Jenny Patoka, Rizwana Ali, and Matthew D. Koci Department of Poultry Science, North Carolina State University, Raleigh, NC Introduction Astroviruses are small round, positive sense RNA viruses, with a distinct 5- or 6- pointed star morphology (1). They are recognized as a leading causes of acute enteritis in infants, the elderly and the immunocompromised (2). Species specific astroviruses are also known to cause enteric disease in other animals including turkeys (3). Astroviruses in general contain a 7-8 kb RNA genome organized into three open reading frames (ORFs), ORF 1a, 1b, and 2 (Fig 1). From the 5’ end of the genome, ORF 1a encodes the nonstructural proteins identified as a serine protease, transmembrane helices, and a nuclear localization signal (4). ORF 1b encodes the RNA-dependent polymerase and ORF 2 encodes the capsid protein (5). How the proteins encoded in the astrovirus genome are involved in causing disease has not been characterized. No specific proteins have been identified as virulence determinants. Generation of technologies and reagents proposed in this project will allow us to study how the different regions of the virus are involved in causing diarrhea. Objective The overall objective of this study is to create a plasmid that contains the full-length genome of TastV-2, for use in reverse genetic experiments. The construct will allow for the construction of a custom recombinant virus to determine how different regions of the virus are involved in disease. Materials and Methods  TAstV2 viral sequence was digested from respective pCR-XL vector using restriction endonucleases following the manufacturer ’ s instructions.  DNA digestion products were electrophoresised on a 1% agarose gel and visualized using ethidium bromide.  Restriction products were identified based on size, extracted from the agarose gel and purified for subsequent cloning reactions.  Recipient vector DNA was dephosphorylated using Shrimp Alkaline Phosphatase (SAP), mixed at a ratio of 1:3 with purified insert DNA and ligated using T4 DNA ligase according to the manufacturer ’ s instructions.  Ligation reactions were transformed into chemically competent E. coli and grown on LB agar media containing ampicillin following the manufacturer ’ s instructions.  Resulting constructs were confirmed using restriction digest reactions and DNA sequencing.  Vector NTI v10 (Invitrogen) was used to analyze DNA sequencing data and to generate vector maps. Figure 1: Schematic of TAstV2 genome and cloning vectors containing overlapping regions of the virus genome. A) Green Arrows represent each of the three reading frames. Boxed area show relative regions of the viral genome cloned. B) Vector map of construct containing TAstV2 genomic sequence corresponding to nucleotide positions C) Vector map of construct containing TAstV2 sequence corresponding to nucleotide positions ORF1a ORF1b ORF2 Figure 3: Construction of a new plasmid containing nucleotide positions of the TAstV2 genome. A) Map of new construct generated by digesting pGEM-pol (Fig 2B) with Apa I and Eco RV. The capsid portion was removed from pCR-XL (Fig 1C) using the same enzymes. Digested DNAs were ligated and used to transform E coli. B) pGEM-pol-cap restriction digest with Eco RI. Visual confirmation of pGEM-pol-cap by electrophoresis and ethidium bromide staining following restriction digestion with Eco RI. M = molecular weight marker. Lane 1 indicates the expected band sizes of 4507bp and 2539bp. 1234M 1kb 2kb 3kb Figure 2: TAstV2 polymerase region subcloned from pCR-XL to pGEM. A) Map of recipient plasmid with positions of restriction sites (Apa I and Bsa A I) used to prepare vector for ORF1b insert. B) Map of new construct formed by digesting ORF1b insert out of pCR-XL (Fig 1B) using Eco RV and Apa I and then ligated into cut pGEM vector. C) Visual confirmation of pGEM-pol by electrophoresis and ethidium bromide staining following restriction digestion with Eco RI and Apa I. M = molecular weight marker. Lanes 1-4 are digestion products from 4 individual clones. Double digestion of all 4 clones yielded the expected band sizes of 2575bp and 2065bp. 1kb 2kb 3kb M 1 A B A C AB C Results Development of Turkey Astrovirus Type 2 Full-Length Infectious Clone Discussion  Astroviruses are the cause of acute enteritis in humans and are responsible for outbreaks of enteric disease in turkey poults.  Little is known about how the proteins encoded in the astrovirus genome are involved in causing disease.  The constructs developed as part of this project is the first step towards creating a reverse genetics system for understanding the viral pathogenesis of turkey astroviruses.  Future experiments: -Link the third viral region, ORF 1a, with the new construct pGEM/pol-cap. -Create infectious RNA and produce the recombinant virus. References 1. C. R. Madeley, B. P. Cosgrove, Lancet 2, 451 (1975). 2. D. K. Mitchell, Pediatr Infect Dis J 21, 1067 (2002). 3. M. D. Koci, S. Schultz-Cherry, Avian Pathol 31, 213 (Jun, 2002). 4. M. M. Willcocks, A. S. Boxall, M. J. Carter, J Gen Virol 80, 2607 (1999). 5. B. Marczinke et al., J Virol 68, 5588 (1994). Acknowledgements Project funded in part by grants from the Undergraduate Research Department and the WISE program. pCR-XL-TOPO/TAstV2pol Lethal Gene Kan R Zeocin R TAstV2 ORF1b Lac Z pUC Origin Eco RV Apa I Bam HI Eco RI pCR-XL-TOPO/TAstV2cap Lethal Gene Kan R Zeocin R Lac Z TAstV2 Cap pUC Origin Bam HI Eco RI Apa I Eco RI pGEM®-5Zf(+) AmpR Lac Z phage f1 origin Apa I Bsa AI Eco RV pGEM/pol AmpR TAstV2 pol Lac Z Apa I Bam HI Eco RI Eco RV pGEM/pol-cap AmpR TAstV Lac Z Apa I Bam HI Eco RV Eco RI B