Development of a New Multispecies Protein A-Based ELISA for Serodiagnosis of Hepatitis E Infection with Validation in Domestic Swine.

Hepatitis E virus (HEV) infects roughly 20 million people worldwide, causing self-limiting acute hepatic disease that can evolve into a chronic course. HEV-3, HEV-4, and HEV-7 genotypes are zoonotic and transmitted to humans by consuming raw or undercooked meat. Here, we developed an indirect ELISA based on the recombinant HEV-3 capsid and performed a seroprevalence study on domestic swine in northeastern Brazil. Our in-house ELISA was initially validated using a subset of 79 sera characterized by concordant results for two distinct commercial ELISA kits. Our ELISA exhibited excellent sensitivity (94%) and specificity (100%), with an area under the curve of 0.99 Further testing, including 212 swine sera, revealed a seroprevalence of 57.5% (95% confidence interval, 50.6-64.3%). Our findings indicate that the novel ELISA test could accurately detect specific anti-HEV antibodies in domestic pigs and should be further validated in humans and other mammals.

Diagnostic Potential for the Detection of Canine Visceral Leishmaniasis of an ELISA Assay Based on the Q5 Recombinant Protein: A Large-Scale and Comparative Evaluation Using Canine Sera with a Positive Diagnosis from the Dual-Path-Platform (DPP) Test.

Dogs are considered the major domestic reservoir for human visceral leishmaniasis, a serious disease caused by the Leishmania infantum parasite. Diagnosis of canine visceral leishmaniasis (CVL) is critical for disease control, with several methods currently available. Among the serological tests, the DPP rapid test and the EIE-LVC, more commonly used in Brazil, are associated with variable sensitivity and specificity. Research with novel recombinant proteins such as the ELISA with the recombinant chimeric protein Q5 may therefore improve the CVL diagnosis. This study aimed to evaluate the true diagnostic potential of Q5 in an ELISA assay using a large number of CVL-suspected sera (406) with a previous positive diagnosis based on the rapid DPP test. Sera from the DPP-positive dogs, also assessed with the EIE-LVC test, were compared with sera from healthy dogs (n = 46) and used for ELISA tests using the recombinant Q5. The resulting data as well as the correlation with the clinical signs and the environmental characteristics of the animals were analyzed using Medal and GraphPad Prism 8.0. Overall, similar levels of lower sensitivity (67-68%) were seen for both the commercial EIE-LVC test and the Q5 ELISA when all assessed sera were considered, but a much greater sensitivity (92%) was seen for those samples from symptomatic dogs only. In contrast, many negative results were observed for the DPP-positive sera from asymptomatic dogs or those with no clinical information available. A selection of those sera were tested yet again in new ELISA assays using a second batch of the recombinant Q5, purified under milder denaturing conditions, as well as using another recombinant protein (Lci13). The results reveal a higher-than-expected incidence of likely false-positive results for DPP, reinforcing the need for other recombinant proteins, such as the chimeric Q5, to be investigated as possible alternatives to the currently used CVL diagnostic methods.

Evolutionary study of potentially zoonotic hepatitis E virus genotype 3 from swine in Northeast Brazil.

Hepatitis E virus (HEV), an emerging virus associated with acute hepatic disease, leads to thousands of deaths worldwide. HEV has already been reported in Brazil; however, there is a lack of epidemiological and molecular information on the genetic variability, taxonomy, and evolution of HEV. It is thus unclear whether hepatitis E is a neglected disease in Brazil or it has low relevance for public health in this country. Here, for the first time, we report the presence of HEV in Northeast Brazil. A total of 119 swine faecal samples were screened for the presence of HEV RNA using real-time polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR; among these, two samples were identified as positive. Molecular evolution analyses based on capsid sequences revealed that the samples had close proximities to HEV sequences belonging to genotype 3 and were genetically related to subtype 3f isolated in humans. Parsimony ancestral states analysis indicated gene flow events from HEV cross-species infection, suggesting an important role of pig hosts in viral spillover. HEV's ability for zoonotic transmission by inter-species host switching as well as its possible adaptation to new animal species remain important issues for human health.

Development and Validation of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of ZIKV in Mosquito Samples from Brazil.

The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.

Evolutionary study of potentially zoonotic hepatitis E virus genotype 3 from swine in Northeast Brazil

Hepatitis E virus (HEV), an emerging virus associated with acute hepatic disease, leads to thousands of deaths worldwide. HEV has already been reported in Brazil; however, there is a lack of epidemiological and molecular information on the genetic variability, taxonomy, and evolution of HEV. It is thus unclear whether hepatitis E is a neglected disease in Brazil or it has low relevance for public health in this country. Here, for the first time, we report the presence of HEV in Northeast Brazil. A total of 119 swine faecal samples were screened for the presence of HEV RNA using real-time polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR; among these, two samples were identified as positive. Molecular evolution analyses based on capsid sequences revealed that the samples had close proximities to HEV sequences belonging to genotype 3 and were genetically related to subtype 3f isolated in humans. Parsimony ancestral states analysis indicated gene flow events from HEV cross-species infection, suggesting an important role of pig hosts in viral spillover. HEV's ability for zoonotic transmission by inter-species host switching as well as its possible adaptation to new animal species remain important issues for human health.

In vitro and in vivo models for studying Zika virus biology.

The emergence and rapid spread of Zika virus (ZIKV) in the Americas has prompted the development of in vitro and in vivo models to understand several aspects of ZIKV biology and boost the development of vaccines and antivirals. In vitro model studies include reverse genetics systems, two-dimensional (2D) cell models, such as primary cells and cell lines, and ex vivo three-dimensional (3D) models derived from skin, brain and placenta. While these models are cost-effective and allow rigorous control of experimental variables, they do not always recapitulate in vivo scenarios. Thus, a number of in vivo models have been developed, including mosquitoes (Aedes sp. and Culex sp.), embryonated chicken eggs, immunocompetent and immunodeficient mice strains, hamsters, guinea pigs, conventional swine and non-human primates. In this review, we summarize the main research systems that have been developed in recent years and discuss their advantages, limitations and main applications.