Flaviviruses hijack the host microbiota to facilitate their transmission.

Flaviviruses, such as Dengue and Zika viruses, infect millions of people worldwide using mosquitos as vectors. In this issue of Cell, Zhang et al. reveal how these viruses manipulate the skin microbiome of infected hosts in a way that increases vector recruitment and viral spread. They propose vitamin A as a way to counteract the virus and decrease transmission.

Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway.

IMPORTANCE: Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.

Impact of alphavirus 3'UTR plasticity on mosquito transmission.

Alphaviruses such as chikungunya and western equine encephalitis viruses are important human pathogens transmitted by mosquitoes that have recently caused large epidemic and epizootic outbreaks. The epidemic potential of alphaviruses is often related to enhanced mosquito transmission. Tissue barriers and antiviral responses impose bottlenecks to viral populations in mosquitoes. Substitutions in the envelope proteins and the presence of repeated sequence elements (RSEs) in the 3'UTR of epidemic viruses were proposed to be specifically associated to efficient replication in mosquito vectors. Here, we discuss the molecular mechanisms that originated RSEs, the evolutionary forces that shape the 3'UTR of alphaviruses, and the significance of RSEs for mosquito transmission. Finally, the presence of RSEs in the 3'UTR of viral genomes appears as evolutionary trait associated to mosquito adaptation and emerges as a common feature among viruses from the alphavirus and flavivirus genera.

Toll-like receptor 4 and lipopolysaccharide from commensal microbes regulate Tembusu virus infection.

Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection. We found that lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria enhanced TMUV proliferation by promoting its attachment. Toll-like receptor 4 (TLR4), a cell surface receptor, can transmit signal from LPS. We confirmed colocalization of TLR4 with TMUV envelope (E) protein as well as their interaction in infected cells. Coherently, TMUV infection of susceptible cells was inhibited by an anti-TLR4 antibody, purified soluble TLR4 protein, and knockdown of TLR4 expression. LPS-enhanced TMUV proliferation could also be blocked by a TLR4 inhibitor. Meanwhile, pretreatment of duck primary cells with TMUV significantly impaired LPS-induced interleukin 6 production. Collectively, our study provides first insights into vector-free transmission mechanisms of flaviviruses.

Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection.

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we showed that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required during flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrated specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical and imaging methodology, we showed that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT proteins, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection, the virus employs members of ESCRT proteins for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Another important ESCRT protein, TSG101, is not required for virus production. The ESCRT, complex, ALIX-CHMP4A, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

Discovery of Novel Crustacean and Cephalopod Flaviviruses: Insights into the Evolution and Circulation of Flaviviruses between Marine Invertebrate and Vertebrate Hosts.

Most described flaviviruses (family Flaviviridae) are disease-causing pathogens of vertebrates maintained in zoonotic cycles between mosquitoes or ticks and vertebrate hosts. Poor sampling of flaviviruses outside vector-borne flaviviruses such as Zika virus and dengue virus has presented a narrow understanding of flavivirus diversity and evolution. In this study, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus, and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and maximum likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor with terrestrial vector-borne flaviviruses than with classical insect-specific flaviviruses. In addition, we identify variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNA interference response. This suggests that Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. Analyses of the mono- and dinucleotide composition of marine flaviviruses compared to that of flaviviruses with known host status suggest that some marine flaviviruses share a nucleotide bias similar to that of vector-borne flaviviruses. Furthermore, we identify crustacean flavivirus endogenous viral elements that are closely related to elements of terrestrial vector-borne flaviviruses. Taken together, these data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expand our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.IMPORTANCE Some flaviviruses are known to cause disease in vertebrates and are typically transmitted by blood-feeding arthropods such as ticks and mosquitoes. While an ever-increasing number of insect-specific flaviviruses have been described, we have a narrow understanding of flavivirus incidence and evolution. To expand this understanding, we discovered a number of novel flaviviruses that infect a range of crustaceans and cephalopod hosts. Phylogenetic analyses of these novel marine flaviviruses suggest that crustacean flaviviruses share a close ancestor to all terrestrial vector-borne flaviviruses, and squid flaviviruses are the most divergent of all known flaviviruses to date. Additionally, our results indicate horizontal transmission of a marine flavivirus between crabs and sharks. Taken together, these data suggest that flaviviruses move horizontally between invertebrates and vertebrates in ocean ecosystems. This study demonstrates that flavivirus invertebrate-vertebrate host associations have arisen in flaviviruses at least twice and may potentially provide insights into the emergence or origin of terrestrial vector-borne flaviviruses.

Usutu virus disease: a potential problem for North America?

Usutu virus is an emerging mosquito-borne flavivirus initially identified in South Africa in 1959 that is now circulating throughout parts of Africa, Europe, and the Middle East. It is closely related to West Nile virus, and has similar vectors, amplifying bird hosts, and epidemiology. Usutu virus infection can occur in humans and may be asymptomatic or cause systemic (e.g., fever, rash, and hepatitis) or neuroinvasive (e.g., meningitis and encephalitis) disease. Given few reported cases, the full clinical spectrum is not known. No anti-viral treatment is available, but it can be largely prevented by avoiding mosquito bites. Because of similar mosquitoes, birds, and climate to Europe, the potential for introduction to North America is possible.

Impact of pyriproxyfen on virus behavior: implications for pesticide-induced virulence and mechanism of transmission.

BACKGROUND: More than 3 years since the last Zika virus (ZIKV) outbreak in Brazil, researchers are still deciphering the molecular mechanisms of neurovirulence and vertical transmission, as well as the best way to control spread of ZIKV, a flavivirus. The use of pesticides was the main strategy of mosquito control during the last ZIKV outbreak. METHODS: We used vesicular stomatitis virus (VSV) tagged with green fluorescent protein (GFP) as our prototypical virus to study the impact of insecticide pyriproxyfen (PPF). VZV-GFP infected and uninfected Jurkat, HeLa and trophoblast cells were treated with PPF and compared to untreated cells (control). Cell viability was determined by the MTT assay. Cell morphology, presence of extracellular vesicles (EVs), virus infection/GFP expression as well as active mitochondrial levels/localization were examined by confocal microscopy. RESULTS: PPF, which was used to control mosquito populations in Brazil prior to the ZIKV outbreak, enhances VSV replication and has cell membrane-altering properties in the presence of virus. PPF causes enhanced viral replication and formation of large EVs, loaded with virus as well as mitochondria. Treatment of trophoblasts or HeLa cells with increasing concentrations of PPF does not alter cell viability, however, it proportionately increases Jurkat cell viability. Increasing concentrations of PPF followed by VSV infection does not interfere with HeLa cell viability. Both Jurkats and trophoblasts show proportionately increased cell death with increased concentrations of PPF in the presence of virus. CONCLUSIONS: We hypothesize that PPF disrupts the lipid microenvironment of mammalian cells, thereby interfering with pathways of viral replication. PPF lowers viability of trophoblasts and Jurkats in the presence of VSV, implying that the combination renders immune system impairment in infected individuals as well as enhanced vulnerability of fetuses towards viral vertical transmission. We hypothesize that similar viruses such as ZIKV may be vertically transmitted via EV-to-cell contact when exposed to PPF, thereby bypassing immune detection. The impact of pesticides on viral replication must be fully investigated before large scale use in future outbreaks of mosquito borne viruses.

The bright and the dark side of human antibody responses to flaviviruses: lessons for vaccine design.

Zika and dengue viruses belong to the Flavivirus genus, a close group of antigenically related viruses that cause significant arthropod-transmitted diseases throughout the globe. Although infection by a given flavivirus is thought to confer lifelong protection, some of the patient's antibodies cross-react with other flaviviruses without cross-neutralizing. The original antigenic sin phenomenon may amplify such antibodies upon subsequent heterologous flavivirus infection, potentially aggravating disease by antibody-dependent enhancement (ADE). The most striking example is provided by the four different dengue viruses, where infection by one serotype appears to predispose to more severe disease upon infection by a second one. A similar effect was postulated for sequential infections with Zika and dengue viruses. In this review, we analyze the molecular determinants of the dual antibody response to flavivirus infection or vaccination in humans. We highlight the role of conserved partially cryptic epitopes giving rise to cross-reacting and poorly neutralizing, ADE-prone antibodies. We end by proposing a strategy for developing an epitope-focused vaccine approach to avoid eliciting undesirable antibodies while focusing the immune system on producing protective antibodies only.

West Nile and Usutu Virus Infections and Challenges to Blood Safety in the European Union.

West Nile virus (WNV) and Usutu virus (USUV) circulate in several European Union (EU) countries. The risk of transfusion-transmitted West Nile virus (TT-WNV) has been recognized, and preventive blood safety measures have been implemented. We summarized the applied interventions in the EU countries and assessed the safety of the blood supply by compiling data on WNV positivity among blood donors and on reported TT-WNV cases. The paucity of reported TT-WNV infections and the screening results suggest that blood safety interventions are effective. However, limited circulation of WNV in the EU and presumed underrecognition or underreporting of TT-WNV cases contribute to the present situation. Because of cross-reactivity between genetically related flaviviruses in the automated nucleic acid test systems, USUV-positive blood donations are found during routine WNV screening. The clinical relevance of USUV infection in humans and the risk of USUV to blood safety are unknown.

Mosquito-borne epornitic flaviviruses: an update and review.

West Nile Virus, Usutu virus, Bagaza virus, Israel turkey encephalitis virus and Tembusu virus currently constitute the five flaviviruses transmitted by mosquito bites with a marked pathogenicity for birds. They have been identified as the causative agents of severe neurological symptoms, drop in egg production and/or mortalities among avian hosts. They have also recently shown an expansion of their geographic distribution and/or a rise in cases of human infection. This paper is the first up-to-date review of the pathology of these flaviviruses in birds, with a special emphasis on the difference in susceptibility among avian species, in order to understand the specificity of the host spectrum of each of these viruses. Furthermore, given the lack of a clear prophylactic approach against these viruses in birds, a meta-analysis of vaccination trials conducted to date on these animals is given to constitute a solid platform from which designing future studies.

A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks.

Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses, such as Japanese encephalitis virus, West Nile virus, and Bagaza virus, is able to transmit vector-independently. To date, why these flaviviruses can be transmitted without mosquito vectors remains poorly understood. To explore the key molecular basis of flavivirus transmissibility, we compared virus replication and transmissibility of an early and a recent TMUV in ducks. The recent TMUV strain FX2010 replicated systemically and transmitted efficiently in ducks, while the replication of early strain MM1775 was limited and did not transmit among ducks. The TMUV envelope protein and its domain I were responsible for tissue tropism and transmissibility. The mutation S156P in the domain I resulted in disruption of N-linked glycosylation at amino acid 154 of the E protein and changed the conformation of "150 loop" of the E protein, which reduced virus replication in lungs and abrogated transmission in ducks. These data indicate that the 156S in the envelope protein is critical for TMUV tissue tropism and transmissibility in ducks in the absence of mosquitos. Our findings provide novel insights on understanding TMUV transmission among ducks.IMPORTANCE Tembusu virus, similar to other mosquito-borne flaviviruses such as WNV, JEV, and BAGV, can be transmitted without the presence of mosquito vectors. We demonstrate that the envelope protein of TMUV and its amino acid (S) at position 156 is responsible for tissue tropism and transmission in ducks. The mutation S156P results in disruption of N-linked glycosylation at amino acid 154 of the E protein and changes the conformation of "150 loop" of the E protein, which induces limited virus replication in lungs and abrogates transmission between ducks. Our findings provide new knowledge about TMUV transmission among ducks.

Molecular detection of viruses causing hemorrhagic fevers in rodents in the south-west of Korea.

Many pathogens causing hemorrhagic fevers of medical and veterinary importance have been identified and isolated from rodents in the Republic of Korea (ROK). We investigated the occurrence of emerging viruses causing hemorrhagic fevers, such as hemorrhagic fever with renal syndrome (HFRS), severe fever with thrombocytopenia syndrome (SFTS), and flaviviruses, from wild rodents. Striped field mice, Apodemus agrarius (n = 39), were captured during 2014-2015 in the south-west of ROK. Using molecular methods, lung samples were evaluated for SFTS virus, hantavirus, and flavivirus, and seropositivity was evaluated in the blood. A high positive rate of hantavirus (46.2%) was detected in A. agrarius lungs by reverse transcription-nested polymerase chain reaction (RT-N-PCR). The monthly occurrence of hantavirus was 16.7% in October, 86.7% in November, and 25% in August of the following year (p < 0.001). Moreover, 17.9% of blood samples were serologically positive for hantavirus antibodies. The most prevalent strain in A. agrarius was Hantaan virus. All samples were positive for neither SFTS virus nor flavivirus. Hantaan virus was detected in 86.7% of A. agrarius in November (autumn), and thus, virus shedding from A. agrarius can increase the risk of humans contracting HFRS. These findings may help to predict and prevent disease outbreaks in ROK.

Astrocytes in Flavivirus Infections.

Virus infections of the central nervous system (CNS) can manifest in various forms of inflammation, including that of the brain (encephalitis) and spinal cord (myelitis), all of which may have long-lasting deleterious consequences. Although the knowledge of how different viruses affect neural cells is increasing, understanding of the mechanisms by which cells respond to neurotropic viruses remains fragmented. Several virus types have the ability to infect neural tissue, and astrocytes, an abundant and heterogeneous neuroglial cell type and a key element providing CNS homeostasis, are one of the first CNS cell types to get infected. Astrocytes are morphologically closely aligned with neuronal synapses, blood vessels, and ventricle cavities, and thereby have the capacity to functionally interact with neurons and endothelial cells. In this review, we focus on the responses of astrocytes to infection by neurotropic flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), which have all been confirmed to infect astrocytes and cause multiple CNS defects. Understanding these mechanisms may help design new strategies to better contain and mitigate virus- and astrocyte-dependent neuroinflammation.

Spondweni Virus in Field-Caught Culex quinquefasciatus Mosquitoes, Haiti, 2016.

Spondweni virus (SPONV) and Zika virus cause similar diseases in humans. We detected SPONV outside of Africa from a pool of Culex mosquitoes collected in Haiti in 2016. This finding raises questions about the role of SPONV as a human pathogen in Haiti and other Caribbean countries.

Host genetic control of mosquito-borne Flavivirus infections.

Flaviviruses are arthropod-borne viruses, several of which represent emerging or re-emerging pathogens responsible for widespread infections with consequences ranging from asymptomatic seroconversion to severe clinical diseases and congenital developmental deficits. This variability is due to multiple factors including host genetic determinants, the role of which has been investigated in mouse models and human genetic studies. In this review, we provide an overview of the host genes and variants which modify susceptibility or resistance to major mosquito-borne flaviviruses infections in mice and humans.

The Many Faces of the Flavivirus NS5 Protein in Antagonism of Type I Interferon Signaling.

The vector-borne flaviviruses cause severe disease in humans on every inhabited continent on earth. Their transmission by arthropods, particularly mosquitoes, facilitates large emergence events such as witnessed with Zika virus (ZIKV) or West Nile virus in the Americas. Every vector-borne flavivirus examined thus far that causes disease in humans, from dengue virus to ZIKV, antagonizes the host type I interferon (IFN-I) response by preventing JAK-STAT signaling, suggesting that suppression of this pathway is an important determinant of infection. The most direct and potent viral inhibitor of this pathway is the nonstructural protein NS5. However, the mechanisms utilized by NS5 from different flaviviruses are often quite different, sometimes despite close evolutionary relationships between viruses. The varied mechanisms of NS5 as an IFN-I antagonist are also surprising given that the evolution of NS5 is restrained by the requirement to maintain function of two enzymatic activities critical for virus replication, the methyltransferase and RNA-dependent RNA polymerase. This review discusses the different strategies used by flavivirus NS5 to evade the antiviral effects of IFN-I and how this information can be used to better model disease and develop antiviral countermeasures.

Evaluating the risk for Usutu virus circulation in Europe: comparison of environmental niche models and epidemiological models.

BACKGROUND: Usutu virus (USUV) is a mosquito-borne flavivirus, reported in many countries of Africa and Europe, with an increasing spatial distribution and host range. Recent outbreaks leading to regional declines of European common blackbird (Turdus merula) populations and a rising number of human cases emphasize the need for increased awareness and spatial risk assessment. METHODS: Modelling approaches in ecology and epidemiology differ substantially in their algorithms, potentially resulting in diverging model outputs. Therefore, we implemented a parallel approach incorporating two commonly applied modelling techniques: (1) Maxent, a correlation-based environmental niche model and (2) a mechanistic epidemiological susceptible-exposed-infected-removed (SEIR) model. Across Europe, surveillance data of USUV-positive birds from 2003 to 2016 was acquired to train the environmental niche model and to serve as test cases for the SEIR model. The SEIR model is mainly driven by daily mean temperature and calculates the basic reproduction number R0. The environmental niche model was run with long-term bio-climatic variables derived from the same source in order to estimate climatic suitability. RESULTS: Large areas across Europe are currently suitable for USUV transmission. Both models show patterns of high risk for USUV in parts of France, in the Pannonian Basin as well as northern Italy. The environmental niche model depicts the current situation better, but with USUV still being in an invasive stage there is a chance for under-estimation of risk. Areas where transmission occurred are mostly predicted correctly by the SEIR model, but it mostly fails to resolve the temporal dynamics of USUV events. High R0 values predicted by the SEIR model in areas without evidence for real-life transmission suggest that it may tend towards over-estimation of risk. CONCLUSIONS: The results from our parallel-model approach highlight that relying on a single model for assessing vector-borne disease risk may lead to incomplete conclusions. Utilizing different modelling approaches is thus crucial for risk-assessment of under-studied emerging pathogens like USUV.

Usutu Virus RNA in Mosquitoes, Israel, 2014-2015.

We identified Usutu virus (USUV) RNA in 6 pools of mosquitoes trapped in northern Israel during 2014-2015. These Israeli strains were most similar to strains identified in Senegal and Germany, which further elucidates common ancestry and evolutionary dynamics of USUV. Our findings suggest that human infection with USUV might occur in Israel.

Characterization of Fitzroy River Virus and Serologic Evidence of Human and Animal Infection.

In northern Western Australia in 2011 and 2012, surveillance detected a novel arbovirus in mosquitoes. Genetic and phenotypic analyses confirmed that the new flavivirus, named Fitzroy River virus, is related to Sepik virus and Wesselsbron virus, in the yellow fever virus group. Most (81%) isolates came from Aedes normanensis mosquitoes, providing circumstantial evidence of the probable vector. In cell culture, Fitzroy River virus replicated in mosquito (C6/36), mammalian (Vero, PSEK, and BSR), and avian (DF-1) cells. It also infected intraperitoneally inoculated weanling mice and caused mild clinical disease in 3 intracranially inoculated mice. Specific neutralizing antibodies were detected in sentinel horses (12.6%), cattle (6.6%), and chickens (0.5%) in the Northern Territory of Australia and in a subset of humans (0.8%) from northern Western Australia.