infiltration of macrophages and altered activation of microglia and astrocytes. We suggest that the innate immune response activated in nGD confers resistance against viral infection of the CNS.

We report the whole-genome sequence of a monkeypox virus strain isolated in Israel. The strain was isolated in 2018 from a patient travelling back from West Africa. The virus was fully sequenced on the Illumina MiSeq and Oxford Nanopore Technologies MinION platforms., (Copyright © 2020 Cohen-Gihon et al.)

We report here the whole-genome sequence of the first camelpox virus case diagnosed in Israel. The strain (Negev2016) was isolated in 2016 from a camel in southern Israel and was sequenced on the Illumina MiSeq and Oxford Nanopore MinION platforms., (Copyright © 2019 Israeli et al.)

We report a case of monkeypox in a man who returned from Nigeria to Israel in 2018. Virus was detected in pustule swabs by transmission electron microscopy and PCR and confirmed by immunofluorescence assay, tissue culture, and ELISA. The West Africa monkeypox outbreak calls for increased awareness by public health authorities worldwide.

Identification and characterization of novel unknown viruses is of great importance. The introduction of high-throughput sequencing (HTS)-based methods has paved the way for genomics-based detection of pathogens without any prior assumptions about the characteristics of the organisms. However, the use of HTS for the characterization of viral pathogens from clinical samples remains limited. Here, we report the identification of a novel Orthobunyavirus species isolated from horse plasma. The identification was based on a straightforward HTS approach. Following enrichment in cell culture, RNA was extracted from the growth medium and rapid library preparation, HTS and primary bioinformatic analyses were performed in less than 12 hours. Taxonomical profiling of the sequencing reads did not reveal sequence similarities to any known virus. Subsequent application of de novo assembly tools to the sequencing reads produced contigs, of which three showed some similarity to the L, M, and S segments of viruses belonging to the Orthobunyavirus genus. Further refinement of these contigs resulted in high-quality, full-length genomic sequences of the three genomic segments (L, M and S) of a novel Orthobunyavirus. Characterization of the genomic sequence, including the prediction of open reading frames and the inspection of consensus genomic termini and phylogenetic analysis, further confirmed that the novel virus is indeed a new species, which we named Ness Ziona virus.

Viral infections of the central nervous system (CNS) lead to a broad range of pathologies. CNS infections with Orthopox viruses have been mainly documented as an adverse reaction to smallpox vaccination with vaccinia virus. To date, there is insufficient data regarding the mechanisms underlying pathological viral replication or viral clearance. Therefore, informed risk assessment of vaccine adverse reactions or outcome prediction is limited. This work applied a model of viral infection of the CNS, comparing neurovirulent with attenuated strains. We followed various parameters along the disease and correlated viral load, morbidity, and mortality with tissue integrity, innate and adaptive immune response and functionality of the blood⁻brain barrier. Combining these data with whole brain RNA-seq analysis performed at different time points indicated that neurovirulence is associated with host immune silencing followed by induction of tissue damage-specific pathways. In contrast, brain infection with attenuated strains resulted in rapid and robust induction of innate and adaptive protective immunity, followed by viral clearance and recovery. This study significantly improves our understanding of the mechanisms and processes determining the consequence of viral CNS infection and highlights potential biomarkers associated with such outcomes., Competing Interests: The authors declare no conflict of interest.

Sindbis virus (SINV) is a mosquito-borne Alphavirus responsible for outbreaks of SINV disease, mainly in north Europe. SINV has been isolated from mosquitoes in Israel since the 1980s but SINV disease outbreaks have never been recorded. To gain better understanding of the kinetics of SINV circulation in Israel, 3008 mosquito pools, collected 2004-2006 and 2013-2015, were tested for SINV and phylogenetic analysis was conducted on partially sequenced SINV-positive pools. Results indicate possible expansion of SINV circulation across Israel in 2013-2015 compared to 2004-2006 with 6.35% (191 pools) of total pools positive for SINV RNA. Phylogenetic analysis showed all sequenced Israeli SINV strains belong to genotype I and form, together with SINV sequences from Saudi Arabia, a distinct Middle Eastern cluster. With high endemicity of SINV and as a major crossroads for bird migration between Africa and Eurasia, Israel provides valuable information on SINV dynamics and pathogenicity.

An outbreak of a disease in camels with skin lesions was reported in Israel during 2016. To identify the etiological agent of this illness, we employed a multidisciplinary diagnostic approach. Transmission electron microscopy (TEM) analysis of lesion material revealed the presence of an orthopox-like virus, based on its characteristic brick shape. The virus from the skin lesions successfully infected chorioallantoic membranes and induced cytopathic effect in Vero cells, which were subsequently positively stained by an orthopox-specific antibody. The definite identification of the virus was accomplished by two independent qPCR, one of which was developed in this study, followed by sequencing of several regions of the viral genome. The qPCR and sequencing results confirmed the presence of camelpox virus (CMLV), and indicated that it is different from the previously annotated CMLV sequence available from GenBank. This is the first reported case of CMLV in Israel, and the first description of the isolated CMLV subtype., Competing Interests: The authors declare no conflict of interest. Founding was based on institutional support.

Declaration of smallpox eradication by the WHO in 1980 led to discontinuation of the worldwide vaccination campaign. The increasing percentage of unvaccinated individuals, the existence of its causative infectious agent variola virus (VARV), and the recent synthetic achievements increase the threat of intentional or accidental release and reemergence of smallpox. Control of smallpox would require an emergency vaccination campaign, as no other protective measure has been approved to achieve eradication and ensure worldwide protection. Experimental data in surrogate animal models support the assumption, based on anecdotal, uncontrolled historical data, that vaccination up to 4 days postexposure confers effective protection. The long incubation period, and the uncertainty of the exposure status in the surrounding population, call for the development and evaluation of safe and effective methods enabling extension of the therapeutic window, and to reduce the disease manifestations and vaccine adverse reactions. To achieve these goals, we need to evaluate the efficacy of novel and already licensed vaccines as a sole treatment, or in conjunction with immune modulators and antiviral drugs. In this review, we address the available data, recent achievements, and open questions., Competing Interests: The authors declare no conflicts of interest.

Eczema vaccinatum is a severe and occasionally lethal complication of smallpox vaccine, characterized by systemic viral dissemination, distant from the initial inoculation site of the vaccine. A major risk factor for eczema vaccinatum is a background of atopic dermatitis, a chronic, common allergic, relapsing disorder, manifested by dry and inflamed skin, itchy rash, Th2 biased immune response and hypersensitivity to various antigens. Unlike the severe manifestations of eczema vaccinatum in humans, current models present only mild symptoms that limits examination of potential therapeutics for eczema vaccinatum. The atopic dermatitis and eczema vaccinatum models we present here, are the first to simulate the severity of the diseases in humans. Indeed, dermatitic mice display persistent severe dermatitis, characterized by dry and inflamed skin with barrier dysfunction, epidermal hyperplasia and significant elevation of serum IgE. By exposing atopic dermatitis mice to ectromelia virus, we generated eczema vaccinatum that mimic the human disease better than known eczema vaccinatum models. Similarly to humans, eczematous mice displayed enlarged and disseminated skin lesions, which correlated with elevated viral load. Cidofovir and antiviral antibodies conferred protection even when treatment started at a late eczematous stage. Moreover, we are the first to demonstrate that despite a severe background of atopic dermatitis, modified vaccinia Ankara virus (MVA) vaccination protects against lethal ectromelia virus exposure. We finally show that protection by MVA vaccination is dependent on CD4 + T cells and is associated with significant activation of CD8 + cytotoxic T cells and induction of humoral immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Zika virus RNA presence in serum, whole-blood and urine samples from six Israeli travellers symptomatic for Zika virus disease was examined. Whole-blood samples were positive for as late as 2 months (58 days) post-symptom onset, longer than for urine (26 days) and serum (3 days). These findings suggest the utility of whole blood in Zika infection diagnosis., (This article is copyright of The Authors, 2016.)

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis.

Poxvirus detection assays are based on morphology, viral antigens and specific nucleic acids, none of which indicates virus viability or infectious capacity. Determination of virus viability is achieved by propagation in cell cultures and subsequent analysis by the mentioned methods, a process that takes days. Thus, presented here the development of a new assay, named PILA (Poxvirus Infection Luciferase Assay), for rapid detection of infectious poxviruses which is a cell-based reporter assay. The assay is composed of two steps: (i) Transfection of cells with a poxvirus specific reporter vector which consists of the early 7.5-kDa-STR promoter, regulating the expression of luciferase gene; (ii) Infection with a poxvirus containing sample. Luciferase activity measured post infection, indicates the presence of infectious poxvirus in the sample. The assay can detect quantities as low as 100 PFU of VACV, six hours post infection. Orthopox virus universality was confirmed by detection of various Orthopoxviruses, and specificity was verified by using pox-specific neutralizing antibodies. The PILA is specific, rapid, simple, and suitable for detecting viable virus. The assay can be utilized for applications such as poxvirus titration, neutralizing assay and drug discovery. The assay was adjusted for live detection assay by using GFP as reporting gene., (Copyright 2010 Elsevier B.V. All rights reserved.)

The mechanism of protection afforded by vaccinia virus (VACV) - the smallpox vaccine - is a key issue for the development of modern vaccines and countermeasures. Antibodies to VACV antigens of the extracellular virion (EV) form play a central role in protection against poxvirus diseases in animal models, and contribute to the protection of immunized humans against poxviruses. B5, a viral EV protein, is conserved among different orthopoxviruses and antibodies to B5 that protect mice against VACV challenge. Antibodies to B5 are primarily responsible for neutralization of vaccinia EVs, yet the mechanism of EV neutralization by antibodies to B5 is not fully understood. The paper under evaluation demonstrates that most of the neutralization in vitro and protection in vivo in a mouse model, by monoclonal human anti-B5 IgGs, is heavily dependent on the ability of the IgGs to bind complement (C3 and C1q). Similarly, IgGs capable of complement binding control complement-dependent cytotoxicity of VACV-infected cells. Human polyclonal antibodies induced by the smallpox vaccine were similarly dependent on complement for EV neutralization and the complement-dependent destruction of infected cells. These findings not only contribute to a better understanding of the mechanism of protection by antibodies, but might also help in the development and evaluation of newly-developed therapeutic and prophylactic antibody-based products against virulent orthopoxviruses, and for the prevention or treatment of smallpox vaccine-related post-vaccinal adverse effects.

Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

Smallpox has been eradicated but stockpiles of the causative infectious agent, variola virus, have been maintained over decades. Today, the threat of accidental or intentional poxvirus release is accompanied by the fact that the existing licensed smallpox vaccines cause rare but severe adverse reactions yet are the only products with approved efficacy against smallpox. New safer vaccines and new strategies of immunization are to be developed to fit to a scenario of emergency smallpox vaccination. However, we still lack knowledge about the pathogen and the mechanisms involved in acquiring protective immunity. Here, we review the history of smallpox vaccines and recent achievements in the development of highly efficacious and safer vaccines and vaccine applications. These include i) assessment of adequate animal models to study pathogenesis and protective immunity, ii) characterization of the immunity elicited by next generation vaccines, and (iii) the investigation of the requirements for rapidly protective vaccination.

Background: Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium., Results: We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection., Conclusion: Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.

Vaccinia virus encodes a 90-kDa protein conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded varphiX174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which (32)P was transferred from [alpha-(32)P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.

Background: Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain., Methods: We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV)., Results: In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses., Conclusions: ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.

Since smallpox eradication by the WHO during the 1980s, potency of new vaccines is compared to vaccines that were used during the eradication campaign. In this work we characterize the tail scarification technique in mice as a model for scarification in humans. Similar to humans, mice develop "clinical take" which is dependent on the vaccination dose. Appearance of anti-Vaccinia IgM is followed by IgG antibodies 10 days post scarification and lasting more then 1(1/2) years. Mice with "clinical take" are 100% protected against lethal respiratory challenge (100LD(50)) of Vaccinia WR indicating that the "clinical take" can serve as a correlate of protective immunity. Reducing the vaccination dose and using Cowpox virus as a more virulent strain, enabled us to draw the limit of the vaccine potency in mice. Similar to humans, in revaccinated mice the development of "clinical take" was inversely correlated to the level of pre-existing antibodies. These results indicate that tail scarification of mice can be used as a model for evaluation of smallpox vaccines. High correlation between "clinical take" and protective immunity allows the use of visual inspection to evaluate vaccine potency.

Hepatitis B virus (HBV) is a 3.2-kb DNA virus that replicates preferentially in the liver. Liver-enriched nuclear receptors (NRs) play a major role in the HBV life cycle, operating as essential transcription factors for viral gene expression. Notably, these NRs are also key players in metabolic processes that occur in the liver, serving as central transcription factors for key enzymes of gluconeogenesis, fatty acid beta-oxidation, and ketogenesis. However, the association between these metabolic events and HBV gene expression is poorly understood. Here we show that peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), a major metabolic regulator and a coactivator of key gluconeogenic genes, robustly coactivates HBV transcription. We further demonstrate that the liver-enriched NR hepatocyte nuclear factor 4alpha that binds HBV plays an important role in this process. Physiologically, we show that a short-term fast that turns on the gluconeogenic program robustly induces HBV gene expression in vivo. This induction is completely reversible by refeeding and depends on PGC-1alpha. We conclude that HBV is tightly regulated by changes in the body's nutritional state through the metabolic regulator PGC-1alpha. Our data provide evidence for nutrition signaling to control viral gene expression and life cycle and thus ascribe to metabolism an important role in virus-host interaction.

Vaccination with vaccinia virus is carried out in order to induce protection against variola virus, the causative agent of smallpox. Serum titer of vaccinia virus-neutralizing antibodies is considered to be well-correlated with in vivo protection. Plaque reduction neutralization test (PRNT) is the gold standard for detecting and quantifying vaccinia virus-neutralizing antibodies in sera of vaccinees. However, PRNT is time and labor consuming, which does not allow large-scale screening needed for a population survey. A simplified, sensitive, standardized, reproducible and rapid method, neutralization tissue-culture enzyme immunoassay (NTC-EIA) was developed for quantitation of neutralizing antibodies against vaccinia virus. The assay consists of the following steps: neutralization of the virus with serially diluted sera, infection of cells in culture and measurement of residual virus replication using an enzyme immunoassay. The assay can be used for animal (rabbit) or human sera. Titer averages obtained using NTC-EIA were highly correlated (R2=0.9994) to those obtained using PRNT. The assay is carried out in 96-well plates and takes only 2 days to complete. With the appropriate setup, it can be automated fully to allow screening of a large number of sera.

The early steps in hepatitis B virus (HBV) infection, a human hepadnavirus, initiates from cell attachment followed by entry and delivery of the genetic information to the nucleus. Despite the fact that these steps determine the virus-related pathogenesis, their molecular basis is poorly understood. Cumulative data suggest that this process can be divided to cell attachment, endocytosis, membrane fusion and post-fusion consecutive steps. These steps are likely to be regulated by the viral envelope proteins and by the cellular membrane, receptors and extracellular matrix. In the absence of animal model for HBV, the duck hepadnavirus DHBV turned out to be a fruitful animal model. Therefore data concerning the early, post-attachment steps in hepadnaviral entry are largely based on studies performed with DHBV in primary duck liver hepatocytes. These studies are now starting to illuminate the mechanisms of hepadnavirus route of cell entry and to provide some new insights on the molecular basis of the strict species specificity of hepadnavirus infection.

Virus infection is initiated by recognition and attachment of the virus to the cell surface. Despite the fact that this interaction determines the virus-related pathogenesis, its molecular basis remained obscure for HBV. This process is mediated primarily by the viral envelope and the cellular receptors. HBV infection is not exceptional in this regard but its putative receptors have not been identified yet. The recent development of protocols to establish HBV susceptible cell lines and unique tools to measure HBV-cell attachment at a single cell resolution set the stage for the study of HBV-host cell interaction. These studies revealed that the QLDPAF epitope of the HBV surface antigen large protein (LHBsAg) plays a major role in this process. Quantitative measurements suggested the presence of a second player in this process and both act synergistically to improve cell attachment. As the step of virus-cell attachment is potentially susceptible to specific inhibitors, understanding the molecular basis of virus-cell attachment can be expected to have therapeutic impacts., (Copyright 2003 John Wiley & Sons, Ltd.)

pX, the hepatitis B virus-encoded transcription coactivator, is involved in viral infection in vivo. pX stimulates the activity of several transcription factors including nuclear factor-kappaB (NF-kappaB), but the mechanism of activation is poorly understood. The IkappaB kinase complex (IKK) mediates activation of NF-kappaB in response to various extracellular stimuli, including inflammatory cytokines like tumor necrosis factor and interleukin 1, human T cell lymphoma virus 1 Tax protein, and tumor promoters like phorbol esters. It is not known whether IKK also mediates activation of NF-kappaB by pX. Here we report that IKK was not essential for activation of NF-kappaB by pX. Expression of pX resulted in the degradation of IkappaBalpha in the absence of its phosphorylation at Ser(32) and Ser(36) residues. Although pX stimulated the activity of cotransfected IKK-beta when it was overexpressed, it failed to activate endogenous IKK. Furthermore, expression of pX stimulated NF-kappaB nuclear translocation and transcriptional activity in IKK-gamma-null fibroblast 5R cells. Our data indicate that pX stimulates NF-kappaB activity through a mechanism that is dependent on IkappaBalpha degradation but not on IKK activation.

A variant polyadenylation signal, which is conserved and employed by mammalian hepadnaviruses, has a sequence resembling that of the TATA box. We report here that this composite box manifests all the promoter characteristics. It binds effectively TATA-binding protein with TFIIB and TFIIA in a synergistic manner. This capacity, however, is lost when the box is converted to a canonical and simple poly(A) signal. Furthermore, we show that it has promoter activity and supports transcription of reporter genes preferentially in liver-derived cells, a characteristic behavior of the hepatitis B virus (HBV) promoters. In addition, we show that the HBV noncanonical poly(A) signal supports transcription initiation from the viral genome, suggesting that it is a genuine promoter, possibly of the polymerase/reverse transcriptase gene. Finally, we found that this deviant poly(A) signal is crucial for HBV replication since a viral mutant with a canonical poly(A) box is impaired in replication. Our data, therefore, raise the interesting and novel possibility that a composite poly(A) box might have a dual function. At the level of DNA it functions as a promoter to initiate transcription, whereas at the level of RNA it serves as a poly(A) signal to process RNA. An interesting outcome of this strategy of gene expression is that it provides a novel mechanism for the synthesis of an approximately genome length transcript.

The transcription program of the hepatitis B virus (HBV) genome is regulated by an enhancer element that binds multiple ubiquitous and liver-enriched transcription activators. HBV transcription and replication are repressed in the presence of p53. Here we describe a novel molecular mechanism that is responsible for this repression. The p53 protein binds to a defined region within the HBV enhancer in a sequence-specific manner, and this, surprisingly, results in p53-dependent transcriptional repression in the context of the whole HBV enhancer. This unusual behavior of the HBV enhancer can be reconstituted by replacing its p53-binding region with the p53-binding domain of the mdm2 promoter. Remarkably, mutation of the EP element of the enhancer reversed the effect of p53 from repression to transcriptional stimulation. Furthermore, EP-dependent modulation of p53 activity can be demonstrated in the context of the mdm2 promoter, suggesting that EP is not only required but is also sufficient to convert p53 activity from positive to negative. Our results imply that the transcriptional effect of DNA-bound p53 can be dramatically modulated by the DNA context and by adjacent DNA-protein interactions.

The distribution of acetylcholinesterase (AChE) in the rat vallate papillary bud was investigated by histochemical electron microscopy. Previous reports of specific AChE activity around subgemmal and intragemmal nerves and between some taste bud cells have been confirmed. In addition we have consistently observed dense precipitation between microvilli in the taste pore. The studies suggest that the neurotransmitter, acetylcholine, may be involved in early events in the taste process which are believed to occur in the pore.

The taste bud of the human fungiform papilla was examined by electron microscopy. Typical type I, type II, and type III cells were found along with contact sites with nerve endings. Vesicles in nerve fibers contacting type I and type II cells suggest that these cells may receive efferent impulses, whereas vesicles and granules in type III cells adjacent to (afferent) nerve fibers support the view that type III cells are sensory receptors. All of these features are virtually indistinguishable from those previously reported in fungiform taste buds of other mammals.