Temperature modulates immune gene expression in mosquitoes during arbovirus infection.

The principal vector of dengue, Zika and chikungunya viruses is the mosquito Aedes aegypti, with its ability to transmit pathogens influenced by ambient temperature. We use chikungunya virus (CHIKV) to understand how the mosquito transcriptome responds to arbovirus infection at different ambient temperatures. We exposed CHIKV-infected mosquitoes to 18, 28 and 32°C, and found that higher temperature correlated with higher virus levels, particularly at 3 days post infection, but lower temperature resulted in reduced virus levels. RNAseq analysis indicated significantly altered gene expression levels in CHIKV infection. The highest number of significantly differentially expressed genes was observed at 28°C, with a more muted effect at the other temperatures. At the higher temperature, the expression of many classical immune genes, including Dicer-2, was not substantially altered in response to CHIKV. The upregulation of Toll, IMD and JAK-STAT pathways was only observed at 28°C. Functional annotations suggested that genes in immune response and metabolic pathways related to energy supply and DNA replication were involved in temperature-dependent changes. Time post infection also led to substantially different gene expression profiles, and this varied with temperature. In conclusion, temperature significantly modulates mosquito gene expression in response to infection, potentially leading to impairment of immune defences at higher temperatures.

Chikungunya Virus Transmission at Low Temperature by Aedes albopictus Mosquitoes.

Aedes albopictus is an important vector of chikungunya virus (CHIKV). In Australia, Ae. albopictus is currently only known to be present on the islands of the Torres Strait but, should it invade the mainland, it is projected to spread to temperate regions. The ability of Australian Ae. albopictus to transmit CHIKV at the lower temperatures typical of temperate areas has not been assessed. Ae. albopictus mosquitoes were orally challenged with a CHIKV strain from either Asian or East/Central/South African (ECSA) genotypes (107 pfu/mL), and maintained at a constant temperature of either 18 °C or 28 °C. At 3- and 7-days post-infection (dpi), CHIKV RNA copies were quantified in mosquito bodies, and wings and legs using real time polymerase chain reaction (qRT-PCR), while the detection of virus in saliva (a proxy for transmission) was performed by amplification in cell culture followed by observation of cytopathic effect in Vero cells. Of the ≥95% of Ae. albopictus that survived to 7 dpi, all mosquitoes became infected and showed body dissemination of CHIKV at both temperatures and time points. Both the Asian and ECSA CHIKV genotypes were potentially transmissible by Australian Ae. albopictus at 28 °C within 3 days of oral challenge. In contrast, at 18 °C none of the mosquitoes showed evidence of ability to transmit either genotype of CHIKV at 3 dpi. Further, at 18 °C only Ae. albopictus infected with the ECSA genotype showed evidence of virus in saliva at 7 dpi. Overall, infection with the ECSA CHIKV genotype produced higher virus loads in mosquitoes compared to infection with the Asian CHIKV genotype. Our results suggest that lower ambient temperatures may impede transmission of some CHIKV strains by Ae. albopictus at early time points post infection.

Vector competence of Australian Aedes aegypti and Aedes albopictus for an epidemic strain of Zika virus.

BACKGROUND: Recent epidemics of Zika virus (ZIKV) in the Pacific and the Americas have highlighted its potential as an emerging pathogen of global importance. Both Aedes (Ae.) aegypti and Ae. albopictus are known to transmit ZIKV but variable vector competence has been observed between mosquito populations from different geographical regions and different virus strains. Since Australia remains at risk of ZIKV introduction, we evaluated the vector competence of local Ae. aegypti and Ae. albopictus for a Brazilian epidemic ZIKV strain. In addition, we evaluated the impact of daily temperature fluctuations around a mean of 28°C on ZIKV transmission and extrinsic incubation period. METHODOLOGY/PRINCIPAL FINDINGS: Mosquitoes were orally challenged with a Brazilian ZIKV strain (8.8 log CCID50/ml) and maintained at either 28°C constant or fluctuating temperature conditions. At 3, 7 and 14 days post-infection (dpi), ZIKV RNA copies were quantified in mosquito bodies, as well as wings and legs, using qRT-PCR, while virus antigen in saliva (a proxy for transmission) was detected using a cell culture ELISA. Despite high body and disseminated infection rates in both vectors, the transmission rates of ZIKV in saliva of Ae. aegypti (50-60%) were significantly higher than in Ae. albopictus (10%) at 14 dpi. Both species supported a high viral load in bodies, with no significant differences between constant and fluctuating temperature conditions. However, a significant difference in viral load in wings and legs between species was observed, with higher titres in Ae. aegypti maintained at constant temperature conditions. For ZIKV transmission to occur in Ae. aegypti, a disseminated virus load threshold of 7.59 log10 copies had to be reached. CONCLUSIONS/SIGNIFICANCE: Australian Ae. aegypti are better able to transmit a Brazilian ZIKV strain than Ae. albopictus. The results are in agreement with the global consensus that Ae. aegypti is the major vector of ZIKV.

Chikungunya virus in Asia - Pacific: a systematic review.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes an acute febrile syndrome and severe, debilitating rheumatic disorders in humans that may persist for months. CHIKV's presence in Asia dates from at least 1954, but its epidemiological profile in the region remains poorly understood. We systematically reviewed CHIKV emergence, epidemiology, clinical features, atypical manifestations and distribution of virus genotypes, in 47 countries from South East Asia (SEA) and the Western Pacific Region (WPR) during the period 1954-2017. Following the Cochrane Collaboration guidelines, Pubmed and Scopus databases, surveillance reports available in the World Health Organisation (WHO) and government websites were systematically reviewed. Of the 3504 records identified, 461 were retained for data extraction. Although CHIKV has been circulating in Asia almost continuously since the 1950s, it has significantly expanded its geographic reach in the region from 2005 onwards. Most reports identified in the review originated from India. Although all ages and both sexes can be affected, younger children and the elderly are more prone to severe and occasionally fatal forms of the disease, with child fatalities recorded since 1963 from India. The most frequent clinical features identified were arthralgia, rash, fever and headache. Both the Asian and East-Central-South African (ECSA) genotypes circulate in SEA and WPR, with ECSA genotype now predominant. Our findings indicate a substantial but poorly documented burden of CHIKV infection in the Asia-Pacific region. An evidence-based consensus on typical clinical features of chikungunya could aid in enhanced diagnosis and improved surveillance of the disease.

Zika Virus in the Male Reproductive Tract.

Arthropod-borne viruses (arboviruses) are resurging across the globe. Zika virus (ZIKV) has caused significant concern in recent years because it can lead to congenital malformations in babies and Guillain-Barré syndrome in adults. Unlike other arboviruses, ZIKV can be sexually transmitted and may persist in the male reproductive tract. There is limited information regarding the impact of ZIKV on male reproductive health and fertility. Understanding the mechanisms that underlie persistent ZIKV infections in men is critical to developing effective vaccines and therapies. Mouse and macaque models have begun to unravel the pathogenesis of ZIKV infection in the male reproductive tract, with the testes and prostate gland implicated as potential reservoirs for persistent ZIKV infection. Here, we summarize current knowledge regarding the pathogenesis of ZIKV in the male reproductive tract, the development of animal models to study ZIKV infection at this site, and prospects for vaccines and therapeutics against persistent ZIKV infection.

Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus.

In an effort to simplify and expand the utility of African horse sickness virus (AHSV) reverse genetics, different plasmid-based reverse genetics systems were developed. Plasmids containing cDNAs corresponding to each of the full-length double-stranded RNA genome segments of AHSV-4 under control of a T7 RNA polymerase promoter were co-transfected in cells expressing T7 RNA polymerase, and infectious AHSV-4 was recovered. This reverse genetics system was improved by reducing the required plasmids from 10 to five and resulted in enhanced virus recovery. Subsequently, a T7 RNA polymerase expression cassette was incorporated into one of the AHSV-4 rescue plasmids. This modified 5-plasmid set enabled virus recovery in BSR or L929 cells, thus offering the possibility to generate AHSV-4 in any cell line. Moreover, mutant and cross-serotype reassortant viruses were recovered. These plasmid DNA-based reverse genetics systems thus offer new possibilities for investigating AHSV biology and development of designer AHSV vaccine strains.

African horse sickness virus induces apoptosis in cultured mammalian cells.

Infection of mammalian cell cultures with African horse sickness virus (AHSV) is known to result in dramatic cytopathic effects (CPE), but no CPE is observed in infected insect cell cultures despite productive virus replication. The basis for this phenomenon has not yet been investigated, but is suggestive of apoptosis being induced following virus infection of the mammalian cells. To investigate whether AHSV can induce apoptosis in infected mammalian cells, Culicoides variipennis (KC) insect cells and BHK-21 mammalian cells were infected with AHSV-9 and analyzed for morphological and biochemical hallmarks of apoptosis. In contrast to KC cells, infection of BHK-21 cells with AHSV-9 resulted in ultrastructural changes and nuclear DNA fragmentation, both of which are associated with the induction of apoptosis. Results also indicated that AHSV-9 infection of BHK-21 cells resulted in activation of caspase-3, a key agent in apoptosis, and in mitochondrial membrane depolarization. Cumulatively, the data indicate that the intrinsic pathway is activated in AHSV-induced apoptosis.

Membrane permeabilization of the African horse sickness virus VP5 protein is mediated by two N-terminal amphipathic α-helices.

The VP5 outer capsid protein of African horse sickness virus (AHSV) is cytotoxic when expressed in Spodoptera frugiperda (Sf-9) cells. Secondary structure analysis of the VP5 amino acid sequence of AHSV-9 identified two N-terminal amphipathic α-helices within the first 43 amino acids. Baculovirus expression of N- and C-terminal truncated VP5 proteins in Sf-9 cells indicated that the N-terminal 43 amino acids correlated with low levels of protein expression and with increased membrane permeabilization and cytotoxicity. Exogenous addition of chemically synthesized VP5 peptides indicated that both N-terminal amphipathic α-helices are required for membrane permeabilization of Sf-9 cells. These findings suggest that AHSV VP5 is a membrane-destabilizing protein.

Silencing of African horse sickness virus VP7 protein expression in cultured cells by RNA interference.

RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of homologous mRNA. Short interfering RNAs (siRNAs) are the mediators of RNAi and represent powerful tools to silence gene expression in mammalian cells including genes of viral origin. In this study, we applied siRNAs targeting the VP7 gene of African horse sickness virus (AHSV) that encodes a structural protein required for stable capsid assembly. Using a VP7 expression reporter plasmid and an in vitro model of infection, we show that synthetic siRNA molecules corresponding to the AHSV VP7 gene silenced effectively VP7 protein and mRNA expression, and decreased production of infectious virus particles as evidenced by a reduction in the progeny virion titres when compared to control cells. This work establishes RNAi as a genetic tool for the study of AHSV and offers new possibilities for the analysis of viral genes important for AHSV physiology.