Extracellular Vesicles in Flaviviridae Pathogenesis: Their Roles in Viral Transmission, Immune Evasion, and Inflammation.

The members of the Flaviviridae family are becoming an emerging threat for public health, causing an increasing number of infections each year and requiring effective treatment. The consequences of these infections can be severe and include liver inflammation with subsequent carcinogenesis, endothelial damage with hemorrhage, neuroinflammation, and, in some cases, death. The mechanisms of Flaviviridae pathogenesis are being actively investigated, but there are still many gaps in their understanding. Extracellular vesicles may play important roles in these mechanisms, and, therefore, this topic deserves detailed research. Recent data have revealed the involvement of extracellular vesicles in steps of Flaviviridae pathogenesis such as transmission, immune evasion, and inflammation, which is critical for disease establishment. This review covers recent papers on the roles of extracellular vesicles in the pathogenesis of Flaviviridae and includes examples of clinical applications of the accumulated data.

Potential for Protein Kinase Pharmacological Regulation in Flaviviridae Infections.

Protein kinases (PKs) are enzymes that catalyze the transfer of the terminal phosphate group from ATP to a protein acceptor, mainly to serine, threonine, and tyrosine residues. PK catalyzed phosphorylation is critical to the regulation of cellular signaling pathways that affect crucial cell processes, such as growth, differentiation, and metabolism. PKs represent attractive targets for drugs against a wide spectrum of diseases, including viral infections. Two different approaches are being applied in the search for antivirals: compounds directed against viral targets (direct-acting antivirals, DAAs), or against cellular components essential for the viral life cycle (host-directed antivirals, HDAs). One of the main drawbacks of DAAs is the rapid emergence of drug-resistant viruses. In contrast, HDAs present a higher barrier to resistance development. This work reviews the use of chemicals that target cellular PKs as HDAs against virus of the Flaviviridae family (Flavivirus and Hepacivirus), thus being potentially valuable therapeutic targets in the control of these pathogens.

Iminosugars: A host-targeted approach to combat Flaviviridae infections.

N-linked glycosylation is the most common form of protein glycosylation and is required for the proper folding, trafficking, and/or receptor binding of some host and viral proteins. As viruses lack their own glycosylation machinery, they are dependent on the host's machinery for these processes. Certain iminosugars are known to interfere with the N-linked glycosylation pathway by targeting and inhibiting α-glucosidases I and II in the endoplasmic reticulum (ER). Perturbing ER α-glucosidase function can prevent these enzymes from removing terminal glucose residues on N-linked glycans, interrupting the interaction between viral glycoproteins and host chaperone proteins that is necessary for proper folding of the viral protein. Iminosugars have demonstrated broad-spectrum antiviral activity in vitro and in vivo against multiple viruses. This review discusses the broad activity of iminosugars against Flaviviridae. Iminosugars have shown favorable activity against multiple members of the Flaviviridae family in vitro and in murine models of disease, although the activity and mechanism of inhibition can be virus-specfic. While iminosugars are not currently approved for the treatment of viral infections, their potential use as future host-targeted antiviral (HTAV) therapies continues to be investigated.

Undersampling: case studies of flaviviral inhibitory activities.

Imbalanced datasets, comprising of more inactive compounds relative to the active ones, are a common challenge in ligand-based model building workflows for drug discovery. This is particularly true for neglected tropical diseases since efforts to identify therapeutics for these diseases are often limited. In this report, we analyze the performance of several undersampling strategies in modeling the Dengue Virus 2 (DENV2) inhibitory activity, as well as the anti-flaviviral activities for the West Nile (WNV) and Zika (ZIKV) viruses. To this end, we build datasets comprising of 1218 (159 actives and 1059 inactives), 1044 (132 actives and 912 inactives) and 302 (75 actives and 227 inactives) molecules with known DENV2, WNV and ZIKV inhibitory activity profiles, respectively. We develop ensemble classifiers for these endpoints and compare the performance of the different undersampling algorithms on external sets. It is observed that data pruning algorithms yield superior performance relative to data selection algorithms. The best overall performance is provided by the one-sided selection algorithm with test set balanced accuracy (BACC) values of 0.84, 0.74 and 0.77 for the DENV2, WNV and ZIKV inhibitory activities, respectively. For the model building, we use the recently proposed GT-STAF information indices, and compare the predictivity of 3 molecular fragmentation approaches: connected subgraphs, substructure and alogp atom types, which are observed to show comparable performance. On the other hand, a combination of indices based on these fragmentation strategies enhances the predictivity of the built ensembles. The built models could be useful for screening new molecules with possible DENV, WNV and ZIKV inhibitory activities. ADMET modelers are encouraged to adopt undersampling algorithms in their workflows when dealing with imbalanced datasets.

Whole-genome sequencing of human Pegivirus variant from an Egyptian patient co-infected with hepatitis C virus: a case report.

BACKGROUND: Human pegivirus (HPgV) is structurally similar to hepatitis C virus (HCV) and was discovered 20 years ago. Its distribution, natural history and exact rule of this viral group in human hosts remain unclear. Our aim was to determine, by deep next-generation sequencing (NGS), the entire genome sequence of HPgV that was discovered in an Egyptian patient while analyzing HCV sequence from the same patient. We also inspected whether the co-infection of HCV and HPgV will affect the patient response to HCV viral treatment. To the best of our knowledge, this is the first report for a newly isolated HPgV in an Egyptian patient who is co-infected with HCV. CASE PRESENTATION: The deep Next Generation Sequencing (NGS) technique was used to detect HCV sequence in hepatitis C patient's plasma. The results revealed the presence of HPgV with HCV. This co-infection was confirmed using conventional PCR of the HPgV 5' untranslated region. The patient was then subjected to direct-acting-antiviral treatment (DAA). At the end of the treatment, the patient showed a good response to the HCV treatment (i.e., no HCV-RNA was detected in the plasma), while the HPgV-RNA was still detected. Sequence alignment and phylogenetic analyses demonstrated that the detected HPgV was a novel isolate and was not previously published. CONCLUSION: We report a new variant of HPgV in a patient suffering from hepatitis C viral infection.

The Small-Compound Inhibitor K22 Displays Broad Antiviral Activity against Different Members of the Family Flaviviridae and Offers Potential as a Panviral Inhibitor.

The virus family Flaviviridae encompasses several viruses, including (re)emerging viruses which cause widespread morbidity and mortality throughout the world. Members of this virus family are positive-strand RNA viruses and replicate their genome in close association with reorganized intracellular host cell membrane compartments. This evolutionarily conserved strategy facilitates efficient viral genome replication and contributes to evasion from host cell cytosolic defense mechanisms. We have previously described the identification of a small-compound inhibitor, K22, which exerts a potent antiviral activity against a broad range of coronaviruses by targeting membrane-bound viral RNA replication. To analyze the antiviral spectrum of this inhibitor, we assessed the inhibitory potential of K22 against several members of the Flaviviridae family, including the reemerging Zika virus (ZIKV). We show that ZIKV is strongly affected by K22. Time-of-addition experiments revealed that K22 acts during a postentry phase of the ZIKV life cycle, and combination regimens of K22 together with ribavirin (RBV) or interferon alpha (IFN-α) further increased the extent of viral inhibition. Ultrastructural electron microscopy studies revealed severe alterations of ZIKV-induced intracellular replication compartments upon infection of K22-treated cells. Importantly, the antiviral activity of K22 was demonstrated against several other members of the Flaviviridae family. It is tempting to speculate that K22 exerts its broad antiviral activity against several positive-strand RNA viruses via a similar mechanism and thereby represents an attractive candidate for development as a panviral inhibitor.

Flaviviridae virus nonstructural proteins 5 and 5A mediate viral immune evasion and are promising targets in drug development.

Infections with viruses in the Flaviviridae family have a vast global and economic impact because of the high morbidity and mortality. The pathogenesis of Flaviviridae infections is very complex and not fully understood because these viruses can inhibit multiple immune pathways including the complement system, NK cells, and IFN induction and signalling pathways. The non-structural (NS) 5 and 5A proteins of Flaviviridae viruses are highly conserved and play an important role in resisting host immunity through various evasion mechanisms. This review summarizes the strategies used by the NS5 and 5A proteins of Flaviviridae viruses for evading the innate immune response by inhibiting pattern recognition receptor (PRR) signalling pathways (TLR/MyD88, IRF7), suppressing interferon (IFN) signalling pathways (IFN-γRs, STAT1, STAT2), and impairing the function of IFN-stimulated genes (ISGs) (e.g. protein kinase R [PKR], oligoadenylate synthase [OAS]). All of these immune evasion mechanisms depend on the interaction of NS5 or NS5A with cellular proteins, such as MyD88 and IRF7, IFN-αRs, IFN-γRs, STAT1, STAT2, PKR and OAS. NS5 is the most attractive target for the discovery of broad spectrum compounds against Flaviviridae virus infection. The methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities of NS5 are the main therapeutic targets for antiviral drugs against Flaviviridae virus infection. Based on our site mapping, the sites involved in immune evasion provide some potential and promising targets for further novel antiviral therapeutics.

Innate Immune Evasion Mediated by Flaviviridae Non-Structural Proteins.

Flaviviridae-caused diseases are a critical, emerging public health problem worldwide. Flaviviridae infections usually cause severe, acute or chronic diseases, such as liver damage and liver cancer resulting from a hepatitis C virus (HCV) infection and high fever and shock caused by yellow fever. Many researchers worldwide are investigating the mechanisms by which Flaviviridae cause severe diseases. Flaviviridae can interfere with the host's innate immunity to achieve their purpose of proliferation. For instance, dengue virus (DENV) NS2A, NS2B3, NS4A, NS4B and NS5; HCV NS2, NS3, NS3/4A, NS4B and NS5A; and West Nile virus (WNV) NS1 and NS4B proteins are involved in immune evasion. This review discusses the interplay between viral non-structural Flaviviridae proteins and relevant host proteins, which leads to the suppression of the host's innate antiviral immunity.

Evaluation of Viremia Frequencies of a Novel Human Pegivirus by Using Bioinformatic Screening and PCR.

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.

Current approaches in antiviral drug discovery against the Flaviviridae family.

Viruses belonging to the Flaviviridae family primarily spread through arthropod vectors, and are the major causes of illness and death around the globe. The Flaviviridae family consists of 3 genera which include the Flavivirus genus (type species, yellow fever virus) as the largest genus, the Hepacivirus (type species, hepatitis C virus) and the Pestivirus (type species, bovine virus diarrhea). The flaviviruses (Flavivirus genus) are small RNA viruses transmitted by mosquitoes and ticks that take over host cell machinery in order to propagate. However, hepaciviruses and pestiviruses are not antropod-borne. Despite the extensive research and public health concern associated with flavivirus diseases, to date, there is no specific treatment available for any flavivirus infections, though commercially available vaccines for yellow fever, Japanese encephalitis and tick-born encephalitis exist. Due to the global threat of viral pandemics, there is an urgent need for new drugs. In many countries, patients with severe cases of flavivirus infections are treated only by supportive care, which includes intravenous fluids, hospitalization, respiratory support, and prevention of secondary infections. This review discusses the strategies used towards the discovery of antiviral drugs, focusing on rational drug design against Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Yellow Fever virus (YFV) and Hepatitis C virus (HCV). Only modified peptidic, nonpeptidic, natural compounds and fragment-based inhibitors (typically of mass less than 300 Da) against structural and non-structural proteins are discussed.

Natural compounds against flaviviral infections.

Arthropod borne flaviviral diseases are a major public health concern in the tropics. However, the majority of cases are associated with Dengue virus (DENV), Yellow Fever virus (YFV), West Nile virus (WNV) and Chikungunya virus (CHIKV) infections. Despite their profound clinical and economic impact among large sections of the population there is a lack of effective treatment against these diseases. A large number of plants are available in nature, which may act as a source for lead molecules against various diseases including arthropod borne flaviviral infections. In this review we discuss various crude extracts as well as purified compounds from natural sources with promising anti-DENV, YFV, WNV and CHIKV activity.

Influence of hepatitis G virus (GB virus C) on the prognosis of HIV-infected women.

This study was undertaken to evaluate the prevalence of GB virus C (GBV-C) viraemia and anti-E2 antibody, and to assess the effect of co-infection with GBV-C and HIV during a 10-year follow-up of a cohort of 248 HIV-infected women. Laboratory variables (mean and median CD4 counts, and HIV and GBV-C viral loads) and clinical parameters were investigated. At baseline, 115 women had past exposure to GBV-C: 57 (23%) were GBV-C RNA positive and 58 (23%) were anti-E2 positive. There was no statistical difference between the groups (GBV-C RNA + /anti-E2 - , GBV-C RNA - /anti-E2 + and GBV-C RNA - /anti-E2 - ) regarding baseline CD4 counts or HIV viral loads (P = 0.360 and 0.713, respectively). Relative risk of death for the GBV-C RNA + /anti-E2 - group was 63% lower than that for the GBV-C RNA - /anti-E2 - group. Multivariate analysis demonstrated that only HIV loads ≥ 100,000 copies/mL and AIDS-defining illness during follow-up were associated with shorter survival after AIDS development. It is likely that antiretroviral therapy (ART) use in our cohort blurred a putative protective effect related to the presence of GBV-C RNA.

2,3-Dihydro-1,2-Diphenyl-substituted 4H-Pyridinone derivatives as new anti Flaviviridae inhibitors.

With the aim of identifying novel lead compounds active against emergent human infectious diseases, a series of 2,3-dihydro-4H-pyridinone derivatives has been prepared and evaluated for antiviral activity. Compounds were evaluated in vitro in cell-based assays for cytotoxicity and against a wide spectrum of viruses. In the antiviral screening, several compounds showed to be fairly active against viruses belonging to the Flaviviridae family. The Pestiviruses (bovine viral diarrhoea virus) were inhibited by 4a cis (CC(50) > 100 µm; EC(50) = 14 µm), compounds 4c cis and 6a showed a significant activity against Flaviviruses (Yellow Fever Virus) (CC(50) > 100 µm; EC(50) = 18µm, CC(50) > 100 µm; EC(50) = 10 µm). Among these, compound 6a displayed great inhibitory activity against Hepaciviruses (hepatitis C virus) in replicon assay [CC(50) > 100 µm; EC(50) (1b) = 4 µm]. In vitro inhibitory activity against the HCV RNA-dependent RNA polymerase (NS5B) of title compounds is discussed. The antiviral screening of viral strains indicated that compound 6a can be selected as promising tool in novel anti-flaviviruses development.

Vaccination and antiviral treatment of neglected diseases caused by flaviviral infections.

Flaviviral infections have a re-emerging impact on the health situation in developing countries with several billions of people living at risk. In the present review, we focus on three members of the genus Flavivirus belonging to the Flaviviridae family. They are transmitted to humans by mosquito bites, namely those viruses leading to Dengue Fever, Yellow Fever and mosquito-borne Japanese encephalitis. All three virus groups have a spherical structure with a diameter of approximately 50 nm. Although sharing a similar genomic structure and intracellular life cycle, they show different clinical manifestations. Infections are incurable, as there is no antiviral treatment available for either of the three viruses. Thus, prevention and vaccination are the best defenses. The most promising vaccines are live attenuated vaccines (LAVs), such as the YF17D strain against Yellow Fever or the SA-14-14-2 strain against Japanese encephalitis. Additionally, recombinant vaccines for Japanese encephalitis are in development. Although Dengue Fever is the most prevalent arthropode-borne flaviviral infection and a lot of research to develop a vaccine against all four Dengue Fever serotypes was undertaken, no vaccine is available on the market yet. Promising tetravalent vaccine candidates are currently undergoing clinical phase trials, including LAVs, recombinant and chimeric candidates as well as non-replicating vaccine approaches. Additionally, encouraging anti-flaviviral approaches target non-structural proteins, virus-specific proteases essential for cellular maturation of viral particles. Peptide inhibitors against the highly conserved NS2B and NS3 proteases are attractive as pan-flaviviral drug candidates.

Prevalence and clinical significance of GB virus type C/hepatitis G virus coinfection in patients with chronic hepatitis C undergoing antiviral therapy.

Coinfection with GBV-C/HGV in patients with chronic hepatitis C (CHC) may influence clinical course and response rates of antiviral therapy. Aim of the study was to investigate the prevalence of GBV-C/HGV/HCV coinfection and its influence on outcome of interferon/ribavirin combination therapy. Three hundred and four patients with CHC [m/f = 211/93, age: 42 (18-65)] were investigated. HGV RNA detection was performed by polymerase chain reaction prior to and 6 months after the end of antiviral therapy. HGV/HCV coinfection could be identified in 37/304 (12.2%) patients with intravenous drug abuse as the most common source of infection (N = 21, (56.8%)). The predominant HCV genotype in coinfected individuals was HCV-3a (HCV-3a: 51.4%, HCV-1: 37.8%, HCV-4: 10.8%). HGV coinfection was more prevalent in patients infected with HCV-3 compared to HCV-1 or HCV-4 [19/45 (42.2%) vs. 14/185 (7.6%) vs. 4/52 (7.7%), P < 0.01]. Patients with HGV/HCV coinfection were younger [35 (18-56) vs. 43 (19-65), years; P < 0.01], and advanced fibrosis (F3-F4) was less frequent (22.2% vs. 42.9%, P < 0.05). A sustained virological response was achieved more frequently in HGV/HCV coinfected patients [26/37 (70.3%)] than in monoinfected patients [120/267 (44.9%), P < 0.01]. HGV RNA was undetectable in 65.7% of the coinfected patients at the end of follow-up. Intravenous drug abuse seems to be a major risk factor for HGV coinfection in patients with chronic hepatitis C. Coinfection with HGV does not worsen the clinical course of chronic hepatitis C or diminish response of HCV to antiviral therapy. Interferon/ribavirin combination therapy also clears HGV infection in a high proportion of cases.

Xanthohumol enhances antiviral effect of interferon alpha-2b against bovine viral diarrhea virus, a surrogate of hepatitis C virus.

Xanthohumol (XN) is a natural compound with multifunctional potentials, including antiviral activity. In this study, the antiviral activity of addition of XN to interferon (IFN)-alpha was examined and compared with each compound alone using bovine viral diarrhea virus (BVDV), a surrogate model of hepatitis C virus (HCV). BVDV E2 protein and the viral RNA level were determined by immunofluorescence and quantitative real-time RT-PCR, respectively. The addition of XN to IFN-alpha significantly improved CPEs induced by the virus and inhibited BVDV E2 protein and viral RNA levels. The interaction between XN and IFN-alpha was significant (P<0.001). XN at 3.13 microg/ml in combination with IFN-alpha at 50 IU/ml showed greater inhibitory effect on the viral RNA level than each compound used alone at 6.25 microg/ml and 100 IU/ml, respectively, indicating synergistic effect on BVDV replication in this combination. The inhibitory activity in all the tested combinations of XN and IFN-alpha was stronger than that of each compound used alone at the corresponding concentration. These results suggest that XN in combination with IFN-alpha exhibited a greater in vitro antiviral effect compared with each compound used alone. Further studies are deserved to investigate the anti-HCV activity of XN and the potential of XN in formulating novel anti-HCV regimen.

New drug targets for hepatitis C and other Flaviviridae viruses.

The Flaviviridae family comprises the genus Flavivirus, Hepacivirus and Pestivirus. These viruses are responsible for considerable human and animal disease and mortality worldwide. Flaviviruses cause a range of acute febrile illnesses along with encephalitic or haemorrhagic diseases. Chronic hepatitis C virus (HCV) infection is the most important hepacivirus human disease and remains a global health threat with nearly 200 million carriers worldwide. Current treatment consists in the use of peginterferon alfa (pegIFN) plus ribavirin (RBV) for 24 to 72 weeks, depending on HCV genotype, baseline viral load and the achievement of rapid virological response during therapy. However, current hepatitis C therapy fails to eradicate HCV in nearly half of treated patients and is hampered by relatively serious adverse events. No effective antiviral therapy is currently available for the treatment of flaviviruses or pestiviruses. Following the relative success of antiretroviral therapy against HIV infection, rapid progresses have been made in the development of specifically targeted antiviral therapies against HCV (STAT-C) and other Flaviviridae agents. Drug discovery for HCV is currently particularly exciting, since inhibitors of the HCV serine protease and the RNA-dependent RNA polymerase have recently entered the late stages of clinical development.

Low frequency of GB virus C viremia in a cohort of HIV-1-infected elite suppressors.

The persistence of GB virus C viremia in patients with chronic HIV-1 infection has been associated with increased survival. Elite suppressors are untreated HIV-1-infected patients who maintain viral loads of below 50 copies/ml. This study found that the frequency of GB virus C viremia in elite suppressors and chronically infected patients with progressive disease was not significantly different. Thus, GB virus C does not appear to explain the nonprogressive course seen in this cohort of elite suppressors.

Prevalence and impact of GBV-C, SEN-V and HBV occult infections in HIV-HCV co-infected patients on HCV therapy.

BACKGROUND/AIMS: It has been suggested that, in HIV-HCV co-infected patients, co-infections with other viruses may affect the response to HCV therapy. We aimed to assess the prevalence of GBV-C, SEN-V and occult HBV infections, their impact on HCV and HIV infections and on the response to HCV therapy in HIV-HCV co-infected patients. METHODS: Three-hundred and sixty eight patients were tested before starting interferon-ribavirin for the presence of occult hepatitis B DNA, GBV-C RNA and SEN-V DNA by using real time PCR. Clinical, immunological, virological, histological characteristics and response to HCV therapy were compared according to the presence or not of each viral co-infection. RESULTS: HBV DNA, GBV-C RNA and SEN-V DNA were found in 5 (1.4%, CI95%: 0.2-2.4%), 104 (29.9%, CI95%: 25.1-34.7%) and 209 patients (57.9%, CI95%: 52.8-63.0%), respectively. GBV-C positive patients had significantly higher CD4 count at baseline, during and after HCV therapy, even after stratification on antiretroviral treatment. No other significant difference was observed according to the presence or not of GBV-C or SEN-V co-infection, in particular regarding virological responses to HCV combination therapy. CONCLUSIONS: There is no reason to withhold HCV therapy in HIV infected patients who have access to HAART, because of occult HBV, GBV-C or SEN-V co-infections.

Impact of GB virus type C infection on mother-to-child HIV transmission in the Women and Infants Transmission Study Cohort.

BACKGROUND: GB virus type C (GBV-C) viraemia is associated with a beneficial outcome in HIV-infected individuals in several though not all studies. GBV-C viraemia was examined in a matched case-control study of 133 HIV-infected pregnant women who transmitted HIV to their infants ('cases') and 266 non-transmitting controls. METHODS: HIV-infected children and controls were pair-matched for high-risk delivery, race and year of delivery. GBV-C status was determined in maternal plasma samples obtained at or within 3 months of delivery. RESULTS: Pregnant women with GBV-C viraemia (11% of those studied) had lower HIV RNA levels (P=0.01) and higher CD4 percentages (P=0.0006) [corrected] than women without GBV-C. A trend towards decreased mother-to-child transmission in the multivariate analysis was observed among GBV-C viraemic women delivering after highly active antiretroviral therapy (HAART) became available [odds ratio (OR) 0.30, 95% confidence interval (CI) 0.08-1.05; P=0.06], but not in women delivering prior to the widespread use of HAART. CONCLUSIONS: GBV-C viraemia was associated with a beneficial effect on CD4 percentage and HIV RNA level in these pregnant women, and was also associated with a trend towards reduced risk of mother-to-child HIV transmission among women after HAART became available. Further studies with larger or multiple cohorts are necessary to assess possible benefits in this population.