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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-CONH 2 )-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.

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., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Zika virus (ZIKV) has been associated with serious health conditions, and an intense search to discover different ways to prevent and treat ZIKV infection is underway. Berberine and emodin possess several pharmacological properties and have been shown to be particularly effective against the entry and replication of several viruses. We show that emodin and berberine trigger a virucidal effect on ZIKV. When the virus was exposed to 160 µM of berberine, a reduction of 77.6% in the infectivity was observed; when emodin was used (40 µM), this reduction was approximately 83.3%. Dynamic light scattering data showed that both compounds significantly reduce the hydrodynamic radius of virus particle in solution. We report here that berberine and emodin, two natural compounds, have strong virucidal effect in Zika virus.

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.

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.

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.

Total DNA extracted from Lb. plantarum ST8Sh was screened for the presence of more than 50 genes related to production of biogenic amines (histidine decarboxylase, tyrosine decarboxylase, and ornithine decarboxylase), virulence factors (sex pheromones, gelatinase, cytolysin, hyaluronidase, aggregation substance, enterococcal surface protein, endocarditis antigen, adhesion of collagen, integration factors), and antibiotic resistance (vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, bacitracin). Lb. plantarum ST8Sh showed a low presence of virulence genes. Only 13 genes were detected (related to sex pheromones, aggregation substance, adhesion of collagen, tetracycline, gentamicin, chloramphenicol, erythromycin, but not to vancomycin, and bacitracin) and may be considered as indication of safety for application in fermented food products. In addition, interaction between Lb. plantarum ST8Sh and drugs from different groups were determined in order to establish possible application of the strain in combination with commercial drugs. Cytotoxicity of the semi-purified bacteriocins produced by Lb. plantarum ST8Sh was depended on applied concentration-highly cytotoxic when applied at 25 μg/mL and no cytotoxicity at 5 μg/mL.

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.

During ZIKV the outbreak in Brazil it was observed an increase of almost 20 times the number of reported cases of microcephaly in newborn babies. There is no vaccine or approved drug available for the treatment and prevention of infections by this virus. EGCG, a polyphenol present in green tea has been shown to have an antiviral activity for many viruses. In view of the need for the development of a drug against a Brazilian strain of ZIKV, we assessed the effect of EGCG on ZIKV entry in Vero E6 cells. The drug was capable of inhibiting the virus entry by at least 1-log (>90%) at higher concentrations (>100μM). The pre-treatment of cells with EGCG did not show any effect on virus attachment. This is the first study to demonstrate the effect of EGCG on ZIKV indicating that this drug might be possibility to be used for prevention of Zika virus infections., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.

Conjugate compounds constitute a new class of molecules of important biological interest mainly for the treatment of diseases such as cancer. The N-terminus region of cationic peptides has been described as important for their biological activity. The aim of this study was to evaluate the lytic peptide Hecate (FALALKALKKALKKLKKALKKAL) and the effect of conjugating this macromolecule with gallic acid (C7H6O5) in terms of structure, anti-cancer activity, and toxicity. An N-terminus GA-Hecate peptide conjugate was synthesized to provide information regarding the relationship between the amino-terminal region and its charge and the secondary structure and biological activity of the peptide; and the effects of gallic acid on these parameters. Peptide secondary structure was confirmed using circular dichroism (CD). The CD measurements showed that the peptide has a high incidence of α-helical structures in the presence of SDS and LPC, while GA-Hecate presented lower incidence of α-helical structures in the same chemical environment. An evaluation of the anti-cancer activity in HeLa cancer cells indicated that both peptides are active, but that coupling gallic acid at the N-terminus decreased the activity of the free peptide. GA-Hecate showed lower activity in non-tumor keratinocyte cells but higher hemolytic activity. Our findings suggest that the N-terminus of Hecate plays an important role in its activity against cervical cancer by affecting it secondary structure, toxicity, and hemolytic activity. This study highlights the importance of the N-terminus in antitumor activity and could provide an important tool for developing new anti-cancer drugs.

Hepatitis C is considered the major cause of cirrhosis and hepatocellular carcinoma. Conventional treatment is not effective against some hepatitis C virus (HCV) genotypes; therefore, new treatments are needed. Coffee and, more recently, caffeine, have been found to have a beneficial effect in several disorders of the liver, including those manifesting abnormal liver biochemistry, cirrhosis and hepatocellular carcinoma. Caffeine acts directly by delaying fibrosis, thereby improving the function of liver cellular pathways and interfering with pathways used by the HCV replication cycle. In the current study, the direct relationship between caffeine and viral replication was evaluated. The Huh-7.5 cell line was used for transient infections with FL-J6/JFH-5'C19Rluc2AUbi and to establish a cell line stably expressing SGR-Feo JFH-1. Caffeine efficiently inhibited HCV replication in a dose-dependent manner at non-cytotoxic concentrations and demonstrated an IC50 value of 0.7263 mM after 48 h of incubation. These data demonstrate that caffeine may be an important new agent for anti-HCV therapies due to its efficient inhibition of HCV replication at non-toxic concentrations.

In this work, we investigated the effect of supplementing fermented milk with quinoa flour as an option to increase probiotic activity during fermented milk production and storage. Fermented milk products were produced with increasing concentrations of quinoa flour (0, 1, 2, or 3g/100g) and submitted to the following analyses at 1, 14, and 28 d of refrigerated storage: postacidification, bacterial viability, resistance of probiotics to simulated gastrointestinal (GI) conditions, and adhesion of probiotics to Caco-2 cells in vitro. The kinetics of acidification were measured during the fermentation process. The time to reach maximum acidification rate, time to reach pH 5.0, and time to reach pH 4.6 (end of fermentation) were similar for all treatments. Adding quinoa flour had no effect on fermentation time; however, it did contribute to postacidification of the fermented milk during storage. Quinoa flour did not affect counts of Bifidobacterium animalis ssp. lactis BB-12 or Lactobacillus acidophilus La-5 during storage, it did not protect the probiotic strains during simulated GI transit, and it did not have a positive effect on the adhesion of probiotic bacteria to Caco-2 cells in vitro. Additionally, the adhesion of strains to Caco-2 cells decreased during refrigerated storage of fermented milk. Although the addition of up to 3% quinoa flour had a neutral effect on probiotic activity, its incorporation to fermented milk can be recommended because it is an ingredient with high nutritive value, which may increase the appeal of the product to consumers., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Respiratory syncytial virus (RSV) is a major cause of acute respiratory disease in infants and young children. Considering that several aspects of the humoral immune response to RSV infection remain unclear, this study aimed to investigate the occurrence, levels, and avidity of total IgG, IgG1, and IgG3 antibodies against RSV in serum samples from children ≤5 years old. In addition, a possible association between antibody avidity and severity of illness was examined. The occurrence and levels of RSV-specific IgG depended on age, with infants <3 months old displaying high levels of antibodies, which were probably acquired from the mother. Children ≥24 months old also showed frequent occurrence and high levels of IgG, which was produced actively during infection. In addition, the avidity assay showed that the avidity of RSV-specific total IgG and IgG1 was lower in infants <3 months old who had acute respiratory disease than in age-matched controls. The avidity of RSV-specific IgG detected in children ≥24 months old with lower respiratory infection was lower than that in children with upper respiratory infection. These results indicate that the presence of high avidity RSV-specific IgG antibodies may lead to better protection against RSV infection in children <3 months old, who may have a lower probability of developing disease of increased severity. In addition, children ≥24 months old with RSV-specific IgG antibodies of low avidity tended to develop more severe RSV illness. These findings may be helpful in establishing vaccination schedules when a vaccine becomes available., (Copyright © 2011 Wiley-Liss, Inc.)

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 adenoviruses (HAdV) are a major cause of acute respiratory diseases (ARD), gastroenteritis, conjunctivitis and urinary infections. Between November 2000-April 2007, a total of 468 nasopharyngeal aspirate samples were collected from children with ARD at the Clinics Hospital of Uberlândia. These samples were tested by immunofluorescence assay (IFA) and 3% (14/468) tested positive for the presence of HAdV. By performing polymerase chain reaction (PCR) to detect HAdV DNA in samples that tested negative or inconclusive for all viruses identifiable by IFA (respiratory syncytial virus, parainfluenza viruses 1, 2 and 3, influenza viruses A and B and HAdV), as well as negative for rhinoviruses by reverse transcription-PCR, additional 19 cases were detected, for a total of 33 (7.1%) HAdV-positive samples. Nucleotide sequences of 13 HAdV samples were analyzed, revealing that they belonged to species B, C and E. Further analyses showed that species C (HAdV-2) was the most prevalent among the sequenced samples. To our knowledge, this is the first report describing the presence of HAdV-4 in Brazil. We also detected an isolate that was 100% identical to a part of the feline adenovirus hexon gene sequence.

The human metapneumovirus (hMPV) is a pathogen of the respiratory tract identified first in the Netherlands in 2001 and since then it has been detected worldwide. The purpose of this study was to identify and characterize hMPV in samples collected from children <5 years presenting with acute respiratory disease (ARD) seen at a public hospital in Uberlândia, in Southeastern Brazil. One hundred fourteen nasopharyngeal aspirates (NPAs) samples that were negative for the presence of nine other respiratory viruses were tested by reverse transcription polymerase chain reaction (RT-PCR) for the presence of hMPV RNA. Fourteen out of 114 (12.3%) samples were positive for presence of hMPV RNA. PCR products, obtained by the amplification of partial nucleotide sequence of gene N, were sequenced and compared with sequences deposited in GenBank. Sequences from eight samples were obtained and all four subtypes were identified. Also, the recently proposed sublineages "a" and "b" of subtype A2 were found; mean age was 21 months old; upper respiratory tract infection (URTI) was the most common clinical symptom; the virus was detected in samples collected from March to November, a period that corresponds to late summer to mid-spring in Brazil. This is the first study to describe the circulation of all hMPV subtypes in Minas Gerais state.