Vector microbiota manipulation by host antibodies: the forgotten strategy to develop transmission-blocking vaccines

The gut microbiota is involved in many aspects of host physiology but its role in body weight and glucose metabolism remains unclear. Here we studied the compositional changes of gut microbiota in diet-induced obesity mice that were conventionally raised or received microbiota transplantation. In conventional mice, the diversity of the faecal microbiota was weakly associated with 1st week weight gain but transferring the microbiota of mice with contrasting weight gain to germfree mice did not change obesity development or feed efficiency of recipients regardless whether the microbiota was taken before or after 10 weeks high fat (HF) feeding. Interestingly, HF-induced glucose intolerance was influenced by microbiota inoculation and improved glucose tolerance was associated with a low Firmicutes to Bacteroidetes ratio. Transplantation of Bacteroidetes rich microbiota compared to a control microbiota ameliorated glucose intolerance caused by HF feeding. Altogether, our results demonstrate that gut microbiota is involved in the regulation of glucose metabolism and the abundance of Bacteroidetes significantly modulates HF-induced glucose intolerance but has limited impact on obesity in mice. Our results suggest that gut microbiota is a part of complex aetiology of insulin resistance syndrome, individual microbiota composition may cause phenotypic variation associated with HF feeding in mice.

Among the vector-borne flaviviruses, West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and Dengue virus (DENV) constitute the most frequently-observed pathogens with significant public health impact in endemic regions throughout the globe. This seroepidemiological study was undertaken to investigate human exposure to DENV, WNV and TBEV, as well as other flaviviruses via various serological assays in the Mediterranean province of Mersin, Turkey, where scarce data is currently present for the circulation of these agent. A total of 920 sera were collected after informed consent from asymptomatic blood donors (all were male; age range: 18-63 yrs, mean age: 35.17 ± 9.56 yrs) were taken between August 2010 and April 2011. All samples were initially screened via a commercial ELISA kit for DENV IgM and IgG. Reactive samples were further evaluated via commercial indirect immunofluorescence tests (IIFTs) for yellow fever virus (YFV) IgG, TBEV IgG and via ELISA for WNV IgG. Moreover, presence of neutralizing antibodies were investigated in all reactive samples via plaque reduction neutralization (PRNT) assay for WNV, whose activity has been detected previously in the region. Samples interpreted as positive for TBEV IgG were further evaluated for specificity by TBEV PRNT assay. DENV IgM reactive samples were also assessed for NS1 antigens and IgM/IgG antibodies via a commercial immunochromatographic assay (ICA). DENV IgM and IgG antibodies were detected in 0.9% (8/920) and 16.6% (153/920) of the samples, respectively. One sample was simultaneously positive for IgM and IgG. WNV PRNT revealed positive results in 85.6% (137/160) of the reactive samples, which indicated frequent WNV exposure and frequent development of cross-reactions in the screening assay. Positive or borderline DENV IgM reactivity was identified in 0.43% (4/920) of the samples, which remained negative for NS1 antigen and antibodies in the ICA. Antibody specificity in two samples, positive for DENV and TBEV IgG in IIFT could not be confirmed by TBEV PRNT. A total of 19 reactive samples (19/920, 2.1%), that comprise seven borderline and six positive DENV IgG positivities as well as six samples with IgG positivity for different virus combinations remained negative after DENV confirmatory and WNV/TBEV PRNT assays. When the samples with borderline results were omitted from the evaluation, 12 samples (12/920, 1.3%) were considered to represent exposure to DENV or an antigenically-similar flavivirus. These findings indicated the activity of and frequent exposure (137/920, 14.9%) to WNV, as previously suggested in the study region. In 1.3% of the samples, probable exposure to DENV or other flaviviruses was revealed and this requires further serosurveillance efforts. WNV must be considered in the etiology of febrile diseases or viral neuroinvasive infections of unexplained etiology in the study area.

Vector-borne flaviviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus, and dengue virus, cause millions of infections in humans. TBEV causes a broad range of pathological symptoms, ranging from meningitis to severe encephalitis or even hemorrhagic fever, with high mortality. Despite the availability of an effective vaccine, the incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuroinflammation in LGTV-infected mice, characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. The type I interferon (IFN) system is important to control viral infections; however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system are poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system, is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection. The first phase is in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). In the second phase, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.

BackgroundThe genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.MethodsA Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.ResultsTwo degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.ConclusionThe assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.

The tick-borne encephalitis virus (TBEV) is one of the most common members of the Orthoflavivirus genus, which comprises the causative agents of severe diseases in humans and animals. Due to the expanding areas of orthoflavivirus infection, its differential diagnosis is highly demanded. Commercial test kits based on inactivated TBEV may not provide reliable differentiation between flaviviruses because of serological crossover in this genus. Application of recombinant domains (sE and dIII) of the TBEV Sukhar-strain protein E as antigens in an ELISA test system allowed us to identify a wide range of antibodies specific to different TBEV strains. We tested 53 sera from human patients with confirmed TBE diagnosis (the efficacy of our test system based on sE protein was 98%) and 56 sera from patients with other orthoflavivirus infections in which no positive ones were detected using our ELISA test system, thus being indicative of its 100% specificity. We also tested mouse and rabbit sera containing antibodies specific to 17 TBEV strains belonging to different subtypes; this assay exhibited high efficacy and differentiation ability in detecting antibodies against TBEV from other orthoflaviviruses such as Omsk hemorrhagic fever, Powassan, yellow fever, dengue, West Nile, Zika, and Japanese encephalitis viruses.

In the last few years, several imported cases of Zika virus (ZIKV) infection were reported in European countries. We report the first imported ZIKV infection case in a Croatian traveler returning from Brazil. The patient presented with a low-grade fever, pruritic rash, general weakness, myalgia, arthralgia and edema of the legs and recovered completely within a week. ZIKV infection was confirmed by detection of IgM/IgG antibodies using enzyme-linked immunosorbent assay (ELISA) and confirmed by plaque-reduction neutralization test (PRNT). ZIKV IgM antibodies cross-reacted with dengue virus (DENV), West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) in ELISA. In indirect immunofluorescence assay (IFA), IgM cross-reactivity was found only with DENV-3. ZIKV IgG antibodies cross-reacted with DENV in both ELISA and IFA. PRNT for DENV was negative. Control serology performed on days 64 and 98 after disease onset showed a decline in cross-reactive heterologous DENV IgG antibodies compared to persistently high titer of homologous ZIKV IgG antibodies.

Flaviviruses are a genus of positive-sense RNA viruses that includes Japanese encephalitis virus, West Nile virus, tick-borne encephalitis virus, yellow fever virus, and dengue viruses. They are transmitted by arthropod vectors and cause significant human disease worldwide. The rapid rise in human infections caused by these viruses has been attributed to increased urbanization, travel, and increased spread of vectors probably linked to climate change. Dengue viruses now cause the most widespread arthropod-borne viral disease infecting humans. Thus, in this chapter we introduce and describe the molecular and cellular biology of flaviviruses using dengue viruses as an example.

ABSTRACTHighly effective prophylactic vaccines for flaviviruses including yellow fever virus, tick-borne encephalitis virus and Japanese encephalitis virus are currently in use. However, the development of a dengue virus (DENV) vaccine has been hampered by the requirement of simultaneous protection against four distinct serotypes and the threat that DENV-specific antibodies might either mediate neutralization or, on the contrary, exacerbate disease through the phenomenon of antibody-dependent enhancement (ADE) of infection. Therefore, understanding the cellular, biochemical and molecular basis of antibody-mediated neutralization and ADE are fundamental for the development of a safe DENV vaccine. Here we summarize current structural and mechanistic knowledge underlying these phenomena. We also review recent results demonstrating that the humoral immune response triggered during natural DENV infection is able to generate neutralizing antibodies binding complex quaternary epitopes only present on the surface of intact virions.

Dengue virus (DENV) is a highly prevalent vector-borne virus that causes life-threatening illnesses to humans worldwide. The development of a tool to control vector populations has the potential to reduce the burden of DENV. Toxic sugar bait (TSB) provides a form of vector control that takes advantage of the sugar-feeding behavior of adult mosquitoes. However, studies on the effect of ingestion of toxins in TSB on vector competence and vectorial capacity for viruses are lacking. This study evaluated vector competence for DENV serotype-1 of Aedes albopictus at 7 and 14 days post-ingestion of TSB formulated with spinosad (of bacteria origin) as an oral toxin. Our results and others were modeled to estimate effects on Ae. albopictus vectorial capacity for DENV. Ingestion of TSB caused a reduction in survival of females, but increased mosquito susceptibility to DENV infection, disseminated infection, and transmission. However, this increase in vector competence was obviated by the reduction in survival, leading to a lower predicted vectorial capacity. The findings of this study highlight the importance of evaluating the net impact of TSB ingestion on epidemiological parameters of vectorial capacity in the context of vector control efforts to reduce the risk of transmission of vector-borne viruses.

Dengue virus (DENV) is the most prevalent mosquito-borne Flavivirus that affects humans worldwide. Aedes albopictus, which is naturally infected with the bacteria Wolbachia, is considered to be a secondary vector of DENV. However, it was responsible for a recent DENV outbreak of unprecedented magnitude in Reunion Island, a French island in the South West Indian Ocean. Moreover, the distribution of the cases during this epidemic showed a spatially heterogeneous pattern across the island, leading to questions about the differential vector competence of mosquito populations from different geographic areas. The aim of this study was to gain a better understanding of the vector competence of the Ae. albopictus populations from Reunion Island for local DENV epidemic strains, while considering their infection by Wolbachia. Experimental infections were conducted using ten populations of Ae. albopictus sampled across Reunion Island and exposed to three DENV strains: one strain of DENV serotype 1 (DENV-1) and two strains of DENV serotype 2 (DENV-2). We analyzed three vector competence parameters including infection rate, dissemination efficiency and transmission efficiency, at different days post-exposition (dpe). We also assessed whether there was a correlation between the density of Wolbachia and viral load/vector competence parameters. Our results show that the Ae. albopictus populations tested were not able to transmit the two DENV-2 strains, while transmission efficiencies up to 40.79% were observed for the DENV-1 strain, probably due to difference in viral titres. Statistical analyses showed that the parameters mosquito population, generation, dpe and area of sampling significantly affect the transmission efficiencies of DENV-1. Although the density of Wolbachia varied according to mosquito population, no significant correlation was found between Wolbachia density and either viral load or vector competence parameters for DENV-1. Our results highlight the importance of using natural mosquito populations for a better understanding of transmission patterns of dengue.

The ongoing Zika virus outbreak, which briefly threatened to put a stop to the 2016 Olympic Games in Brazil, has again focused attention on the threat of vector‐borne diseases. One efficient strategy to battle such diseases, now underway in Brazil to fight the Zika virus, is to kill the insects that carry the pathogen. Vector control has been practised with varying effectiveness for at least half a century, especially for malaria; the Brazil outbreak again highlights the need for integrating existing methods including the use of insecticides, vaccines, drugs and low technology such as sanitation to keep insect vectors at bay. In addition, new approaches are being developed based on sophisticated methods such as genome editing of the insect vector and deliberate infection of mosquitoes with the Wolbachia bacteria. However, attempts to scale up these new methods from field trials to deployment have been slowed down or halted by environmental concerns or public resistance against genetically modified organisms. The Zika outbreak in South America has stimulated cross‐border collaboration of public health authorities and increased pressure to use new integrative approaches that are essential for combatting vector‐borne diseases. The irony is that the Zika virus itself has a minimal global impact compared with other insect‐borne pathogens, but it has attracted enormous publicity because of the risk of causing microcephaly in unborn children, combined with the coincidence of the outbreak during the Olympic year. The exact risk for the foetus is as yet unknown [1], but it is clear that Zika is otherwise a relatively mild disease with no symptoms in the majority of cases (http://www.cdc.gov/zika/symptoms/index.html). By contrast, other mosquito‐borne diseases, including malaria, yellow fever, dengue and chikungunya (CHIKV), are major causes of both mortality and morbidity, responsible for hundreds of millions of cases each year and millions of deaths …

In dengue-endemic regions like Vietnam, viral haemorrhagic fevers (VHFs) with overlapping clinical presentations often lead to misdiagnoses. This study investigated alternative VHF pathogens in patients clinically suspected of dengue but negative by standard diagnostics during a 2016 outbreak in central Vietnam. Among 198 hospitalized patients, 52 dengue virus (DENV)-negative cases were tested using enzyme-linked immunosorbent assays for immunoglobulin G (IgG)/IgM against seven VHFs and molecular assays for pan-flaviviruses and hantaviruses. Positive amplicons were sequenced and subjected to phylogenetic analysis. High flavivirus IgG seropositivity was observed, especially for Japanese encephalitis virus (96%), DENV, tick-borne encephalitis virus and West Nile virus (92% each), with notable cross-reactivity. IgM responses were specific, with 37% of patients positive for anti-JEV and 33% for anti-DENV. Pan-flavivirus reverse transcription polymerase chain reaction confirmed DENV-1 RNA in four patients. One patient tested positive for hantaviral RNA and seropositive for both IgG and IgM. Sequencing and phylogenetic analysis identified Orthohantavirus seoulense (SEOV) as the causative agent, clustering with SEOV strains from Vietnam and Indonesia. The detection of SEOV highlights co-circulation of rodent-borne viruses during arboviral outbreaks. This study highlights significant serological cross-reactivity among flaviviruses and underdiagnosed circulation of hantaviruses such as SEOV. Enhanced multimodal diagnostic surveillance of pathogens remains essential.

Mosquito-borne viral diseases and potential transmission blocking vaccine candidates

BackgroundDengue is an arboviral disease caused by dengue virus (DENV), whose main vectors are the mosquitoes Aedes aegypti and Aedes albopictus. A. aegypti is the only DENV vector in Cape Verde, an African country that suffered its first outbreak of dengue in 2009. However, little is known about the variation in the level of vector competence of this mosquito population to the different DENV serotypes. This study aimed to evaluate the vector competence of A. aegypti from the island of Santiago, Cape Verde, to four DENV serotypes and to detect DENV vertical transmission.MethodsMosquitoes were fed on blood containing DENV serotypes and were dissected at 7, 14 and 21 days post-infection (dpi) to detect the virus in the midgut, head and salivary glands (SG) using RT-PCR. Additionally, the number of copies of viral RNA present in the SG was determined by qRT-PCR. Furthermore, eggs were collected in the field and adult mosquitoes obtained were analyzed by RT-PCR and the platelia dengue NS1 antigen kit to detect transovarial transmission.ResultsHigh rates of SG infection were observed for DENV-2 and DENV-3 whereas for DENV-1, viral RNA was only detected in the midgut and head. DENV-4 did not spread to the head or SG, maintaining the infection only in the midgut. The number of viral RNA copies in the SG did not vary significantly between DENV-2 and DENV-3 or among the different periods of incubation and the various titers of DENV tested. With respect to DENV surveillance in mosquitoes obtained from the eggs collected in the field, no samples were positive.ConclusionAlthough no DENV positive samples were collected from the field in 2014, it is important to highlight that the A. aegypti population from Santiago Islands exhibited different degrees of susceptibility to DENV serotypes. This population showed a high vector competence for DENV-2 and DENV-3 strains and a low susceptibility to DENV-1 and DENV-4. Viral RNA copies in the SG remained constant for at least 21 dpi, which may enhance the vector capacity of A. aegypti and suggests the presence of a mechanism modulating virus replication in the SG.

Comparison of ELISA and immunoassays for measurement of IgG and IgM antibody to West Nile virus in human sera against virus neutralisation