Arboviruses and their Vectors

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.

Virus-host interactions play important roles in virus infection and host cellular response. Several viruses, including dengue virus (DENV), usurp host chaperones to support their amplification and survival in the host cell. We investigated the interaction of nonstructural protein 1 (NS1) of DENV with three endoplasmic reticulum-resident chaperones (i.e. GRP78, calnexin and calreticulin) to delineate their functional roles and potential binding sites for protein complex formation. GRP78 protein showed prominent association with DENV NS1 in virus-infected Huh7 cells as evidenced by co-localization and co-immunoprecipitation assays. Further studies on the functional interaction of GRP78 protein were performed by using siRNA-mediated gene knockdown in a DENV replicon transfection system. GRP78 knockdown significantly decreased intracellular NS1 production and delayed NS1 secretion but had no effect on viral RNA replication. Dissecting the important domain of GRP78 required for DENV NS1 interaction showed co-immunoprecipitation of DENV NS1 with a full-length and substrate-binding domain (SBD), but not an ATPase domain, of GRP78, confirming their interaction through SBD binding. Molecular dynamics simulations of DENV NS1 and human GRP78 complex revealed their potential binding sites through hydrogen and hydrophobic bonding. The majority of GRP78-binding sites were located in a β-roll domain and connector subdomains on the DENV NS1 structure involved in hydrophobic surface formation. Taken together, our findings demonstrated the roles of human GRP78 in facilitating the intracellular production and secretion of DENV NS1 as well as predicted potential binding sites between the DENV NS1 and GRP78 complex, which could have implications in the future development of target-based antiviral drugs.

Arboviruses are viral pathogens that are transmitted from an animal reservoir to humans via an arthropod vector. These viruses result in a large burden of disease worldwide and show a propensity for establishing new endemic foci in geographically distant regions. The potential impact of arboviruses in Central Asia is unclear due to the scarcity of reports available in English; however, the collation of available data shows that numerous important human viruses are circulating in the region. Pathogens such as Crimean–Congo haemorrhagic fever virus, tick-borne encephalitis virus and Tahyna virus are likely to be responsible for numerous cases of human disease in Central Asia on an annual basis. There is evidence that pathogens such as West Nile virus and sandfly fever virus have resulted in sporadic outbreaks of human disease across the region; these events appear to be triggered by a significant change in the abundance of local arthropod vectors or events altering the contact between humans and local arthropod populations, such as conflict or natural disasters. In addition, there are several under-researched arboviruses that could result in a significant disease, including Karshi virus, Issyk-Kul virus and Syr-Darya Valley fever virus. This review provides the first comprehensive assessment of emerging arboviruses in Central Asia. Further research is required to assess the full impact of arboviruses on human health in the region and to monitor potential spread. Up-to-date information regarding arbovirus endemicity will allow for the development and distribution of rapid diagnostics, the implementation of bite-prevention strategies in at-risk areas and improved travel recommendations.

Dengue virus (DENV) replication between mosquito and human hosts is hypothesized to be associated with viral determinants that interact in a differential manner between hosts. However, the understanding of inter-host viral determinants that drive DENV replication and growth between hosts is limited. Through the use of clinical isolates, we identified an amino acid variation of Ala, Met and Val at position 116 of DENV-1 NS4B. While the proportion of virus with the NS4B-116V variant remained constantly high in serial passages in a mosquito cell line, populations of the NS4B-116M and NS4B-116A variants became dominant after serial passages in mammalian cell lines. Using recombinant DENV-1 viruses, the Val to Ala or Met alteration at position NS4B-116 (rDENV-1-NS4B-116A and rDENV-1-NS4B-116M) resulted in enhanced virus growth in human cells in comparison to the clone with Val at NS4B-116 (rDENV-1-NS4B-116V). However, the reverse phenomenon was observed in a mosquito cell line. Additionally, in a human cell line, differential levels of IFN-α/β and IFN-stimulated gene expressions (IFIT3, IFI44L, OAS1) suggested that the enhanced viral growth was dependent on the ability of the NS4B protein to hamper host IFN response during the early phase of infection. Overall, we identified a novel and critical viral determinant at the pTMD3 of NS4B region that displayed differential effects on DENV replication and fitness in human and mosquito cell lines. Taken together, the results suggest the importance of the NS4B protein in virus replication and adaptation between hosts.

Southeastern Brazil has been suffering a rapid expansion of a severe sylvatic yellow fever virus (YFV) outbreak since late 2016, which has reached one of the most populated zones in Brazil and South America, heretofore a yellow fever-free zone for more than 70 years. In the current study, we describe the complete genome of 12 YFV samples from mosquitoes, humans and non-human primates from the Brazilian 2017 epidemic. All of the YFV sequences belong to the modern lineage (sub-lineage 1E) of South American genotype I, having been circulating for several months prior to the December 2016 detection. Our data confirm that viral strains associated with the most severe YF epidemic in South America in the last 70 years display unique amino acid substitutions that are mainly located in highly conserved positions in non-structural proteins. Our data also corroborate that YFV has spread southward into Rio de Janeiro state following two main sylvatic dispersion routes that converged at the border of the great metropolitan area comprising nearly 12 million unvaccinated inhabitants. Our original results can help public health authorities to guide the surveillance, prophylaxis and control measures required to face such a severe epidemiological problem. Finally, it will also inspire other workers to further investigate the epidemiological and biological significance of the amino acid polymorphisms detected in the Brazilian 2017 YFV strains.

We studied minor variants within two tick-borne encephalitis virus (TBEV) populations with a common ancestor: the mouse brain-adapted variant EK-328c and the tick-adapted variant M. High-throughput sequencing with custom amplicons from RT-PCR viral RNA was performed on Illumina MiSeq 2*250 paired-end v2 chemistry. Using the LowFreq program (default settings) and Sanger-sequenced consensus as a reference, variants with an abundance of 1 % and above within the studied populations were identified. Using the obtained data in the context of our previous studies, we concluded that TBEV variants, which are different from the major population phenotype and can become a major part of the viral population under favourable environmental conditions, can exist at abundances of less than 1 % in the long-term. The comparison of our data with the literature allowed us to conclude that the laboratory variant EK-328c and variant M have similar SNV counts to TBEV variants from natural populations and some fast-evolving RNA viruses.

Toll-like receptors and RNA helicases are involved in the control of RNA virus infection through production of type I interferons (IFNs). To delineate the relative contributions of these signalling pathways in the innate immune response and the control of Zika virus (ZIKV) pathogenesis, the impact of a deficiency in TRIF and/or IPS-1 adaptor proteins was investigated in mice. Mice were infected intravenously with ZIKV and monitored for clinical signs for 14 days. Groups of mice were sacrificed on days 1, 3 and 7 post-infection (p.i.) and viral RNA was measured by digital droplet PCR in serum, spleen, brain and eyes. Some mice were sacrificed at 12 h p.i. for determination of the levels of IFN-α/–β (ELISA), cytokines/chemokines (Luminex) and total/phosphorylated IRF3 and IRF7 (Western blotting). All groups of mice infected with ZIKV exhibited no clinical signs of infection. However, IPS-1−/− and TRIF−/−xIPS-1−/− mice developed higher viraemia than WT and TRIF−/− groups on days 1, 3 and 7. TRIF−/−xIPS-1−/− mice presented higher viral RNA levels in spleen, brain and eyes over time than TRIF−/−, IPS-1−/− and WT groups. At 12 h, IFN-α/-β and cytokine/chemokine levels in spleen were significantly decreased in IPS-1−/− and TRIF−/−xIPS-1−/− compared to WT and TRIF−/−. On day 1 p.i., IFN-β levels were significantly reduced in spleen of TRIF−/−xIPS-1−/− mice compared to all other groups. These data suggest that IPS-1 plays a more important role than TRIF in the early type I IFN response and that both IPS-1 and TRIF are involved at later stages of ZIKV infection.

Dengue virus NS1 is a glycoprotein of 46–50 kDa that is conserved among flaviviruses, associates as a dimer to cell membranes and is secreted as a hexamer to the extracellular milieu. Recent evidence showed that NS1 is secreted efficiently from infected mosquito cells. To explore the secretory route of NS1 in mosquito cells, infected cells were treated with brefeldin A (BFA) and methyl-beta-cyclodextrin (MβCD). The results showed that MβCD, but not BFA, significantly reduced the release of NS1. Moreover, silencing the expression of caveolin-1 (CAV1; a key component of the caveolar system that transports cholesterol inside the cell), but not SAR1 (a GTPase that participates in the classical secretory pathway), also results in a significant reduction of the secretion of NS1. These results indicate that NS1 is released from mosquito cells via an unconventional secretory route that bypasses the Golgi complex, with the participation of CAV1. In agreement with this notion, differences were observed in the glycosylation status between secreted NS1 and E proteins. Classical mechanics and docking simulations suggested highly favoured interactions between the caveolin-binding domain present in NS1 and the scaffolding domain of CAV1. Finally, proximity ligation assays showed direct interaction between NS1 and CAV1 in infected mosquito cells. These findings are in line with the lipoprotein nature of secreted NS1 and provide new insights into the biology of NS1.

Every year, tick-borne encephalitis virus (TBEV) causes severe central nervous system infection in 10 000 to 15 000 people in Europe and Asia. TBEV is maintained in the environment by an enzootic cycle that requires a tick vector and a vertebrate host, and the adaptation of TBEV to vertebrate and invertebrate environments is essential for TBEV persistence in nature. This adaptation is facilitated by the error-prone nature of the virus’s RNA-dependent RNA polymerase, which generates genetically distinct virus variants called quasispecies. TBEV shows a focal geographical distribution pattern where each focus represents a TBEV hotspot. Here, we sequenced and characterized two TBEV genomes, JP-296 and JP-554, from questing Ixodes ricinus ticks at a TBEV focus in central Sweden. Phylogenetic analysis showed geographical clustering among the newly sequenced strains and three previously sequenced Scandinavian strains, Toro-2003, Saringe-2009 and Mandal-2009, which originated from the same ancestor. Among these five Scandinavian TBEV strains, only Mandal-2009 showed a large deletion within the 3′ non-coding region (NCR), similar to the highly virulent TBEV strain Hypr. Deep sequencing of JP-296, JP-554 and Mandal-2009 revealed significantly high quasispecies diversity for JP-296 and JP-554, with intact 3′NCRs, compared to the low diversity in Mandal-2009, with a truncated 3′NCR. Single-nucleotide polymorphism analysis showed that 40 % of the single-nucleotide polymorphisms were common between quasispecies populations of JP-296 and JP-554, indicating a putative mechanism for how TBEV persists and is maintained within its natural foci.

During virus multiplication, the viral genome is recognized and recruited for replication based on specific cis-acting elements. Here, we dissected the important cis-acting sequence elements in Semliki Forest virus RNA by using a trans-replication system. As the viral replicase is expressed from a separate plasmid, the template RNA can be freely modified in this system. We show that the cis-acting element at the beginning of the non-structural protein 1 (nsP1) coding region together with the end of the 3′ UTR are the minimal requirements for minus-strand synthesis. To achieve a high level of replication, the native 5′ UTR was also needed. The virus-induced membranous replication compartments (spherules) were only detected when a replication-competent template was present with an active replicase and minus strands were produced. No translation could be detected from the minus strands, suggesting that they are segregated from the cytoplasm. Minus strands could not be recruited directly to initiate the replication process. Thus, there is only one defined pathway for replication, starting with plus-strand recognition followed by concomitant spherule formation and minus-strand synthesis.

Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the molecular mechanisms involved in CHIKV infection. A critical step in the life cycle of CHIKV is fusion of the viral membrane with a host cell membrane. Here, we elucidate this process using ensemble-averaging liposome–virus fusion studies, in which the fusion behaviour of a large virus population is measured, and a newly developed microscopy-based single-particle assay, in which the fusion kinetics of an individual particle can be visualised. The combination of these approaches allowed us to obtain detailed insight into the kinetics, lipid dependency and pH dependency of hemifusion. We found that CHIKV fusion is strictly dependent on low pH, with a threshold of pH 6.2 and optimal fusion efficiency below pH 5.6. At this pH, CHIKV fuses rapidly with target membranes, with typically half of the fusion occurring within 2 s after acidification. Cholesterol and sphingomyelin in the target membrane were found to strongly enhance the fusion process. By analysing our single-particle data using kinetic models, we were able to deduce that the number of rate-limiting steps occurring before hemifusion equals about three. To explain these data, we propose a mechanistic model in which multiple E1 fusion trimers are involved in initiating the fusion process.

The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.