Noncytopathic Virus Research Articles

Clonal Exhaustion as a Mechanism to Protect Against Severe Immunopathology and Death from an Overwhelming CD8 T Cell Response

The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of the immune response. Here, we demonstrate, with three different inocula of lymphocytic choriomeningitis virus (LCMV), how the race between virus replication and T cell responses can result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence, and little immunopathology. An intermediate dose only partially exhausted the T cell responses and resulted in significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This suggests that for non-cytopathic viruses like LCMV, hepatitis C virus, and hepatitis B virus, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death.

Structural and functional insights into alphavirus polyprotein processing and pathogenesis

Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1-4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus.

Apoptosis in Bovine Viral Diarrhea Virus (BVDV)–Induced Mucosal Disease Lesions

Cattle persistently infected with a noncytopathic Bovine viral diarrhea virus (BVDV) are at risk of developing fatal "mucosal disease" (MD). The authors investigated the role of various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD. Therefore, they compared the expression of caspase-3, caspase-8, caspase-9, and Bcl-2L1 (Bcl-x) in tissues of 6 BVDV-free control animals, 7 persistently infected (PI) animals that showed no signs of MD (non-MD PI animals), and 11 animals with MD and correlated the staining with the localization of mucosal lesions. Caspase-3 and -9 staining were markedly stronger in MD cases and were associated with mucosal lesions, even though non-MD PI animals and negative controls also expressed caspase-9. Conversely, caspase-8 was not elevated in any of the animals analyzed. Interestingly, Bcl-x also colocalized with mucosal lesions in the MD cases. However, Bcl-x was similarly expressed in tissues from all 3 groups, and thus, its role in apoptosis needs to be clarified. This study clearly illustrates ex vivo that the activation of the intrinsic, but not the extrinsic, apoptosis pathway is a key element in the pathogenesis of MD lesions observed in cattle persistently infected with BVDV. However, whether direct induction of apoptosis in infected cells or indirect effects induced by the virus are responsible for the lesions observed remains to be established.

New PARP Gene with an Anti-Alphavirus Function

Alphaviruses represent a highly important group of human and animal pathogens, which are transmitted by mosquito vectors between vertebrate hosts. The hallmark of alphavirus infection in vertebrates is the induction of a high-titer viremia, which is strongly dependent on the ability of the virus to interfere with host antiviral responses on both cellular and organismal levels. The identification of cellular factors, which are critical in orchestrating virus clearance without the development of cytopathic effect, may prove crucial in the design of new and highly effective antiviral treatments. To address this issue, we have developed a noncytopathic Venezuelan equine encephalitis virus (VEEV) mutant that can persistently replicate in cells defective in type I interferon (IFN) production or signaling but is cleared from IFN signaling-competent cells. Using this mutant, we analyzed (i) the spectrum of cellular genes activated by virus replication in the persistently infected cells and (ii) the spectrum of genes activated during noncytopathic virus clearance. By applying microarray-based technology and bioinformatic analysis, we identified a number of IFN-stimulated genes (ISGs) specifically activated during VEEV clearance. One of these gene products, the long isoform of PARP12 (PARP12L), demonstrated an inhibitory effect on the replication of VEEV, as well as other alphaviruses and several different types of other RNA viruses. Additionally, overexpression of two other members of the PARP gene superfamily was also shown to be capable of inhibiting VEEV replication.

Accelerated and Improved Quantification of Lymphocytic Choriomeningitis Virus (LCMV) Titers by Flow Cytometry

Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms.

Interferon stimulated genes, CXCR4 and immune cell responses in peripheral blood mononuclear cells infected with bovine viral diarrhea virus

Non-cytopathic bovine viral diarrhea virus (ncpBVDV) induces immune responses mediated by chemokines and interferon (IFN) stimulated genes (ISGs). Cultured bovine peripheral blood mononuclear cells (PBMC) from ncpBVDV-naïve cattle were used herein to demonstrate that BVDV infection modulates chemokine receptor 4 (CXCR4), CXCL12, IFN-I, ISGs and selected immune cell marker (CD4, CD8, CD14) mRNAs, and that these acute responses to viral infection are reflected in PBMC cultured with serum from heifers carrying fetuses persistently infected (PI) with ncpBVDV. Infection of PBMC with ncpBVDV increased IFN-β, ISG15, RIG-I, CXCR4, CXCL12, and CD8 mRNA concentrations after 32h. Culture of PBMC with uterine vein serum from acutely infected heifers, inoculated with ncpBVDV during early gestation to generate PI fetuses, also increased the concentration of CXCR4, RIG-I and ISG15 mRNAs. In vitro PBMC treatment with ncpBVDV or uterine vein serum from acutely infected pregnant heifers activates chemokine, ISG and immune cell responses.

Hepatitis B Precore Protein: Pathogenic Potential and Therapeutic Promise

Hepatitis B virus (HBV), a small and economically packaged double-stranded DNA virus, represents an enormous global health care burden. In spite of an effective vaccine, HBV is endemic in many countries. Chronic hepatitis B (CHB) results in the development of significant clinical outcomes such as liver disease and hepatocellular carcinoma (HCC), which are associated with high mortality rates. HBV is a non-cytopathic virus, with the host's immune response responsible for the associated liver damage. Indeed, HBV appears to be a master of manipulating and modulating the immune response to achieve persistent and chronic infection. The HBV precore protein or hepatitis B e antigen (HBeAg) is a key viral protein involved in these processes, for instance though the down-regulation of the innate immune response. The development of new therapies that target viral proteins, such as HBeAg, which regulates of the immune system, may offer a new wave of potential therapeutics to circumvent progression to CHB and liver disease.

Quantification and Determination of Spread Mechanisms of Bovine Viral Diarrhoea Virus in Blood and Tissues from Colostrum-Deprived Calves During an Experimental Acute Infection Induced by a Non-Cytopathic Genotype 1 Strain

To detect and monitor the sequential changes in virus levels, a reverse transcription quantitative real-time polymerase chain reaction assay using a TaqMan probe was carried out on frozen blood and tissues samples collected from calves experimentally infected with a non-cytopathic Bovine viral diarrhoea virus (BVDV) genotype 1 strain. Blood samples were collected among days 1-14 post-inoculation (p.i). On day 3 p.i, viral RNA was detected in blood samples from six of the eight inoculated animals. Viral RNA was detected in all remaining inoculated animals between 5 and 12 days p.i. The levels of viral RNA increased along the experiment, with a maximal peak between 6 and 9 days p.i. Analysis of virus load in tissues collected from calves euthanized on days 3, 6, 9 and 14 p.i displayed that BVDV was detected on day 3 p.i, being especially abundant in tonsils and ileocaecal valve, highlighting the role of tonsils as the main earliest viral replication sites as well as the principal source for virus spread to other lymphoid tissues and visceral organs. Coinciding with the highest viraemia levels, the highest viral loads were recorded at 9 days p.i. in tonsils, ileal lymph nodes, distal ileum and spleen, showing the main role of these secondary lymphoid organs in the pathogenic mechanisms of BVDV. However, virus levels in the liver and lung increased only towards the end of the infection. This fact could influence in the appearance of bovine respiratory diseases because of the capacity of BVDV for enhancing susceptibility to secondary infections.

Characterization of Apoptosis Pathways (Intrinsic and Extrinsic) in Lymphoid Tissues of Calves Inoculated with Non-cytopathic Bovine Viral Diarrhoea Virus Genotype-1

Previous studies have shown that activation of effector caspase-3 is associated with the apoptosis of lymphocytes occurring during infection with bovine viral diarrhoea virus (BVDV); however, the regulation of the apoptosis pathways that induce cell death via activation of effector caspase-3 has not yet been clarified. The aim of this study was to examine immunohistochemically the expression of cleaved caspase (CCasp)-8 (initiator caspase of the extrinsic pathway), CCasp9 (initiator caspase of the intrinsic pathway) and Bcl-2 (an anti-apoptotic marker) in gut-associated lymphoid tissue (GALT) of the ileum from calves inoculated with a non-cytopathic strain of BVDV genotype-1. CCasp8 had similar expression to that of CCasp3. In interfollicular T-cell areas there was moderate apoptosis and evidence of moderate activation of initiator caspase-8. In B-cell follicles there was marked lymphocyte apoptosis and evidence of intense caspase-8 activation, highlighting the potentially major role of the extrinsic pathway in lymphocyte apoptosis in the GALT during BVDV infection. Additionally, there was a significant decrease in the number of CCasp9(+) cells from the start of the experiment and this was linked to inactivation of caspase-9. Therefore, the intrinsic pathway may play only a minor role in the induction of lymphocyte apoptosis. Finally, the observed overexpression of Bcl-2 protein could play a major role in protecting lymphocytes in the T-cell areas against apoptosis, while low levels of Bcl-2 expression could be associated with the follicular lymphocyte apoptosis occurring during BVDV infection.

T cell responses in experimental viral retinitis: Mechanisms, peculiarities and implications for gene therapy with viral vectors

T lymphocytes play a decisive role in the course and clinical outcome of viral retinal infection. This review focuses on aspects of the adaptive cellular immune response against viral pathogens in the retina. Two distinct models to study adaptive cell mediated immune responses in viral retinitis are presented: (i) experimental retinitis induced by murine cytomegalovirus (MCMV), where the immune system prevents necrotizing damage to the retina and (ii) retinitis induced by the non-cytopathic lymphocytic choriomeningitis virus (LCMV), where the retinal microanatomy is compromised not by the virus, but by the immune response itself. From these studies it is clear that, in the context of viral infections, the cytotoxic T cell response against a pathogen in the retina does not differ from that seen in other organs, and that once such a response has been initiated, clearing of virus from retinal tissue has priority over preservation of retinal architecture and function. Furthermore, implications drawn from these models for gene therapy in retinal diseases are discussed.

Development of Defective and Persistent Sendai Virus Vector: A UNIQUE GENE DELIVERY/EXPRESSION SYSTEM IDEAL FOR CELL REPROGRAMMING

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research.

Clinical Implications of Chemokines in Acute and Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV), a non-cytopathic positive-stranded RNA virus, is one of the most common causes of chronic liver diseases such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Upon HCV infection, the majority of patients fail to clear the virus and progress to chronic hepatitis C. Chemokines are small chemotactic cytokines that direct the recruitment of immune cells and coordinate immune responses upon viral infection. Chemokine production during acute HCV infection contributes to the recruitment of immune cells with antiviral effector functions and subsequent viral clearance. In chronic HCV infection, however, continuous production of chemokines due to persistent viral replication might result in incessant recruitment of inflammatory cells to the liver, giving rise to persistence of chronic inflammation and liver injury. In this review, we will summarize the roles of chemokines in acute and chronic settings of HCV infection and the clinical relevance of chemokines in the treatment of hepatitis C.

Maternal and Fetal Response to Fetal Persistent Infection with Bovine Viral Diarrhea Virus*

Infection of naïve pregnant cows with non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) results in transplacental infection of the fetus. Infection of the pregnant cow with ncp BVDV late in gestation (after day 150) results in transient infection (TI), as both the dam and fetus can mount an immune response to the virus. In contrast, if the fetus is infected with ncp BVDV early in gestation (before day 150), the fetal immune system is undeveloped and unable to recognize the virus as foreign. This results in induction of immune tolerance to the infecting BVDV strain and persistent infection (PI). Infection of naïve pregnant heifers with ncp BVDV2 on day 75 was hypothesized to induce differential gene expression in white blood cells of the dams and their fetuses, adversely affecting development and antiviral immune responses in PI fetuses. Gene expression differed in maternal blood cells in the presence of PI versus uninfected fetuses. PI adversely affected fetal development and antiviral responses, despite protective immune responses in the dam. Fetal PI with BVDV alters maternal immune function, compromises fetal growth and immune responses, and results in expression of maternal blood biomarkers that can be used to identify cows carrying PI fetuses.

Innate Antiviral Immune Responses to Hepatitis B Virus

Hepatitis B virus (HBV) is a major cause of acute and chronic hepatitis in humans. As HBV itself is currently viewed as a non-cytopathic virus, the liver pathology associated with hepatitis B is mainly thought to be due to immune responses directed against HBV antigens. The outcome of HBV infection is the result of complex interactions between replicating HBV and the immune system. While the role of the adaptive immune response in the resolution of HBV infection is well understood, the contribution of innate immune mechanisms remains to be clearly defined. The innate immune system represents the first line of defense against viral infection, but its role has been difficult to analyze in humans due to late diagnosis of HBV infection. In this review, we discuss recent advances in the field of innate immunity to HBV infection.

Quantitation of virus using laser-based scanning of near-infrared fluorophores replaces manual plate reading in a virus titration assay

A method was developed for quantitation of a virus titration assay for minimally cytopathic and noncytopathic viruses that utilizes laser-based scanning of near-infrared (NIR) fluorophores. This automated method bypasses the need for manual plate reading thus eliminating human bias and error. The image data is translated by LI-COR's Odyssey software into numerical data which is used directly in the virus titer calculations.

The Host–pathogen Interaction during HBV Infection: Immunological Controversies

HBV is a hepatotropic and non-cytopathic virus that causes more than one million deaths annually from liver cirrhosis and hepatocellular carcinoma. As the virus itself is non-cytopathic, it is widely accepted that both viral control and liver pathology are mediated by the host immune system. Until recently, the focus has been on the crucial role of adaptive immune responses in controlling HBV infection, but the potential contribution of the innate system is now an important area of controversy. Unanswered questions include whether and when HBV can trigger components of innate immunity, and whether HBV can actively suppress the induction of innate immunity. We discuss the data available from animal models and human HBV infection addressing the role of innate immunity in the first part of this review. In the second part, we address the immunopathogenesis of the inflammatory events that characterize chronic hepatitis B. The mechanisms thought to be responsible for liver inflammation, namely the intrahepatic recruitment of inflammatory cells, which is orchestrated by chemokines, have been described; however, the underlying immunological triggers are much less clear. The prevailing idea is that liver inflammation results from a recovery of HBV-specific T-cells directly causing liver injury, but this scenario is supported by scanty experimental data. By contrast, recent findings raise the possibility of a contribution from innate components, such as natural killer cells.

Molecular characterization of RNA and protein synthesis during a one-step growth curve of bovine viral diarrhoea virus in ovine (SFT-R) cells

The aim of this study was to determine the kinetics of noncytopathic bovine viral diarrhoea virus (BVDV) multiplication and synthesis of BVDV specific RNA and proteins in ovine cells (SFT-R) during a one-step growth curve. The virus titre and RNA level were determined by focus-forming assay and real time RT-PCR. The RNA synthesis was detected by Northern blot while synthesis of E2 and NS3 proteins was assayed by immunohistochemistry and Western blot. The results showed that synthesis of viral RNA is initiated at 4h, NS3 and E2 proteins are detectable at 6-7h and the replication cycle is complete at 10-12h. Additionally, we provide evidence that NS2-3 protein was cleaved in ovine cells early during infection and in proliferated leukocytes of acutely infected sheep. This study showed that synthesis of BVDV RNA and proteins in ovine cells occurs at similar times as found in bovine cells.

The molecular basis of the failed immune response in chronic HBV: Therapeutic implications

There is a pressing need to develop new immunotherapeutic interventions in chronic hepatitis B virus (HBV) infection in order to limit the high costs and risks of toxicity or viral resistance associated with maintenance antiviral treatment. Here we review recent advances in our understanding of the molecular defects underlying the profound T cell depletion characteristic of these patients. We propose that T cells are driven to apoptosis by the combination of a persistent, high antigen load and excessive inhibitory signals encountered in the hepatic microenvironment. The feasibility of boosting sustained antiviral control by targeted reversal of key tolerising mechanisms is discussed.

Non-Cytopathic Bovine Viral Diarrhea Virus Infection Inhibits Differentiation of Bovine Neural Stem/progenitor Cells into Astrocytes in Vitro

Bovine viral diarrhea virus (BVDV) causes fetal brain malformations in ruminants when the fetuses are infected transplacentally in mid-pregnancy. In both cytopathic and non-cytopatic virus infections, viral lytic infection in actively replicating cells and interruption of vascular integrity have been suggested as the pathogenesis, but functional disturbance of infected neural developing cells has been unclear. In this study, we examined the effect of infection with non-cytopathic BVDV2 on the differentiation of neural stem/precursor cells isolated from the bovine fetus. In the process of differentiation to three types of neural cells, neurons, astrocytes and oligodendrocytes, virus infection significantly and selectively inhibited the differentiation of neural stem/precursor cells into the astrocytic lineage. This inhibition is possibly important for the pathogenesis of congenital brain malformations associated with non-cytopathic BVDV infection.

Hepatitis B Virus Activates Deoxynucleotide Synthesis in Nondividing Hepatocytes by Targeting the R2 Gene

Hepatitis B virus (HBV) causes liver diseases from acute hepatitis to cirrhosis and liver cancer. Currently, more than 350 million people are chronic HBV carriers, with devastating prognosis. HBV is a small enveloped noncytopathic virus, containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells. Infected individuals (acute and chronic) secrete about 10(7) to 10(11) virions per day to the bloodstream, with each infected cell releasing 50-300 viruses per day. HBV infects nondividing hepatocytes and replicates by reverse-transcribing the pregenomic RNA to DNA in the host cells. The level of deoxyribonucleotide triphosphates (dNTPs) in nondividing cells is too low to support viral replication and enable the high yield of secreted virions. Here, we report production of dNTPs by viral-dependent transcription activation of R2, the key component of ribonucleotide reductase (RNR), and show that this process is critical for the HBV life-cycle. This was found in an established HBV-positive cell line and was reproduced by HBV DNA-transduced cells, in both culture and mice. Furthermore, the viral hepatitis B X protein is essential in activating R2 expression by blocking access of Regulatory factor x1, a repressor of the R2 gene. Our findings demonstrate that the hepatitis B X protein is critical in infecting nonproliferating hepatocytes, which contain a low dNTP level. In addition, we provide molecular evidence for a new mechanism of HBV-host cell interaction where RNR-R2, a critical cell-cycle gene, is selectively activated in nonproliferating cells. This mechanism may set the stage for formulating a new category of anti-HBV drugs.