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Specific objectives of the proposed experiments are; determine the timeline for viral persistence in hemocytes vs. whole body levels; identification of hemocyte subtypes associated with virus; follow the fate and persistence of viruses in hemocytes transferred from exposed to naive oysters; determine if low pH virus tolerance correlates with total oyster and hemocyte persisitence; visualize virus localization patterns within phagocytic hemocytes with emphasis on the phagolysosomes; and inhibiting phagocytic activities of hemocytes and effects of immunomodulation on virus persistence in hemocytes. While bivalves are readily recognized as bioconcentrators of both fecal bacteria and fecal viruses, it is not well recognized that enteric viruses are selectively retained, or are, at least resistant to shellfish process that result in the purging or digestion of fecal bacteria. It is for this reason that viral contamination of shellfish poses a serious human health problem that is not easily monitored and is not amenable to resolution by conventional depuration methods. Hypothesis; Since oyster hemocytes play central roles in processing foreign particles, we suggest that the activities of hemocytes are important determinants of virus retention in the host and we think that enteric viruses are largely resistant to the digestive activities of hemocytes. Consequently the overall goal is to identify mechanisms that control uptake and persistence of pathogenic viruses in shellfish, concentrating on the roles of hemocytes in these processes. <P>This proposal directly addresses the current RFP Priority Area 1; to investigate human enteric viruses, Vibrio spp. Salmonella spp. Listeria or other microbial toxins associated with seafood (oysters). In order to rationally approach the problem, this proposal outlines investigation of the physiological uptake and retention of human enteric viruses by a representative bivalve, the oyster Crassostrea virginica. The information obtained will make a significant contribution toward the existing body of knowledge on shellfish physiology, immunology, and virology. The proposal attempts to not only provide mechanistic understanding of virus retention, but collectively the information obtained from this basic research proposal should aid in the development of practical measures useful in reducing the uptake or retention of human pathogenic viruses by edible shellfish.
NON-TECHNICAL SUMMARY: Bivalve mollusks are active filter feeders that remove and sequester pathogenic human enteric viruses from fecally-contaminated waters (Glass et al. 2001; Butt 2004). As a consequence, bivalves can act as disease vectors for enteric viruses [noroviruses (NV) and hepatitis A virus (HAV)]. These viruses have been shown to persist for several weeks within shellfish in some cases. As the part of bivalve digestive processes, small food particles are delivered to primitive macrophage-like blood cells, called hemocytes, which breakdown food. We hypothesize that viruses, after being taken up by the digestive system, ultimately are sequestered within these hemocytes and that acid-tolerant enteric viruses readily persist within these cells. A major intended benefit of this preliminary study will be to develop an in-depth mechanistic understanding of virus retention in the oyster. The role of hemocytes in uptake and retention of NV and HAV, will be determined and quantified. To a lesser extent Norovirus surrogates will also be employed, such as feline calcivirus (FCV), murine norovirus (MNV), and some less common human picornavirus such as parecho, echo, and coxsackie viruses, for experiments comparing the acid tolerance and virus persistence. State of the art methodology will be employed (image analysis, RT PCR, flow cytometry, fluorescent probes) to allow detailed analysis of the problem. The information obtained will make a significant contribution towards the existing body of knowledge on shellfish physiology, immunology, and virology. Collectively, the basic research gained here should aid in the development of practical measures useful in reducing the uptake or retention of human pathogenic viruses by edible shellfish.<P>APPROACH: Timeline for viral persistence in hemocytes vs. whole body levels. Identification of virus can readily be accomplished by use of plaque assay or HAV and MNV (Richards and Watson 2001), and real time RT PCR techniques (Richards et al. 2004). Hemocytes can be extracted from shellfish by use of a larger gauge needle and syringe, and low speed centrifugation to pellett issues, or by pouring oyster hemolymph through glass wool for hemocytes to adhere (Anderson 2001). Extraction of infectious HAV and murine norovirus from contaminated shellfish is done by blending oysters in isotonic basic (pH 9.5) phosphate buffer, followed by centrifugation and neutralization of the supernatant with HCl followed by plague essay (Richards and Watson 2001). Cell monolayers are infected followed by overlay with tissue culture media containing one percent Agarose. After incubation, the resultant plaques are visualized by either crystal violet or neutral read staining. Extraction of viral RNA from shellfish and specific tissues and cell types is accomplished using the GPTT protocol (Kingsley et al. 2001). After centrifugation, the pellet is dissolved in a mixture of guanidinium isothiocynate and phenol (TRI reagent) that inactivates RNases, frees the viral RNA from the virus capsid, and separates total RNA from the DNA and protein fractions. The total RNA is then purified by hybridization to magnetic beads which purify only poly A containing messenger RNA and viral RNA. Qualitative detection is accomplished by traditional one step RT PCR with appropriate primers, SYBR Green one step RT PCR kit and a Cephied thermocycler (Richards et al. 2004). Identification of hemocyte subtypes associated with virus. Two predominant classes of hemocytes (granulocytes and hyalinocytes) have been identified in oysters(Cheng 1984). The granulocytes are thought to be more phagocytic and more active in antimicrobial defenses. Oyster hemocytes will be differentiated by the method of Hegaret et al.(2003), which uses a combination of the DNA binding fluorochrome SYBR Green and internal cell complexity. Mixed hemocyte populations are fixed in formalin and incubated with SYBR Green prior to analysis by BD FACS Calibur Flow Cytometry. Viral persistence in hemocytes transferred to naive hosts. Shellfish do not show histocompatability differences; therefore, tissue grafts and hemocytes from the same species of shellfish are generally not rejected. We will perform transfer experiments where virus contaminated hemocytes from one oyster are transferred by injection into the open circulatory system of a second oyster. If the transfer is successful and the naive oyster becomes contaminated for extended periods, this would offer further evidence that hemocytes are a site of persistence. Correlation of low pH tolerance with total oyster and hemocyte persistence. Certain enteric viruses are less tolerant to low pH than others. We will determine if the length of persistence within correlates with pH sensitivities of these different viruses. If so, this would offer evidence that the virus is persisting within the acidic phagolysosomes of the hemocytes.