Antiviral Evaluation of New Synthetic Bioconjugates Based on GA-Hecate: A New Class of Antivirals Targeting Different Steps of Zika Virus Replication.

Re-emerging arboviruses represent a serious health problem due to their rapid vector-mediated spread, mainly in urban tropical areas. The 2013-2015 Zika virus (ZIKV) outbreak in South and Central America has been associated with cases of microcephaly in newborns and Guillain-Barret syndrome. We previously showed that the conjugate gallic acid-Hecate (GA-FALALKALKKALKKLKKALKKAL-CONH2)-is an efficient inhibitor of the hepatitis C virus. Here, we show that the Hecate peptide is degraded in human blood serum into three major metabolites. These metabolites conjugated with gallic acid were synthesized and their effect on ZIKV replication in cultured cells was evaluated. The GA-metabolite 5 (GA-FALALKALKKALKKL-COOH) was the most efficient in inhibiting two ZIKV strains of African and Asian lineage at the stage of both virus entry (virucidal and protective) and replication (post-entry). We also demonstrate that GA-metabolite 5 does not affect cell growth after 7 days of continuous treatment. Thus, this study identifies a new synthetic antiviral compound targeting different steps of ZIKV replication in vitro and with the potential for broad reactivity against other flaviviruses. Our work highlights a promising strategy for the development of new antivirals based on peptide metabolism and bioconjugation.

Early infection of Zika virus in the male reproductive system of AG129 mice: molecular and immunohistochemical evaluation.

Sexual transmission of Zika virus (ZIKV), an important arbovirus, and the virus persistence in semen raise several questions about how and where it circulates in the male reproductive system (MRS). Several studies reported detection of the virus in testes, epididymis, and prostate at 5 days post-infection (dpi) or more in animal models. In the present study, we investigated the interactions of ZIKV with mouse MRS using the AG129 strain, a ZIKV permissive immunodeficient mouse strain, at two dpi. Viral RNA was detected in blood, testes, epididymis, and prostatic complexes (prostate and seminal vesicles). Immunohistochemical (IHC) analyses, based on the envelope protein, showed an early infection in organs of MRS since ZIKV positive antigens were detected in cells within or surrounding blood vessels, Sertoli, and germ cells in testes and epithelial cells in epididymis and prostate. Positive antigens for NS5 protein, the virus RNA-dependent RNA polymerase, were also detected by IHC in these organs and circulating leukocytes, suggesting that the virus replicates in these sites as early as 2 days post-infection. Analysis of the early stages of ZIKV infection in MRS may improve the current knowledge about this issue and contribute to the development of therapies directed to the infection at this site.

30th Brazilian Society for Virology 2019 Annual Meeting-Cuiabá, Mato Grosso, Brazil.

The 30th meeting of the Brazilian Society for Virology (SBV) was held, for the first time in its 30 years of existence, in Cuiabá, the capital of Mato Grosso State, Central Western Brazil, a tropical region between the three richest biomes in the world: Amazon Florest, Cerrado and Pantanal. In recent years, the field of virology has been built in the State. The aim of this report is to support participants and virologists to receive the most up-to-date information about the meeting, which occurred from 16 to 19 October 2019. National and international speakers gave SBV the opportunity to learn about their experience on their virology fields, sharing recent scientific findings, compiling conferences, round table presentations and work presentations in oral and poster sessions. The meeting held over 300 attendants, who were also involved on oral and poster presentations, showing a great variety of recent unpublished studies on environmental, basic, animal, human, plant and invertebrate virology. In addition, SBV offered the Helio Gelli Pereira award for the best research studies in each field presented during the meeting. The 30th meeting of SBV was very productive and has also encouraged scientific partnership and collaboration among virologists worldwide.

Hierarchical assessment of host factors influencing the spontaneous resolution of hepatitis C infection.

BACKGROUND: Hepatitis C virus (HCV) infection is associated with chronic liver disease, resulting in cirrhosis and hepatocellular carcinoma. Approximately 20% of HCV infections are spontaneously resolved. Here, we assessed the hierarchical relevance of host factors contributing to viral clearance. METHODS: DNA samples from 40 resolved infections and 40 chronic HCV patients paired by age were analyzed. Bivariate analysis was performed to rank the importance of each contributing factor in spontaneous HCV clearance. RESULTS: Interestingly, 63.6% of patients with resolved infections exhibited the protective genotype CC for SNP rs12979860. Additionally, 59.3% of patients with resolved infections displayed the protective genotype TT/TT for SNP ss469415590. Moreover, a ranking of clearance factors was estimated. In order of importance, the IL28B CC genotype (OR 0.197, 95% CI 0.072-0.541) followed by the INFL4 TT/TT genotype (OR 0.237, 95% CI 0.083-0.679), and female gender (OR 0.394, 95% CI 0.159-0.977) were the main predictors for clearance of HCV infection. CONCLUSIONS: HCV clearance is multifactorial and the contributing factors display a hierarchical order. Identifying all elements playing role in HCV clearance is of the most importance for HCV-related disease management. Dissecting the relevance of each contributing factor will certainly improve our understanding of the pathogenesis of HCV infection.

GA-Hecate antiviral properties on HCV whole cycle represent a new antiviral class and open the door for the development of broad spectrum antivirals.

In recent years, synthetic peptides have been considered promising targets for drug development that possess low side-effects, are cost-effective and are susceptible to rational design. Hecate was initially described as a potent bacterial inhibitor and subsequently as an anticancer drug with functions related to its lipid interaction property. Viruses, such as hepatitis C virus (HCV), have a lipid-dependent life cycle and could be affected by Hecate in many ways. Here, we assessed modifications on Hecate's N-terminus region and its effects on HCV and hepatotoxicity. Gallic acid-conjugated Hecate was the most efficient Hecate-derivative, presenting high potential as an antiviral and inhibiting between 50 to 99% of all major steps within the HCV infectious cycle. However, the most promising aspect was GA-Hecate's mechanism of action, which was associated with a balanced lipid interaction with the viral envelope and lipid droplets, as well as dsRNA intercalation, allowing for the possibility to affect other ssRNA viruses and those with a lipid-dependent cycle.

Heat shock proteins HSPB8 and DNAJC5B have HCV antiviral activity.

Hepatitis C is a disease caused by the hepatitis C virus (HCV), and an estimated 3% of the world population is infected with the virus. During replication, HCV interacts with several cellular proteins. Studies have shown that several heat shock proteins (HSPs) have an altered expression profile in the presence of the virus, and some HSPs interact directly with HCV proteins. In the present study, we evaluated the expression levels of heat shock proteins in vitro in the presence and absence of HCV. The differential expression of 84 HSPs and chaperones was observed using a qPCR array, comparing HCV uninfected and infected Huh7.5 cells. To validate qPCR array, the differentially expressed genes were tested by real-time PCR in three different HCV models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all three viral models. We silenced HSPB8 expression and observed an increase in viral replication. In contrast, when we increased the expression of HSPB8, a decrease in the HCV replication rate was observed. The same procedure was adopted for DNAJC5B, and HCV showed a similar replication pattern as that observed for HSPB8. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and that DNAJC5B has the same function, with more relevant results for genotype 3. We also evaluated the direct interactions between HCV and HSP proteins, and the IP experiments showed that the HCV NS4B protein interacts with HSPB8. These results contribute to a better understanding of the mechanisms involved in HCV replication.

Inhibition of hepatitis C virus using siRNA targeted to the virus and Hsp90.

Hepatitis C (HCV) is a viral disease affecting millions of people worldwide, and persistent HCV infection can lead to progressive liver disease with the development of liver cirrhosis and hepatocellular carcinoma. During treatment for hepatitis C, the occurrence of viral resistance is common. To reduce the occurrence of resistance, new viral treatments should target both viral and cellular factors. Many interactions occur between viral and host proteins during the HCV replication cycle and might be used for the development of new therapies against hepatitis C. Heat shock protein 90 (Hsp90) plays a role in the folding of cellular and viral proteins and also interacts with HCV proteins. In the present study, we knocked down the expression of the Hsp90 gene and inhibited viral replication using siRNA molecules. Reducing the expression of Hsp90 successfully decreased HCV replication. All siRNA molecules specific to the viral genome showed the efficient inhibition of viral replication, particularly siRNA targeted to the 5'UTR region. The combination of siRNAs targeting the viral genome and Hsp90 mRNA also successfully reduced HCV replication and reduced the occurrence of viral resistance. Moreover, these results suggest that an approach based on the combination of cellular and viral siRNAs can be used as an effective alternative for hepatitis C viral suppression.

Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent.

Human rhinoviruses (HRV) are usually associated with mild respiratory symptoms in children. However, some studies have found that HRV can cause severe disease, especially when the patient is co-infected with a second virus. In this study, 532 nasopharyngeal aspirates (NPAs) were collected over a nine-year period from children at the Clinics Hospital of Uberlândia. The collected NPAs were then tested for HRV RNA using the reverse transcription-polymerase chain reaction. Eighty-three specimens from children diagnosed with lower respiratory tract illness (LRTI) were positive for HRV RNA and were then tested for the presence of eight other respiratory viruses. A second virus was detected in 37.3% (31/83) of the samples. The most frequent clinical diagnosis was bronchiolitis, followed by other LRTI and then pneumonia. The frequency of severe disease in children infected with more than one virus was not significantly different from the frequency of severe disease in children infected with HRV alone. Children infected with both HRV and parainfluenza virus (1.5 m.o.) were significantly younger than those infected by HRV alone (5.0 m.o.) (p = 0.0454). Overall, these results suggest that infection with a second virus does not lead to a higher frequency of severe syndromes in children presenting with LRTI.

Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent

Human rhinoviruses (HRV) are usually associated with mild respiratory symptoms in children. However, some studies have found that HRV can cause severe disease, especially when the patient is co-infected with a second virus. In this study, 532 nasopharyngeal aspirates (NPAs) were collected over a nine-year period from children at the Clinics Hospital of Uberlândia. The collected NPAs were then tested for HRV RNA using the reverse transcription-polymerase chain reaction. Eighty-three specimens from children diagnosed with lower respiratory tract illness (LRTI) were positive for HRV RNA and were then tested for the presence of eight other respiratory viruses. A second virus was detected in 37.3 percent (31/83) of the samples. The most frequent clinical diagnosis was bronchiolitis, followed by other LRTI and then pneumonia. The frequency of severe disease in children infected with more than one virus was not significantly different from the frequency of severe disease in children infected with HRV alone. Children infected with both HRV and parainfluenza virus (1.5 m.o.) were significantly younger than those infected by HRV alone (5.0 m.o.) (p = 0.0454). Overall, these results suggest that infection with a second virus does not lead to a higher frequency of severe syndromes in children presenting with LRTI.