Congenital CMV infection in a Brazilian neonatal intensive care unit: high prevalence among twin newborns.

BACKGROUND: Cytomegalovirus (CMV) is one of the most important pathogens associated with congenital infection worldwide. Most congenital CMV-infected infants are asymptomatic at birth; however, some can develop delayed sequelae, especially hearing loss. METHODS: This study aimed to investigate the prevalence of congenital CMV infection in a neonatal intensive care unit in a low-income region of Brazil. The objectives extended to identifying associated factors, assessing the clinical status of infected newborns, and undertaking a two-year follow-up to discern potential long-term consequences in the affected infants. This cross-sectional prospective study enrolled newborns up to three weeks of life requiring intensive medical care. We employed a convenience sampling method to include 498 newborns and 477 mothers in the study. Categorical variables underwent analysis employing Fisher's exact test, whereas the examination of continuous variables involved the Mann‒Whitney test. RESULTS: CMV DNA was detected in saliva/urine samples from 6 newborns (1.21%), confirming congenital infection. We noted a significantly greater incidence (OR: 11.48; 95% CI: 2.519-52.33; p = 0.0094) of congenital infection among twins (7.14%) than among nontwins (0.66%). The twin patients exhibited discordant infection statuses, suggesting that only one of the babies tested positive for CMV. Most of the infected children were born to mothers who initiated sexual activity at a younger age (p = 0.0269). Only three out of the six newborns diagnosed with CMV infection underwent comprehensive clinical assessments and received continuous follow-up until they reached two years of age. Only one of the children had weight and height measurements below the norm for their age, coupled with developmental delays. CONCLUSIONS: The prevalence of congenital CMV infection among newborns admitted to the NICU was low and similar to that in the general population. However, we found a significantly greater incidence of congenital CMV infection in twins than in singletons. Interestingly, the twin-infected patients exhibited discordant infection statuses, suggesting that CMV was present in only one of the babies. We also found that most of the infected children were born to mothers who initiated sexual activity at a younger age. Diagnostic accessibility and comprehensive surveillance programs are imperative for effectively managing and preventing congenital CMV infections.

Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential.

Revisiting the dengue epidemic of 2011 in Paraguay: molecular epidemiology of dengue virus in the Asuncion metropolitan area.

BACKGROUND: Dengue is one of the most important re-emerging viral diseases and the most common human arthropod-borne viral infection worldwide. Any of the four Dengue virus serotypes (DENV-1 to 4) can cause asymptomatic infections or clinical manifestations that range in severity from a mild, self-limited illness, to a severe disease characterized by a shock syndrome that can lead to death. Paraguay suffers periodic epidemic outbreaks of dengue since 1988 when the DENV-1 was introduced in the country. Epidemics caused by all four serotypes have been reported and the country. Although dengue is endemic in Paraguay, few studies have described the molecular epidemiology of DENV in the country, which is important to understand the local and global spread, as well as the evolution of this pathogen. METHODS: This was a cross-sectional study of a convenience sample. Suspected dengue patients of any age were recruited from the Emergency Laboratory of the Central Hospital of the Institute of Social Welfare, Asuncion, Paraguay, from February to June of 2011. A DENV antigen test was used to confirm the infection. The protein E gene sequences of isolated viruses were sequenced for phylogenetic analysis. RESULTS: Dengue was confirmed in 55.1% of the participants (n = 98/178). The most frequent clinical findings were fever, headache, and myalgia. Identity analyses of the protein E gene sequence of 56 viruses isolated showed the circulation of DENV-1 (n = 45) and DENV-2 (n = 11) in the Asuncion metropolitan area in 2011. Molecular epidemiology analyses suggest that DENV-1 was introduced into Paraguay from Argentina, while the DENV-2 from Brazil, replacing previous virus lineages. CONCLUSIONS: We have analyzed the molecular epidemiology of DENV-1 and DENV-2 isolated in Paraguay in 2011. We found strong evidence that DENV-1 was introduced into Paraguay from Argentina, while the DENV-2 from Brazil, replacing previous virus lineages. Molecular epidemiology studies are of great interest to analyze the dynamic of DENV spread, which are useful for early implementation of containment measures to reduce the risk of explosive epidemics caused by this virus.

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

Corrigendum to "Immunocompetent Mice Model for Dengue Virus Infection".

[This corrects the article DOI: 10.1100/2012/525947.].

Genetic characterization of Cacipacoré virus from ticks collected in São Paulo State, Brazil.

Cacipacoré virus (CPCV) is a potential emerging virus classified in the genus Flavivirus, family Flaviviridae. In the present study, we present the genetic characterization of a CPCV isolated from ticks (Amblyomma cajennense) collected from a sick capybara (Hydrochoerus hydrochaeris) in São Paulo State, Brazil. The CPCV isolate shares the typical genomic organization of flaviviruses with 10,857 nucleotides in length and a single open reading frame of 10,284 nucleotides encoding a polyprotein of 3,427 amino acids. Phylogenetic analysis revealed that CPCV is unique, as a potentially tick-borne virus, in the Japanese encephalitis virus serogroup.

High prevalence of clinically unsuspected dengue disease among children in Ribeirao Preto city, Brazil.

The aim of this study was to analyze the characteristics of Dengue virus (DENV)-infected children and the accuracy of dengue diagnosis based on clinical presentations. The inclusion criteria were children ≥1-year-old presenting febrile illness with 1-7 days of onset. Children (n = 110) aged 2-15 years were included in this study. DENV infection was confirmed with virological tests using serum, salvia, and/or urine samples. The attending pediatricians classified 56/110 (50.91%) of the children as suspected dengue cases. The DENV infection was confirmed by specific laboratory tests in 52/56 (92.9%) of the suspected dengue cases but also in 44/54 (81.5%) of the unsuspected dengue cases; total of 96/110 (87.27%) confirmed dengue cases. The clinical diagnosis gave an overall sensitivity of 54.2% (52/96) and a specificity of 71.4% (10/14). The positive predictive value of the clinical diagnosis was 92.8% and negative predictive value was 18.5%. After the third day of onset of symptoms, the DENV genome detection rate was similar in serum and saliva samples, suggesting that saliva samples represent an alternative to blood samples for early dengue diagnosis. Vaccination against Yellow fever virus did not influence the antibody response against DENV-1, DENV-2, and DENV-3, which circulated during the study period. Although the signs and symptoms were compatible with dengue, the attending pediatricians did not suspect the disease in several children. Therefore, the inclusion of virological tests for early diagnosis in the protocols for dengue surveillance would help in the implementation of prompt treatment of patients and epidemic containment strategies. J. Med. Virol. 88:1711-1719, 2016. © 2016 Wiley Periodicals, Inc.

A real-time RT-PCR for rapid detection and quantification of mosquito-borne alphaviruses.

Mosquito-borne alphaviruses are widely distributed throughout the world, causing important human illnesses. Therefore, the development of methods to enable early diagnosis of infections by alphavirus is essential. We show here the development and evaluation of a quantitative real-time RT-PCR using genus-specific primers to the nsP1 viral gene of all mosquito-borne alphaviruses. The specificity and sensitivity of the assay were tested using seven alphaviruses and RNA transcribed from Venezuelan equine encephalitis virus. The detection limits of real-time RT-PCR ranged from 10 to 76 copies per ml. The melting temperature (TM) values for amplification of the alphavirus genomes were 83.05 °C and 85.28 °C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with TM peaks of 84.83 to 85.28 °C and encephalitic alphaviruses of 83.34 °C to 84.68 °C could be discriminated both diseases. Real-time RT-PCR may prove very useful for the screening and preliminary diagnosis in outbreaks and surveillance studies as well as for measuring the viral load in pathogenesis studies.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus.

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Differential replicative ability of clinical dengue virus isolates in an immunocompetent C57BL/6 mouse model.

BACKGROUND: Several experimental animal models have been used to study the pathogenesis of dengue disease; however, most of the studies used laboratory-adapted viruses, which lack the virulence of viruses circulating in humans. The aim of this study was to analyze the ability of clinical Dengue virus (DENV) isolates (D2/BR/RP/RMB/09 and D3/BR/SL3/02) to infect immunocompetent C57BL/6 mice. METHODS: Two strategies of intraperitoneal infection, which were based on the concept of the antibody dependent enhancement phenomenon, were used. In one strategy, the animals were inoculated with macrophages infected in vitro with dengue viruses, which were incubated with enhancing antibodies, and in the other strategy, the animals were inoculated with a complex of enhancing antibodies and dengue viruses. RESULTS: The D3/BR/SL3/08 isolate showed a higher ability of infection (virus RNA was more frequently detected in the serum and in several organs) in the experimental model compared to both the D2/BR/RP/RMB/2009 isolate and a laboratory adapted DENV-1 strain (Mochizuki strain), regardless of the infection strategy used. The main features of the D3/BR/SL3/08 isolate were its neuroinvasiveness and the induction of an extended period of viremia. Enhancing antibodies did not influence on the infection of animals when macrophages were used, but the level of viremia was increased when they were used as a complex with a D3/BR/SL3/02 isolate. DISCUSSION: We showed that DENV isolates could infect immunocompetent C57BL/6 mice, which have has been previously used to study some aspect of dengue disease when infected with laboratory adapted strains. DENV genome was detected in the same organs found in humans when autopsy and biopsy samples were analyzed, showing that C57BL/6 mice reproduce some aspects of the DENV tropism observed in humans. The main difference observed between the D3/BR/SL3/02 and D2/BR/RP/RMB/2009 clinical isolates was the neuroinvasive ability of the first one. Neuroinvasiveness has been described in some DENV infected cases and is common for other members of the Flavivirus genus. CONCLUSIONS: These results suggest that C57BL/6 mice can be used as an experimental model to evaluate virulence differences among DENV clinical isolates.

Detection of DENV-4 genotype I from mosquitoes collected in the city of Manaus, Brazil.

BACKGROUND: Dengue epidemics have been reported in Brazil since 1981. In Manaus, a large city in the Amazon region, dengue is endemic with all four-virus serotypes (DENV-1, -2, -3, and -4) simultaneously causing human disease. In 2008, during a surveillance of dengue virus in mosquitoes in the district of Tancredo Neves in Manaus, 260 mosquitoes of Aedes genus were captured, identified and grouped into pools of 10 mosquitoes. FINDINGS: RNA extracts of mosquito pools were tested by a RT-Hemi-Nested-PCR for detection of flaviviruses. One amplicon of 222 bp, compatible with dengue virus serotype 4, was obtained from a pool of Aedes aegypti. The nucleotide sequence of the amplicon indicated that the mosquitoes were infected with DENV-4 of genotype I. This virus of Asian origin has been described in Manaus in 2008 infecting acute febrile illness patients. CONCLUSION: This is the first report of dengue virus serotype 4 genotype I infecting Aedes aegypti in the Americas.

Performance evaluation of Biofire Film Array Respiratory Panel 2.1 for SARS-CoV-2 detection in a pediatric hospital setting.

The highly contagious nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), requires rapid diagnostic tests to prevent the virus from spreading within hospitals and communities. Reverse transcription followed by the polymerase chain reaction (RT-PCR) test is the gold standard for detecting SARS-CoV-2 infections but is time-consuming, labor-intensive, and restricted to centralized laboratories. There is a growing need to develop and implement point-of-care and rapid tests for SARS-CoV-2 detection to address these limitations. We aimed to evaluate the performance of BioFire Film Array Respiratory Panel 2.1 (BioFire FA-RP2.1) for SARS-CoV-2 detection in a pediatric hospital setting. The BioFire FA-RP2.1 test provides rapid results and can identify several viral and bacterial infections in a single test. This prospective, cross-sectional, diagnostic accuracy study enrolled participants ranging from 0 to 18 years of age, seeking medical consultation for any reason, who had been in contact with individuals confirmed to have COVID-19 or managed at the hospital for medical or surgical reasons. We employed a systematic sampling technique to ensure a representative sample. The study included 339 participants with a median age of 5 years. The BioFire FA-RP2.1 test detected SARS-CoV-2 in 18.6% of cases, while the reference RT-PCR test in 14% of cases. The BioFire FA-RP2.1 sensitivity and specificity for SARS CoV-2 detection were 98% and 94%, respectively. The positive probability coefficient (LR+) was 18. The agreement between the two tests was 0.80. In addition, the BioFire FA-RP2.1 test detected coinfection with two viruses in 7,6% of cases. The BioFire FA-RP2.1 is a reliable solution to meet pediatric healthcare needs and improve prognosis in the post-pandemic era thanks to its friendly interface and rapid testing process.

Linear epitope mapping in the E and NS1 proteins of dengue and Zika viruses: Prospection of peptides for vaccines and diagnostics.

The arrival of the Zika virus (ZIKV) in dengue virus (DENV)-endemic areas has posed challenges for both differential diagnosis and vaccine development. Peptides have shown promise in addressing these issues. The aim of this study was to identify the linear epitope profile recognized by serum samples from dengue and Zika patients in the E and NS1 proteins of DENV and ZIKV. This cross-sectional study included individuals of all ages with laboratory-confirmed DENV and ZIKV infections, who were selected through convenience sampling. The serum samples from dengue and Zika patients detected epitopes evenly distributed across the viral proteins in a peptide microarray platform. However, several epitopes were located within "epitope hotspots", characterized by clusters of peptides recognized in more than 30% of the sub-arrays analyzed using individual or pooled serum samples. The serum samples from dengue and Zika patients showed a high level of cross-reactivity with peptides in the DENV and ZIKV proteins. Analysis using an additional peptide microarray platform, which contained peptides selected based on the results of the initial screening, revealed that two DENV and one ZIKV peptide, highly specific to their related viruses, were located within the epitope hotspots; however, they presented low detection rates (32.5, 35.0, and 28.6%, respectively). In addition, two DENV peptides detected at similarly high rates by both dengue and Zika patients were also found within the epitope hotspots. These hotspots contain several immunodominant epitopes that are recognized by a larger number of individuals when compared to 15-amino acid (aa) sequence peptides. Thus, epitope hotspots may have greater potential to serve as antigens in diagnostic tests and vaccine development than peptides composed of only 15 amino acids.

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and ß-thalassemia major and healthy blood donors.

Human Parvovirus B19 (B19V) is a recognized cause of life-threatening conditions among patients with hemoglobinopathies. This study investigates B19V infection in patients with sickle cell disease and ß-thalassemia using different experimental approaches. A total of 183 individuals (144 with sickle cell disease and 39 with ß-thalassemia major) and 100 healthy blood donors were examined for B19V using anti-B19V IgG enzyme immunoassay, quantitative PCR, DNA sequencing, and phylogenetic analysis. Viremia was documented in 18.6% of patients and 1% of donors, and was generally characterized by low viral load (VL); however, acute infections were also observed. Anti-B19V IgG was detected in 65.9% of patients with sickle cell disease and in 60% of donors, whereas the patients with thalassemia exhibited relatively low seroreactivity. The seroprevalence varied among the different age groups. In patients, it progressively increased with age, whereas in donors it reached a plateau. Based on partial NS1 fragments, all isolates detected were classified as subgenotype 1A with a tendency to elicit genetically complex infections. Interestingly, quasispecies occurred in the plasma of not only patients but also donors with even higher heterogeneity. The partial NS1 sequence examined did not exhibit positive selection. Quantitation of B19V with a conservative probe is a technically and practically useful approach. The extensive spread of B19V subgenotype 1A in patients and donors and its recent introduction into the countryside of the São Paulo State, Brazil were demonstrated; however, it is difficult to establish a relationship between viral sequences and the clinical outcomes of the infection.

Detection of dengue virus in sera of Brazilian blood donors.

BACKGROUND: Dengue is the most important arboviral disease in the world. Dengue viruses (DENVs) have produced huge outbreaks in Brazil in the past 25 years with more than 5 million reported cases. During these epidemics, asymptomatic individuals infected with DENV could donate blood and serve as a source of virus dissemination in the community. Here, we studied the circulation of DENV in healthy individuals during an epidemic outbreak. STUDY DESIGN AND METHODS: The study included 500 serum samples from healthy blood donors collected at the Hemotherapy Center of Ribeirão Preto, Brazil, during a dengue outbreak. The presence of DENV RNA in the serum samples was screened by real-time reverse transcription-polymerase chain reaction (PCR). The virus serotype was determined by a heminested PCR procedure. A partial fragment of the NS5 gene sequence was used for phylogenetic analysis. RESULTS: DENV RNA was detected in the serum sample of 2 of 500 (0.4%) individuals. Both of them were infected with DENV-3 Genotype III, a virus that has been circulating in Brazil in the past decade. CONCLUSION: Individuals with asymptomatic DENV infection can be blood donors and serve as a source of virus dissemination in the community. Further studies are needed to determine the risk of recipient infection by DENV as a result of transfusion in Brazil, especially during epidemic periods.

Phylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics.

BACKGROUND: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. RESULTS: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n = 9) and Paraguay (n = 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. CONCLUSION: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.

Immunocompetent mice model for dengue virus infection [corrected].

Dengue fever is a noncontagious infectious disease caused by dengue virus (DENV). DENV belongs to the family Flaviviridae, genus Flavivirus, and is classified into four antigenically distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The number of nations and people affected has increased steadily and today is considered the most widely spread arbovirus (arthropod-borne viral disease) in the world. The absence of an appropriate animal model for studying the disease has hindered the understanding of dengue pathogenesis. In our study, we have found that immunocompetent C57BL/6 mice infected intraperitoneally with DENV-1 presented some signs of dengue disease such as thrombocytopenia, spleen hemorrhage, liver damage, and increase in production of IFNγ and TNFα cytokines. Moreover, the animals became viremic and the virus was detected in several organs by real-time RT-PCR. Thus, this animal model could be used to study mechanism of dengue virus infection, to test antiviral drugs, as well as to evaluate candidate vaccines.

Linear and Continuous Flavivirus Epitopes From Naturally Infected Humans.

This manuscript is an up-to-date review of experimentally validated linear and continuous epitopes identified from arbovirus members of the Flavivirus genus. We summarized 153 immunoreactive peptides from the Dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus described in studies published from 1989 to 2020. We included peptides from structural (envelope, capsid, and pre-membrane) and nonstructural (Ns1-5) viral proteins that demonstrated relevant immunoreactivity with antibodies from naturally infected or vaccinated humans. We included peptides that demonstrated relevant reactivity features, such as indicators of disease severity related to immunological or immunopathological outcomes, differential or group diagnostic markers, immunotherapy candidates, and potential for vaccine formulation. The majority of immunoreactive peptides were described for DENV probably due to its long-lasting impact on human health and the lack of efficient vaccines and therapeutic methods. Immune landscape data regarding linear immunoreactive and continuous flavivirus peptides are still scarce, and a complete and more detailed map remains to be elucidated. Therefore, this review provides valuable data for those investigating the antibody response against flavivirus infection.

Chikungunya virus entry is strongly inhibited by phospholipase A2 isolated from the venom of Crotalus durissus terrificus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever, a globally spreading mosquito-borne disease. There is no approved antiviral or vaccine against CHIKV, highlighting an urgent need for novel therapies. In this context, snake venom proteins have demonstrated antiviral activity against several viruses, including arboviruses which are relevant to public health. In particular, the phospholipase A2CB (PLA2CB), a protein isolated from the venom of Crotalus durissus terrificus was previously shown to possess anti-inflammatory, antiparasitic, antibacterial and antiviral activities. In this study, we investigated the multiple effects of PLA2CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2CB on the CHIKV replicative cycle, and suggest that PLA2CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells.

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection.

Dengue is the most important arbovirus disease in tropical and sub-tropical countries, and can be caused by infection with any of the four-dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real-time RT-PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real-time RT-PCR for early diagnosis of dengue virus infection.