Methods for assessing the tolerance and resistance of different types of cocoa to cocoa swollen-shoot virus

Different types of cocoa were compared for their resistance to infection and tolerance of infection with the virulent strain A of cocoa swollen-shoot virus, inoculated either by mealybugs or manually. The method of infection did not affect the ranking of the cocoa types for resistance, except at high percentages of infection, or for tolerance. In comparison with other methods, manual inoculation simplifies the screening of cocoa types for resistance and tolerance to virus infection, and provides a more sensitive test for resistance.     

Natural history and treatment of hepatitis B virus and hepatitis C virus coinfection

Hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection is not uncommon as a result of similar routes of infection. Patients who are coinfected represent a unique group with diverse serologic profiles. Combined chronic hepatitis B and C leads to more severe liver disease and an increased risk of hepatocellular carcinoma. Furthermore, coinfected patients represent a treatment challenge. No standard recommendations exist for treatment of viral hepatitis due to dual HBV/HCV infection, and therefore treatment must be individualized based on patient variables such as serologic and virologic profiles, patient's prior exposure to antiviral treatment, and the presence of other parenterally transmitted viruses such as hepatitis D virus and human immunodeficiency virus. The natural history and treatment of patients with HBV and HCV coinfection is reviewed.     

Novel mutations in a tissue culture-adapted hepatitis C virus strain improve infectious-virus stability and markedly enhance infection kinetics

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 × 10(8) 50% tissue culture infective doses (TCID(50))/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.     

Epstein-Barr virus LF2: an antagonist to type I interferon

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway, which is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Despite its association with significant human health problems, activities of Epstein-Barr virus (EBV), a human tumor-inducing herpesvirus, to evade host IFN-mediated innate immunity have not been well characterized. To search for EBV genes that block IFN signal transduction, we carried out a screening of EBV open reading frames for their abilities to block IFN-α/β-mediated luciferase expression upon Sendai virus infection. This screening demonstrates that EBV LF2 tegument protein specifically interacts with the central inhibitory association domain of IRF7, and this interaction leads to inhibition of the dimerization of IRF7, which suppresses IFN-α production and IFN-mediated immunity. This demonstrates a novel immune evasion mechanism of EBV LF2 in blocking cellular IRF7-mediated innate immunity.     

Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor

HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4''-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside.     

Seoul virus infection increases aggressive behaviour in male Norway rats

In natural populations of rodents, males are more likely to engage in aggression and be infected with hantaviruses than females. Whether the relationship between hantavirus infection and aggression is due to host- or parasite-mediated mechanisms is unknown. The aim of this study was to determine whether hantavirus infection causes an increase in aggression in male rats and whether these behavioural changes are due to infection of the central nervous system or peripheral tissues. Male laboratory rats were infected with Seoul virus and tested for aggression in a resident-intruder paradigm 15 and 30 days postinoculation (p.i.). Males tested 30 days p.i. (i.e. during the persistent phase of infection) spent more time engaged in aggression than either uninfected males or males tested during the acute phase of infection (i.e. 15 days p.i.). Males that engaged in aggression for a longer duration had more virus present in lung, kidney and testis than males that spent less time engaged in aggression. Infected males shed virus in saliva, faeces, and urine; virus shedding, however, was not correlated with aggression and neither wounding nor transmission of virus to intruder males occurred during behavioural tests. Infection with Seoul virus did not alter either testosterone or corticosterone concentrations. Seoul virus antigens were not detected in the brains of infected rats. These data suggest that hantavirus infection leads to elevated aggression in infected males and may be a by-product of increased virus replication in peripheral tissues.     

Age-related differences in amplification of covalently closed circular DNA at early times after duck hepatitis B virus infection of ducks

Inoculation of 3-day-old (3D) or 3-week-old (3W) ducklings with duck hepatitis B virus results in chronic or transient infection, respectively. We previously showed that rapid production of neutralizing antibody following inoculation of 3W ducklings prevents virus from spreading in the liver and leads to a transient infection (Y.-Y. Zhang and J. Summers, J. Virol. 78:1195-1201, 2004). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses.     

Pathology of Equine Influenza virus (H3N8) in Murine Model

Equine influenza viruses (EIV)—H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×10^6.24 EID₅₀ with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.     

Analysis of the virus dynamics model reveals that early treatment of HCV infection may lead to the sustained virological response

Considerable progress has been made towards understanding hepatitis C virus, its pathogenesis and the effect of the drug therapy on the viral load, yet around 50% of patients do not achieve the sustained virological response (SVR) by the standard treatment. Although several personalized factors such as patients' age and weight may be important, by mathematical modeling we show that the time of the start of the therapy is a significant factor in determining the outcome. Toward this end, we first performed sensitivity analysis on the standard virus dynamics model. The analysis revealed four phases when the sensitivity of the infection to drug treatment differs. Further, we added a perturbation term in the model to simulate the drug treatment period and predict the outcome when the therapy is carried out during each of the four phases. The study shows that while the infection may be difficult to treat in the late phases, the therapy is likely to result in SVR if it is carried out in the first or second phase. Thus, development of newer and more sensitive screening methods is needed for the early detection of the infection. Moreover, the analysis predicts that the drug that blocks new infections is more effective than the drug that blocks the virus production.     

A microfluidic cell chip for virus isolation via rapid screening for permissive cells

Virus identification is a prerequisite not only for the early diagnosis of viral infectious diseases but also for the effective prevention of epidemics. Successful cultivation is the gold standard for identifying a virus, according to the Koch postulates. However, this requires screening for a permissive cell line, which is traditionally time-, reagent- and labor-intensive. Here, a simple and easy-to-operate microfluidic chip, formed by seeding a variety of cell lines and culturing them in parallel, is reported for use in virus cultivation and virus-permissive host-cell screening. The chip was tested by infection with two known viruses, enterovirus 71 (EV71) and influenza virus H1N1. Infection with EV71 and H1N1 caused significant cytopathic effects (CPE) in RD and MDCK cells, respectively, demonstrating that virus cultivation based on this microfluidic cell chip can be used as a substitute for the traditional plate-based culture method and reproduce the typical CPE caused by virus infection. Using this microfluidic cell chip method for virus cultivation could make it possible to identify an emerging virus in a high-throughput, automatic, and unprecedentedly fast way.     

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis

West Nile virus (WNV), a mosquito-borne single-stranded (ss)RNA flavivirus, causes human disease of variable severity. We investigated the involvement of Toll-like receptor (Tlr) 3, which recognizes viral double-stranded (ds)RNA, on WNV infection. Tlr3-deficient (Tlr3(-/-)) mice were more resistant to lethal WNV infection and had impaired cytokine production and enhanced viral load in the periphery, whereas in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type mice. Peripheral WNV infection led to a breakdown of the blood-brain barrier and enhanced brain infection in wild-type but not in Tlr3(-/-) mice, although both groups were equally susceptible upon intracerebroventricular administration of the virus. Tumor necrosis factor-alpha receptor 1 signaling is vital for blood-brain barrier compromise upon Tlr3 stimulation by dsRNA or WNV. Collectively, WNV infection leads to a Tlr3-dependent inflammatory response, which is involved in brain penetration of the virus and neuronal injury.     

Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.     

Synthesis and anti-viral activity of a series of sesquiterpene lactones and analogues in the subgenomic HCV replicon system

Hepatitis C virus (HCV) infection is a severe liver disease that often leads to liver cirrhosis and hepatocellular carcinoma (HCC). Current therapy is inadequate to conquer this viral disease. In this study, we identified parthenolide (1), an active component in feverfew, a popular remedy for fever and migraine, as a lead compound with an EC50 value of 2.21 microM against HCV replication in a subgenomic RNA replicon assay system. Parthenolide is able to potentiate the interferon alpha-exerted anti-HCV effect. Several commercially available sesquiterpene lactones (2-5) structurally analogous to parthenolide and a series of synthesized Michael-type adducts of parthenolide (12-18) also exhibit micromolar concentrations for anti-HCV activities. Structure-activity relationship was elucidated to reveal that the spatial arrangement of the terpenoid skeleton fused with an alpha-methylene-gamma-lactone moiety produces maximal anti-HCV activity. In addition, a strong anti-HCV potency indicates a possibility of secondary amino adducts (12-18) converting back to parthenolide or being replaced by the nucleophilic residues of proteins inside cells. This work shows that screening of natural products is a viable and fast way for identifying novel molecular diversity as potential drug leads.     

Increased hematopoiesis in mice soon after infection by Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV), an erythroleukemogenic replication-competent retrovirus, induces leukemia in its host after a long latency. However, the early effects of infection may determine the pathway that eventually leads to malignant transformation. To determine how F-MuLV affects host cell proliferation soon after infection, BALB/c mice were inoculated with virus and then were assayed for susceptibility to appropriately pseudotyped spleen focus-forming virus (SFFV) as an indicator of erythropoietic activity. Twelve-week-old mice exposed to F-MuLV for 9 days were more susceptible (by a factor of 30) to superinfection by SFFV than were nonviremic mice. To test whether increased susceptibility was the result of increased hematopoietic activity, hematopoietic progenitors from the spleens of F-MuLV-infected mice were enumerated with a clonal culture assay. Nine days after inoculation with F-MuLV, the numbers of colony-forming progenitors increased by a factor of 4. Morphological analysis of the cultured colonies showed that erythroid, granulocytic, monocytic, and mixed granulocytic-monocytic progenitors all had increased. Thus, F-MuLV more rapidly induced a generalized increase in hematopoiesis than has previously been reported. The splenic hyperplasia induced by F-MuLV soon after infection may explain its ability to accelerate leukemogenesis in mice also infected by the polytropic Friend mink cell focus-forming virus.     

On the relative fitness of early and late stage Simian immunodeficiency virus isolates

Simian immunodeficiency virus (SIV) has been shown to evolve from a relatively slowly replicating and mildly cytopathic virus early in the infection (SIVMneCL8) to a faster replicating and more cytopathic virus at later stages of the infection (SIVMne170). It has recently been demonstrated that the early and mildly cytopathic variant SIVMneCL8 out-competed the late and highly cytopathic strain SIVMne170 in cell culture experiments, because the fitness disadvantage derived from the higher cytopathicity was not matched by a sufficient increase in the viral replication rate. However, in another set of experiments where the life span of cells in culture was artificially limited, the late and more cytopathic virus won the competition, because under this condition cytopahticity was not an important determinant of viral fitness. It was hypothesized that the limited life span experiment reflected the immune-mediated high turnover environment in vivo more accurately, and that the presence of immune responses accounts for the selection of the cytopathic strain SIVmne170 during later stages of the infection. This paper investigates the effect of immune responses, in particular cytotoxic T lymphocyte (CTL) responses, on the competition dynamics between these two SIV strains with the help of mathematical models. Model analysis and parameter estimates derived from previously published data on SIV growth kinetics suggest that the SIV-specific CTL response might not be the driving force that leads to the selection of the cytopathic strain SIVMne170 during later stages of the infection. This implies that more complex evolutionary mechanisms might have to be invoked in order to explain the emergence of these strains in vivo. One possibility is that the ability of multiple virus particles to infect the same cell (coinfection) might be a pre-requisite for the emergence of the cytopathic strain SIVMne170 as the disease progresses.     

Microdevices for examining immunological responses of single cells to HIV

More than 60 million people in the world have been diagnosed with HIV infections since the virus was recognized as the causative agent of AIDS in the 1980s. Even though more than half of the infected patients have died, effective disease treatment and prevention measures have not been established. ART (antiretroviral therapy) is the only proven HIV treatment that sustains the suppression of patient viraemia. Current routine approaches to treat HIV infections are targeted at developing vaccines that will induce humoral or cell memory immune responses. However, developing an effective vaccine has been challenging because the HIV mutates rapidly, which allows the virus to evade immune surveillances established against the previous strain. In addition, the virus is able to quickly establish a reservoir and treatment is difficult because of the general lack of knowledge about HIV immune response mechanisms. This review introduces common disease symptoms and the progression of HIV infection with a brief summary of the current treatment approaches. Different cellular immune responses against HIV are also discussed, with emphasis on a nanotechnology research that has focused on probing T-cell response to HIV infection. Furthermore, we discuss recent noteworthy nanotechnology updates on T-cell response screening that is focused on HIV infection. Finally, we review potential future treatment strategies based on the correlations between T-cell response and HIV infection.     

Modeling the role of macrophages in HIV persistence during antiretroviral therapy

HIV preferentially infects activated CD4+ T cells. Current antiretroviral therapy cannot eradicate the virus. Viral infection of other cells such as macrophages may contribute to viral persistence during antiretroviral therapy. In addition to cell-free virus infection, macrophages can also get infected when engulfing infected CD4+ T cells as innate immune sentinels. How macrophages affect the dynamics of HIV infection remains unclear. In this paper, we develop an HIV model that includes the infection of CD4+ T cells and macrophages via cell-free virus infection and cell-to-cell viral transmission. We derive the basic reproduction number and obtain the local and global stability of the steady states. Sensitivity and viral dynamics simulations show that even when the infection of CD4+ T cells is completely blocked by therapy, virus can still persist and the steady-state viral load is not sensitive to the change of treatment efficacy. Analysis of the relative contributions to viral replication shows that cell-free virus infection leads to the majority of macrophage infection. Viral transmission from infected CD4+ T cells to macrophages during engulfment accounts for a small fraction of the macrophage infection and has a negligible effect on the total viral production. These results suggest that macrophage infection can be a source contributing to HIV persistence during suppressive therapy. Improving drug efficacies in heterogeneous target cells is crucial for achieving HIV eradication in infected individuals.

     

Zika virus NS5 protein potential inhibitors: an enhanced in silico approach in drug discovery

The re-emerging Zika virus (ZIKV) is an arthropod-borne virus that has been described to have explosive potential as a worldwide pandemic. The initial transmission of the virus was through a mosquito vector, however, evolving modes of transmission has allowed the spread of the disease over continents. The virus has already been linked to irreversible chronic central nervous system conditions. The concerns of the scientific and clinical community are the consequences of Zika viral mutations, thus suggesting the urgent need for viral inhibitors. There have been large strides in vaccine development against the virus but there are still no FDA approved drugs available. Rapid rational drug design and discovery research is fundamental in the production of potent inhibitors against the virus that will not just mask the virus, but destroy it completely. In silico drug design allows for this prompt screening of potential leads, thus decreasing the consumption of precious time and resources. This study demonstrates an optimized and proven screening technique in the discovery of two potential small molecule inhibitors of ZIKV Methyltransferase and RNA dependent RNA polymerase. This in silico ‘per-residue energy decomposition pharmacophore’ virtual screening approach will be critical in aiding scientists in the discovery of not only effective inhibitors of Zika viral targets, but also a wide range of anti-viral agents.     

Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses

Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3beta and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.