La Crosse virus interactions with bacteria isolated from mosquito digestive tracts

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1 La Crosse virus interactions with bacteria isolated from mosquito digestive tracts Erica Weston and Jennifer Ridge Department of Biology Radford University Radford, VA

2 Introduction Aedes albopictus (Skuse) is a potential vector for many arboviruses, including La Crosse virus (LACV), the leading cause of pediatric encephalitis in North America. La Crosse virus (LACV) is a member of the California serogroup, Genus Orthobunyavirus, Family Bunyaviridae.

3 La Crosse virus Leading cause of pediatric encephalitis in North America, >90% of cases reported in children under 15 years old. Case fatality ratio of <1% ~70 cases reported yearly Specific antivirals and vaccines against LACV do not currently exist.

4 Transmission Transovarial and Venereal Transmission Cycle Amplification Cycle Dead-end hosts, including humans and pets Vectors

5 Mosquito infections Pathogens entering the midgut must interact with both the midgut and resident bacteria The microorganisms present may be genetically manipulated to prevent establishment of a viral infection in the vector

6 Hypothesis Bacteria resident in mosquito tissues can reduce infectivity of La Crosse virus

7 Methods Dissect midguts & diverticula Plate on nutrient agar Isolate individual colonies Amplify 16S rrna gene Sequence Identify using BLAST Incubate with LACV Inoculate Vero monolayers Compare plaque numbers

8 Bacterial isolates BLAST results Isolate Accession Bases a Best match b Accession % identity AaMG1 GQ Erwinia quercina AJ AaMG2 GQ Vagococcus salmoninarium Y AaMG3 GQ Kluyvera cryocrescens AM AaMG4 GQ Enterobacter ludwigii EF AaMG5 GQ Enterobacter ludwigii EF AaMG6 Not sequenced AaMG7 GQ Pseudomonas rhodesiae FJ AaMG8 GQ Enterobacter ludwigii EF AaMG9 GQ Enterobacter ludwigii EF AaMG10 GQ Pantoea agglomerans FJ AaMG11 GQ Bacillus megaterium GQ AaMG14 GQ Chryseobacterium aquaticum AM AaMG18 GQ Erwinia quercina AJ AaMG19 GQ Roseomonas cervicalis AF AaMG20 GQ Pedobacter agri EF AaD1 GQ Curtobacterium flaccumfaciens EU AaD2 Not sequenced AaD3 GQ Leuconostoc mesenteroides FJ AaD4 GQ Bacillus subtilis EU AaD5 GQ Paenibacillus borealis AJ AaD6 Not sequenced AaD7 GQ Brenneria quercina EF AaD8 GQ Leuconostoc mesenteroides FJ AaD9 GQ Vagococcus salmoninarium AM AaD10 GQ Brenneria salicis NR_ AaD11 GQ Erwinia persicinus AJ a Total number of nucleotides sequenced in the Aedes albopictus isolates used in BLAST search. b Highest scoring BLAST result with a full binomial name. Other unidentified isolates may have scored higher.

9 Bacterial diversity The 14 bacteria from the midgut belong to the following bacterial families: Enterobacteriaceae (8 isolates) Enterococcaceae (1) Pseudomonadaceae (1) Bacillaceae (1) Flavobacteriaceae (1) Acetobacteraceae (1) Sphingobacteriaceae (1) The 9 bacteria from the diverticula fall into the following families: Enterobacteriaceae (3 isolates) Bacillaceae (1) Microbacteriaceae (1) Paenibacillaceae (1) Enterococcaceae (1) order Lactobacillales (2)

10 Inhibition of LACV infectivity

11 Bacteria and virus interactions Of the 24 isolates tested, 16 showed a significant reduction in the number of plaques that formed on Vero cells compared to virus not exposed to bacteria. The enteric bacterium E. coli was used as a control and did not significantly inhibit infectivity of LACV

12 Discussion To our knowledge, this is the first study to demonstrate an interaction between bacteria isolated from a mosquito and a virus transmitted by that mosquito. However, we have only demonstrated inhibition of LACV in a cell culture assay, not in living mosquitoes. Is a 44% reduction useful?

13 Future studies Bacterial inhibition of virus infectivity in mosquitoes coinfected with these bacteria and LACV Isolate specific molecule(s) that have antiviral effect

14 Acknowledgments Support was provided by: The RU Department of Biology Seed grant from RU Sponsored Programs Sally Paulson provided the La Crosse virus