Evaluation of platforms to detect Zika and West Nile virus from honeycards

Transcription

1 059 - Evaluation of platforms to detect Zika and West Nile virus from honeycards in Florida PI: Nathan Burkett-Cadena Florida Medical Entomology Laboratory University of Florida IFAS 00 9 th St. SE Vero Beach, FL nburkettcadena@ufl.edu Co-PI: Barry Alto Florida Medical Entomology Laboratory University of Florida IFAS 00 9 th St. SE Vero Beach, FL nburkettcadena@ufl.edu FL DACS Contract # 059 Final Report: August 15, 017

2 The objective project 059 was to test the sensitivity and specificity of two commercially available platforms (Genie II and RAMP) for detecting West Nile virus (WNV) and Zika virus (ZIKV) from honey-cards. In BSL conditions, vector mosquitoes (Aedes aegypti for ZIKV and Culex quinquefasciatus for WNV) were orally infected with respective virus and their saliva collected onto honey cards (Fig 1). Honey cards were held over varying lengths of time (1, and 7 days) and tested by Genie II, RAMP and RT-PCR for WNV and ZIKV. The goal of the research is to determine whether these commercially available arbovirus detection platforms can be used to test honey cards for mosquito-borne viruses. This research will have immediate impact on the ability of mosquito control districts in Florida to perform timely surveillance for mosquito-borne virus transmission, which will translate into an improved ability to protect Florida from these deadly pathogens. This project is complete. WNV-inoculated honey cards (honey cards also referred to as cards ) produced positive real time RT-PCR results for all 5 replicates of titers (.8,.8 and 4.8 log10 PFU/mL) and time points (1,, and 7 days) (Fig ). For cards inoculated with.8 or 4.8 log10 PFU/mL WNV, there were no statistically significant differences between means at days 1,, or 7 (.8 log10 PFU/mL: F,1 = 1.17, p = 0.4; 4.8 log10 PFU/ml: F,11 = 1.88, p = 0.0). Cards inoculated with.8 log10 PFU/mL produced statistically significantly higher CT values when incubated for 7 days compared to cards incubated for 1 or days (F,1 = 8.59, p = 0.005). LAMP results for the WNV-inoculated cards are found in Table 1. All cards (5/5) inoculated with the highest dose (4.8 log10 PFU/mL) and held for 1 or days were positive by the LAMP assay; by day 7, 4/5 replicates inoculated with the highest dose were detectable. Four of 5 cards inoculated with.8 and.8 log10 PFU/mL after 1 day of incubation were positive by LAMP; longer incubation periods resulted in fewer positive cards detected. None of the WNV inoculated cards produced positive results ( 50) in the RAMP WNV assay. ZIKAV-inoculated cards also produced positive real time RT-PCR results (CT 8) at all 5 replicates of titers (.6, 4.6 and 5.6 log10 PFU/mL) and time points (Fig ). Cards inoculated with 4.6 and 5.6 log10 PFU/mL and held for or 7 days produced statistically significantly higher mean CTs when compared to cards held for 1 day (4.6 log10 PFU/mL: (F,1 = 9.7, p = 0.00; 5.6 log10 PFU/ml: (F,1 = 4.4, p = 0.04). There was no statistically significant difference in mean CTs in cards inoculated with.6 log10 PFU/ml at any time point (F,11 = 1.818, p = 0.1). LAMP results for the ZIKA-inoculated cards are found in Table 1. For all titers, the number of replicates detectable by LAMP decreased as incubation periods increased. After 7 days incubation, few cards (/5) inoculated with 4.6 and 5.6 log10 PFU/mL and none of the cards inoculated with.6 log10 PFU/mL were detectable by the LAMP assay. Of the 5 mosquitoes that had disseminated WNV infections and individually fed on honey cards (indicated by the presence of blue dye in their crop), a total of 18 (7%) expectorated virus that was detectable on the honey cards by real time RT-PCR (Table ). By time point, 6.5% (5/8), 80% (8/10), and 71.4% (5/7) of cards held for 1,, or 7 days, respectively, were detectable by real time RT-PCR. There was no significant difference in mean CTs of cards collected on days 1,, or 7 (F,15 = , p = 0.861). Two of the 5 (8%) infected mosquitoes with disseminated infections expectorated virus that was detectable by the LAMP assay, both detected on day.

3 Of the 1 ZIKAV-positive mosquitoes that had disseminated infections and fed on honey cards, a total of 4 (.%) expectorated virus that was detectable by real time RT-PCR (Table ). The mean CTs of cards held for 1 day were statistically significantly higher than cards held for days (p = 0.008). No virus was detected in cards held for 7 days by real time RT-PCR. The ZIKA LAMP assay did not detect virus from any of the mosquito-fed honey cards. These results suggest that to maximize the detection of virus, honey cards should be left in the traps no longer than 1 day if using the LAMP assays, and no longer than days if using real time RT-PCR. The WNV RAMP assay, which is an option available to mosquito control districts for pool-based surveillance, is not recommended for honey card surveillance. Future studies may determine the minimum number of infectious mosquitoes required to feed on a honey card that would be reliably detected by the LAMP or RAMP assays. Real time RT-PCR proved to be the most sensitive of the assays and the most resistant to viral RNA degradation over time. While a positive honey card result would unequivocally indicate that there are positive mosquitoes in the population, negative results would be harder to interpret. If honey cards are used as an initial screening method, a an algorithm may be adopted in which mosquitoes from traps that produce negative cards are pooled and tested by methods optimized for pool-based testing to avoid false-negative results, either due to assay sensitivity limits or if trapped positive mosquitoes did not feed on the substrate.

4 Table 1. Loop-mediated amplification (LAMP) detection of WNV and ZIKAV RNA from artificially inoculated honey cards (n = 5) after incubation for 1,, and 7 days at 8 C and 60-80% RH. No. positive cards per days incubation Inoculation Day Day Day 7 titer (log 10 PFU/ml) 1 WNV.8 4 (80%) (60%) (60%).8 4 (80%) (60%) (40%) (100%) (100%) (80%) ZIKAV.6 5 (100%) (80%) (100%) (40%) (60%) (80%) 4 (40%) (40%) 0 Table. Real time RT-PCR and Loop-mediated amplification (LAMP) viral RNA detection from honey cards fed upon by mosquitoes with disseminated WNV (n = 5) and ZIKAV (n = 1) infections. Cards were incubated for 1,, or 7 days at 8 C and 60-80% RH. No. cards positive by real-time RT-PCR No. cards positive by LAMP Day 1 Day Day 7 Total cards Day 1 Day Day 7 Total cards WNV 5/8 (6.5%) 8/10 (80%) 5/7 (71.4%) Mean 4.9 (1.49) 4.8 (1.96) 4.1 (.5) CT (SD) ZIKAV /4 (50%) /4 (50%) 0/4 Mean N/A CT (.08) (0.85) (SD) 18/5 (7%) 4/1 (.%) 0/8 0/4 /10 (0%) 0/4 0 /7 0/4 /5 (8%) 0/1

5 Figure 1.

6 Fig. Real time RT-PCR detection of WNV RNA from artificially inoculated honey cards (n = 5) after incubation for 1,, and 7 days at 8 C and 60-80% RH. Fig. Real time RT-PCR detection of ZIKAV RNA from artificially inoculated honey cards (n = 5) after incubation for 1,, and 7 days at 8 C and 60-80% RH.