Histopathological changes at the mosquito bite and CHIKV injected sites.

<p>Biopsies obtained from mouse ear samples were fixed in 10% neutral formalin and paraffin embedded. Four to five millimeter sections were made and H&E stained. Slides were observed for cellular recruitment at the mosquito bite site or CHIKV injection site. Yellow arrows in the images point to eosinophils. A-uninfected mice (naïve); B- uninfected mosquito bite site (3hpf); C- uninfected mosquito bite site (6 hpf); D-CHIKV infected mosquito bite site (3 hpf); E-CHIKV infected mosquito bite site (6 hpf); F-medium injected site, G-needle injected CHIKV site(3 hpi); H-needle injected CHIKV site (6 hpi). <i>N = 3</i> per group.</p

Differential expression of cytokines and TLR-3 induced by CHIKV infected mosquito bites.

<p>Relative fold differences were calculated by considering uninfected mosquito bites as naïve. (UIM- uninfected mosquito bites; CIM- CHIKV infected mosquito bites).</p><p>*- P≤0.05; **- p≤0.001.</p

Comparison between uninfected mosquito (UIM) bites and CHIKV infected mosquito (CIM) bites.

<p>Uninfected and CHIKV infected <i>Ae. aegypti</i> ware allowed to feed upon mouse ears, and total RNA was extracted from biopsies at the indicated times. Real-time RT-PCR was performed to measure expression of the indicated cytokine mRNAs. RNA extracted from ears of mice not exposed to mosquitoes were considered as naïve and assigned an arbitrary value of 1.0, and changes in mosquito-induced cytokine gene expression are expressed as the ratio between mosquito-fed and naïve samples. GAPDH gene was used as a normalizing control. The asterisk denotes a statistically significant difference between the means of naïve and experimental groups (*-<i>P</i>≤0·05; **- <i>P</i>≤0·001). <i>N</i> = 3 per group.</p

Comparison between CHIKV infected mosquito (CIM) bites and needle injected CHIKV.

<p>CHIKV infected <i>Ae. aegypti</i> were allowed to feed on mouse ears and total RNA was extracted from biopsies at the indicated times. In parallel, total RNA was extracted from mouse ear biopsies at sites of needle inoculation of CHIKV or medium without virus. Real-time RT-PCR was performed to measure expression of the indicated cytokine mRNAs. RNA extracted from ears of mice not exposed to mosquitoes was considered as naïve for CHIKV infected mosquito bite tissue samples. Medium-inoculated mouse biopsy samples were considered naive for needle inoculated CHIKV samples. Naive samples were assigned an arbitrary value of 1.0, and changes in mosquito-induced cytokine gene expression were expressed as the ratio between mosquito-fed and naïve samples. GAPDH gene was used as a normalizing control. The asterisk denotes a statistically significant difference between the means of naïve and experimental groups (*-<i>P</i>≤0·05; **- <i>P</i>≤0·001). <i>N</i> = 3 per group.</p

Primers used for real-time RT-PCR.

<p>Primers used for real-time RT-PCR.</p

Mosquito TRAPS.

<p>(A) CDC light with Octanol, (B) Modified Trinidad 17, baited with one hamster and (C) EVS baited with CO₂.</p

Full genome sequence database available for recognised species of genus <i>Orbivirus</i> and their genome coding assignments.

<p><b>Pol</b> = Polymerase, <b>OC1</b> = Outer capsid protein 1 (VP2 of BTV), <b>T2</b> = Inner core protein (T2 symmetry), <b>Cap</b> = Capping enzyme, <b>Tup</b> = Tubule forming protein or Tubular protein (NS1), <b>OC2</b> = Outer capsid protein 2 (VP5 of BTV), <b>T13</b> = Outer core protein (T13 symmetry), <b>ViP</b> = Viral inclusion body protein (NS2), <b>Hel</b> = Helicase protein. NCR and G+C content were calculated for full genome sequences.</p

Characteristics of Corriparta virus (CORV-MRM1) (AUS1960/01) genome segments (dsRNA) and their encoded proteins.

<p>Pol = RNA polymerase; OC = outer capsid protein; Cap = capping enzyme (guanylyltransferase); Hel = helicase enzyme; T2 = protein with T = 2 symmetry; T13 = Protein with T = 13 symmetry; ViP = viral inclusion body matrix protein; TuP = tubule protein. OC1 and OC2 refer to the larger and smaller outer capsid proteins respectively.</p

Mosquito TRAPS.

<p>(A) CDC light with Octanol, (B) Modified Trinidad 17, baited with one hamster and (C) EVS baited with CO₂.</p

Agarose gel (1%) electrophoresis for dsRNAs of CORV, BTV and EHDV.

<p>The purified genomic RNA of LIB2007/07 (a western-topotype isolate of BTV serotype 1 (BTV-1w)) - lane 1: AUS1982/05 (an eastern-topotype isolate of EHDV serotype 8 (EHDV-8e)) - lane 2: and CORV-MRM1 (AUS1960/01) - lane 3, were analysed by 1% agarose gel electrophoresis (AGE), then stained with ethidium bromide, visualised and photographed under UV illumination. The genome segment numbers (encoded proteins) are indicated at the side of the gel.</p