The Synthetic Peptide GA-Hecate and Its Analogs Inhibit Multiple Steps of the Chikungunya Virus Infection Cycle In Vitro.

Chikungunya virus (CHIKV) belongs to the Alphavirus genus and is responsible for significant outbreaks worldwide. Currently, there is no approved antiviral therapy against CHIKV. Bioactive peptides have great potential for new drug development. Here, we evaluated the antiviral activity of the synthetic peptide GA-Hecate and its analogs PSSct1905 and PSSct1910 against CHIKV infection. Initial screening showed that all three peptides inhibited the CHIKV replication cycle in baby hamster kidney fibroblast cells (BHK-21) and human hepatocarcinoma epithelial cells (Huh-7). GA-Hecate and its analog PSSct1905 were the most active, demonstrating suppression of viral infection by more than 91%. The analog PSSct1905 exhibited a protective effect in cells against CHIKV infection. We also observed that the analogs PSSct1905 and PSSct1910 affected CHIKV entry into both cell lines, inhibiting viral attachment and internalization. Finally, all tested compounds presented antiviral activity on the post-entry steps of CHIKV infection in all cells evaluated. In conclusion, this study highlights the potential of the peptide GA-Hecate and its analogs as novel anti-CHIKV compounds targeting different stages of the viral replication cycle, warranting the development of GA-Hecate-based compounds with broad antiviral activity.

The Dimeric Peptide (KKYRYHLKPF)2K Shows Broad-Spectrum Antiviral Activity by Inhibiting Different Steps of Chikungunya and Zika Virus Infection.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are important disease-causing agents worldwide. Currently, there are no antiviral drugs or vaccines approved to treat these viruses. However, peptides have shown great potential for new drug development. A recent study described (p-BthTX-I)2K [(KKYRYHLKPF)2K], a peptide derived from the Bothropstoxin-I toxin in the venom of the Bothrops jararacussu snake, showed antiviral activity against SARS-CoV-2. In this study, we assessed the activity of this peptide against CHIKV and ZIKV and its antiviral action in the different stages of the viral replication cycle in vitro. We observed that (p-BthTX-I)2K impaired CHIKV infection by interfering with the early steps of the viral replication cycle, reducing CHIKV entry into BHK-21 cells specifically by reducing both the attachment and internalization steps. (p-BthTX-I)2K also inhibited the ZIKV replicative cycle in Vero cells. The peptide protected the cells against ZIKV infection and decreased the levels of the viral RNA and the NS3 protein of this virus at viral post-entry steps. In conclusion, this study highlights the potential of the (p-BthTX-I)2K peptide to be a novel broad-spectrum antiviral candidate that targets different steps of the replication cycle of both CHIKV and ZIKV.

Detection of DENV-2 and ZIKV coinfection in southeastern Brazil by serum and urine testing.

PURPOSE: Aedes aegypti mosquito-borne diseases have a significant impact on public health in Brazil. In this study, we investigated the presence of the Zika virus (ZIKV) and dengue virus (DENV) in serum and urine samples from symptomatic participants who attended an Emergency Care Unit located in a city in the northwestern region of São Paulo between February 2018 and April 2019. METHODS: Serum and urine samples were collected from participants suspected of having arbovirus infection. After the extraction of viral RNA, viral detection was performed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) (One-Step RT-qPCR). RESULTS: A total of 305 participants participated in this study. A total of 283 blood and 270 urine samples were collected. Of 305 patients, 36.4% (111/305) were positive for ZIKV, 43.3% (132/305) for DENV2, and 0.3% (1/305) for DENV1. Coinfection with ZIKV/DENV2 was observed in 13.1% of participants. If only serum samples were used, ZIKV detection would have decreased to 23.3% (71/305). Of all the participants included in the study, only one was suspected of having ZIKV infection based on clinical diagnosis, and the remaining participants were suspected of having DENV. CONCLUSION: By testing serum and urine samples, we increased the detection of both viruses and detected considerable levels of ZIKV and DENV-2 coinfection when compared to other studies. Additionally, we detected an unnoticed ZIKV outbreak in the city. These findings highlight the importance of the molecular diagnosis of arboviruses to aid public health surveillance and management strategies.

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests.

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Detection of Zika virus in urine from randomly tested individuals in Mirassol, Brazil.

PURPOSE: Studies show that around 80% of Zika virus (ZIKV) infections are asymptomatic. The present study tested urine samples from volunteers, unsuspected of arboviral infection, which attended an emergency care unit (ECU) in Mirassol, Brazil, from March 2018 to April 2019. METHODS: The volunteers were divided into two groups. The first group was composed of outpatients who were not suspected to have an arbovirus infection. This first group was subdivided into two subgroups: outpatients with and without arbovirus-like symptoms. The second group consisted of companions of outpatients treated at the ECU. The second group was also subdivided into two subgroups: totally asymptomatic individuals and those who had arbovirus-like symptoms. RNA was extracted from urine samples, followed by RT-qPCR for ZIKV. RESULTS: We found that 11% (79/697) of the samples tested positive for ZIKV-RNA. Among the ZIKV-RNA-positive individuals, 16.5% (13/79) were companions, of which 61.5% (8/13) were totally asymptomatic and 38.5% (5/13) reported symptoms that could be suggestive of arbovirus infection. In addition, 83.5% (66/79) of the ZIKV-RNA-positive individuals were outpatients without a clinical diagnosis of arbovirus. Of these undiagnosed ZIKV-RNA-positive outpatients, 47% (31/66) had no arbovirus-related symptoms. CONCLUSION: Our study shows the effectiveness of urine as a non-invasive sample to detect the incidence of ZIKV infection. We also highlight the importance of ZIKV molecular diagnosis to aid public health surveillance and prevention of congenital Zika syndrome and other ZIKV-associated diseases.

Biochemical characteristics and potential application of a novel ethanol and glucose-tolerant ß-glucosidase secreted by Pichia guilliermondii G1.2.

ß-glucosidases are glycoside hydrolases that-particularly those from filamentous fungi-have been extensively explored in cellulose fiber saccharification and wine quality improvement. However, these enzymes from yeast have been poorly studied. In this study, an ethanol-glucose tolerant ß-glucosidase that is secreted by Pichia guilliermondii (current name Meyerozyma guilliermondii) was purified and characterized. This enzyme exhibited an estimated molecular mass of 97 kDa and the highest activity between pH 3.5-5.5 and 55 °C. The ß-glucosidase was also tolerant to acetone, ethanol, isopropanol, and methanol up to 30% and glucose at 1 M. It was also stable up to 55 °C for 80 min, maintaining 70% of its initial activity and in a wide pH range (pH 3-10). The enzyme exhibited 90-100% of its initial activity for 72 h at 20, 25, and 30 °C in presence of 10% ethanol at pH 3.5, which is a similar condition to winemaking. Studies that identify new enzymes and describe their purification are required for oenology applications. The ß-glucosidase described herein is a promising candidate for use in the preparation of wine. Additionally, its tolerance to glucose is an important biochemical property that adds value to this enzyme and enables it to be used during the final saccharification process.