Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery.

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

Risk of infection with arboviruses in a healthy population in Pakistan based on seroprevalence.

Infectious diseases caused by arboviruses are a public health concern in Pakistan. However, studies on data prevalence and threats posed by arboviruses are limited. This study investigated the seroprevalence of arboviruses in a healthy population in Pakistan, including severe fever with thrombocytopenia syndrome virus (SFTSV), Crimean-Congo hemorrhagic fever virus (CCHFV), Tamdy virus (TAMV), and Karshi virus (KSIV) based on a newly established luciferase immunoprecipitation system (LIPS) assays, and Zika virus (ZIKV) by enzyme-linked immunosorbent assays (ELISA). Neutralizing activities against these arboviruses were further examined from the antibody positive samples. The results showed that the seroprevalence of SFTSV, CCHFV, TAMV, KSIV, and ZIKV was 17.37%, 7.58%, 4.41%, 1.10%, and 6.48%, respectively, and neutralizing to SFTSV (1.79%), CCHFV (2.62%), and ZIKV (0.69%) were identified, as well as to the SFTSV-related Guertu virus (GTV, 0.83%). Risk factors associated with the incidence of exposure and levels of antibody response were analyzed. Moreover, co-exposure to different arboviruses was demonstrated, as thirty-seven individuals were having antibodies against multiple viruses and thirteen showed neutralizing activity. Males, individuals aged ≤40 years, and outdoor workers had a high risk of exposure to arboviruses. All these results reveal the substantial risks of infection with arboviruses in Pakistan, and indicate the threat from co-exposure to multiple arboviruses. The findings raise the need for further epidemiologic investigation in expanded regions and populations and the necessity to improve health surveillance in Pakistan.

Simultaneous detection of arboviruses by a multiplex RT-qPCR assay in Tocantins, a northern state of Brazil.

In Brazil, Dengue, Zika and Chikungunya viruses constitute a major threat to the public health system. Simultaneous circulation of these arboviruses occurs in many regions of the world due to the expansion of transmission vectors. The infection by these arboviruses triggers similar symptoms during their acute phase. However, in some cases, severe symptoms may occur, leading to different types of disabilities and even death. In this context, considering the similarity of the symptoms, the problems caused by the infection of these arboviruses, and the increasing risk of coinfection in humans, the differential diagnosis of these infections is essential for clinical management and epidemiological investigation. Thus, this study aimed to identify, through diagnosis via Quantitative Polymerase Chain Reaction with Reverse Transcription, arbovirus coinfection in patients from the Tocantins state (Northern Brazil). A total of 495 samples were analyzed, three from which were determined to be a coinfection of Dengue and Chikungunya viruses. The data obtained here indicate the co-circulation and coinfection by Dengue and Chikungunya viruses in the Tocantins state. These results highlight the importance of monitoring the circulation of these arboviruses for the development of health actions that aim their prevention and combat, as well as their clinical and therapeutic management.

Insect ribosome-rescuer Pelo-Hbs1 complex on sperm surface mediates paternal arbovirus transmission.

Arboviruses can be paternally transmitted by male insects to offspring for long-term persistence, but the mechanism remains largely unknown. Here, we use a model system of a destructive rice reovirus and its leafhopper vector to find that insect ribosome-rescuer Pelo-Hbs1 complex expressed on the sperm surface mediates paternal arbovirus transmission. This occurs through targeting virus-containing tubules constituted by viral nonstructural protein Pns11 to sperm surface via Pns11-Pelo interaction. Tubule assembly is dependent on Hsp70 activity, while Pelo-Hbs1 complex inhibits tubule assembly via suppressing Hsp70 activity. However, virus-activated ubiquitin ligase E3 mediates Pelo ubiquitinated degradation, synergistically causing Hbs1 degradation. Importantly, Pns11 effectively competes with Pelo for binding to E3, thus antagonizing E3-mediated Pelo-Hbs1 degradation. These processes cause a slight reduction of Pelo-Hbs1 complex in infected testes, promoting effective tubule assembly. Our findings provide insight into how insect sperm-specific Pelo-Hbs1 complex is modulated to promote paternal virus transmission without disrupting sperm function.

Prevalence of arboviruses in sickle cell disease patients from two different regions of Brazil, the North and Northeast.

Sickle Cell Disease (SCD) is a hereditary disease characterized by extravascular and intravascular hemolysis and clinical variability, from mild pain to potentially life-threatening. Arboviruses include mainly Zika (ZIKV), Chikungunya (CHKV), and Dengue (DENV) virus, and are considered a public and social health problem. The present cross-sectional observational study aimed to investigate the prevalence of arbovirus infection in SCD patients from two Brazilian cities, Salvador and Manaus located in Bahia and Amazonas states respectively. A total of 409 individuals with SCD were included in the study, and 307 (75.06 %) patients tested positive for DENV-IgG, 161 (39.36 %) for ZIKV-IgG, and 60 (14.67 %) for CHIKV-IgG. Only one individual was positive for DENV-NS1 and another for DENV-IgM, both from Salvador. No individuals had positive serology for ZIKV-IgM or CHIKV-IgM. Arbovirus positivity by IgG testing revealed that the SCD group presented high frequencies in both cities. Interestingly, these differences were only statistically significant for ZIKV-IgG (p = 0.023) and CHIKV-IgG (p = 0.005) among SCD patients from Manaus. The reshaping of arbovirus from its natural habitat by humans due to disorderly urban expansion and the ease of international Mobility has been responsible for facilitating the spread of vector-borne infectious diseases in humans. We found the need for further studies on arboviruses in this population to elucidate the real association and impact, especially in acute infection. We hope that this study will contribute to improvements in the personalized clinical follow-up of SCD patients, identifying the influence of arbovirus infection in severe disease manifestations.

Seroprevalence of Arboviruses in a Malaria Hyperendemic Area in Southern Mali.

Diagnostics for febrile illnesses other than malaria are not readily available in rural sub-Saharan Africa. This study assessed exposure to three mosquito-borne arboviruses-dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV)-in southern Mali. Seroprevalence for DENV, CHIKV, and ZIKV was analyzed by detection of IgG antibodies and determined to be 77.2%, 31.2%, and 25.8%, respectively. Among study participants, 11.3% were IgG-positive for all three arboviruses. DENV had the highest seroprevalence rate at all sites; the highest seroprevalence of CHIKV and ZIKV was observed in Bamba. The seroprevalence for all three arboviruses increased with age, and the highest seroprevalence was observed among adults older than 50 years. The prevalence of Plasmodium spp. in the cohort was analyzed by microscopy and determined to be 44.5% (N = 600) with Plasmodium falciparum representing 95.1% of all infections. This study demonstrates the co-circulation of arboviruses in a region hyperendemic for malaria and highlights the needs for arbovirus diagnostics in rural sub-Saharan Africa.

Guillain-Barré syndrome associated with arboviruses in the state of Pernambuco in 2016 / Síndrome de Guillain-Barré associada à arboviroses no estado de Pernambuco em 2016 / Síndrome de Guillain-Barré asociado a arbovirosis en el Estado de Pernambuco en 2016

Abstract Introduction: In 2016, Brazil presented an increase in the notifications related to neurological syndromes with previous register of a febrile illness compatible with arboviruses, including Guillain-Barré syndrome (GBS). Such disease is responsible for the most frequent occurrence of flaccid paralysis in the world, causing bilateral ascending muscle weakness that might affect the respiratory tract. Objective: To investigate the incidence of Guillain-Barré syndrome associated with arboviruses in Pernambuco in 2016 and to describe the confirmed/probable cases. Method: This is an observational retrospective study of GBS cases with a background of infection from dengue, chikungunya, or Zika virus notified to the Pernambuco Health State Secretariat (SES-PE). Cases notified from January 1st to December 31st, 2016 were included, which were considered as possible GBS at the time of notification. The suspect cases were investigated and classified as confirmed/probable by the positive laboratory test result, excluding the possibility of infection. Results: Forty-three suspect cases of GBS after previous arbovirus infection were notified. From these, 23 were classified as confirmed/probable for the etiological agents chikungunya and/or dengue. The mean age of the affected individuals was 37.22 ± 21.29 years and they were mostly female (56.5%). The annual incidence of GBS after arbovirus infection was 0.24 cases per 100 thousand inhabitants in the state. The neurological condition was mainly characterized by the presence of movement (91.3%) and walking (78.3%) alterations. Conclusion: The findings of this research confirm the current set of evidence and show the likelihood of GBS being a severe neurological complication of these arboviruses.

Resumo Introdução: No Brasil, em 2016, houve aumento das notificações relacionadas às síndromes neurológicas com registro anterior de doença febril compatível com infecção por arboviroses, dentre elas a Síndrome de Guillain- Barré (SGB). Trata-se da ocorrência mais frequente de paralisia flácida no mundo, causa fraqueza muscular bilateral ascendente e pode atingir as vias respiratórias. Objetivo: Analisar a incidência da Síndrome de Guillain- Barré associada à arboviroses em Pernambuco no ano de 2016 e descrever os casos confirmados/prováveis. Método: Trata-se de um estudo observacional retrospectivo dos casos de SGB com histórico de infecção prévia por dengue, chikungunya ou vírus Zika, notificados à Secretaria Estadual de Saúde de Pernambuco (SES-PE). Foram incluídos casos notificados de 1º de janeiro a 31 de dezembro de 2016 considerados suspeitos de SGB no momento da notificação. Os casos suspeitos foram investigados e classificados como confirmados/prováveis através de resultado de exame laboratorial positivo, excluída a possibilidade de infecção. Resultados: Foram notificados 43 casos suspeitos de SGB após infecção prévia por arbovírus. Destes, 23 foram classificados como confirmados/prováveis para os agentes etiológicos chikungunya e/ou dengue, sendo a média de idade igual a 37,22 ± 21,29 anos e maioria do sexo feminino (56,5%). A incidência anual de SGB após infecção por arbovírus foi de 0,24 casos por 100 mil habitantes no estado. O quadro neurológico foi caracterizado prioritariamente pela presença de alterações motoras (91,3%) e na marcha (78,3%). Conclusão: Os achados da presente pesquisa acrescentam ao conjunto de evidências sobre a possibilidade de a SGB se apresentar como uma grave complicação neurológica dessas arboviroses.

Resumen Introducción: En Brasil, en 2016, hubo aumento de las notificaciones relacionadas con los síndromes neurológicos con registro anterior de enfermedad febril compatible con infección por arbovirosis, entre ellas el Síndrome de Guillain-Barré (SGB). Se trata de la aparición más frecuente de parálisis flácida en el mundo, causa debilidad muscular bilateral ascendente y puede alcanzar las vías respiratorias. Objetivo: Analizar la incidencia del Síndrome de Guillain-Barré asociada a los arbovirosis en Pernambuco en el año 2016 y describir los casos confirmados / probables. Método: Se trata de un estudio observacional retrospectivo de los casos de SGB con antecedentes de infección previa por dengue, chikungunya o virus Zika, notificados a la Secretaría Estatal de Salud de Pernambuco (SES-PE). Se incluyeron casos notificados del 1 de enero al 31 de diciembre de 2016 considerados sospechosos de SGB en el momento de la notificación. Los casos sospechosos fueron investigados y clasificados como confirmados / probables a través del resultado de un examen de laboratorio positivo, excluida la posibilidad de infección por otros agentes virales. Resultados: Se han notificado 43 casos sospechosos de SGB tras la infección previa por arbovirus. De estos, 23 fueron clasificados como confirmados / probables para los agentes etiológicos chikungunya y / o dengue, siendo la media de edad igual a 37,22 ± 21,29 años y la mayoría del sexo femenino (56,5%). La incidencia anual de SGB tras infección por arbovirus fue de 0,24 casos por 100 mil habitantes en el estado. El cuadro neurológico fue caracterizado prioritariamente por la presencia de alteraciones motoras (91,3%) y en la marcha (78,3%). Conclusión: Los hallazgos de la presente investigación añaden al conjunto de evidencias sobre la posibilidad de que la SGB se presente como una grave complicación neurológica de esas arbovirosis.

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia)

BACKGROUND In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. OBJECTIVES This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. METHODS Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. FINDINGS A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). MAIN CONCLUSION The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon.

Eco-epidemiologia dos arbovírus na área de influência da rodovia Cuiabá-Santarém (BR 163), Estado do Pará, Brasil / Arbovirus eco-epidemiology in the area affected by the Cuiabá-Santarém Highway (BR-163), Pará State, Brazil

O presente estudo descreve os aspectos eco-epidemiológicos sobre arbovírus nos Municípios de Novo Progresso e Trairão, Estado do Pará, Brasil, na área de influência da BR 163. Anticorpos IH foram detectados para diferentes arbovírus, com reações monotípicas para os VMAY e VORO, dois importantes arbovírus associados a epidemias na Amazônia. Anticorpos IgM para o VORO e VMAY foram detectados em soros humanos, sugerindo infecção recente por esses arbovírus. Duas cepas do VDEN-3 foram isoladas de pacientes febris residentes em Novo Progresso e identificadas como genótipo III. Em termos gerais, os dados obtidos sugerem uma área propícia para a circulação e manutenção de arbovírus e uma população pouco imunizada. Portanto, é importante um monitoramento dinâmico das populações locais e de imigrantes e de animais silvestres quanto à presença de anticorpos e isolamentos de arbovírus, o que permitirá um efetivo controle das infecções por esses agentes virais em residentes da área da rodovia dentro do território paraense.

The current study describes the eco-epidemiological aspects of arbovirus diseases in the municipalities (counties) of Novo Progresso and Trairão, Para State, Brazil, in the area affected by highway BR-163. Hemagglutination inhibition (HI) antibodies to different arboviruses were detected, with monotypic reactions to MAYV and OROV, two important arboviruses associated with epidemics in the Amazon. IgM antibodies to OROV and MAYV were found in human sera, suggesting recent infections by these viruses. Two DENV-3 strains were isolated from febrile patients in Novo Progresso and identified as genotype III strains. In general, the data suggest that the area displays ideal conditions for maintenance and circulation of arboviruses, plus a population with low immunization levels. Dynamic surveillance of local immigrants and wild animals is thus important, focusing on antibody prevalence and isolation of arboviruses, thereby allowing effective control of infections by these viral agents in the resident population along highway BR-163 in Pará State.