Marie Riepenhoff-Talty

Age-Dependent Rotavirus-Enterocyte Interactions

Abstract The nature of rotavirus-intestinal cellular interaction was examined in duodenal and jejunal enterocytes obtained from groups of adult and suckling mice orally inoculated with murine rotavirus (MRV). The techniques of immunofluorescence (IFA), electron microscopy (EM), and resetting of MRV-coated sheep erythrocytes (SRBC) were employed for these studies. Immunofluorescence studies demonstrated the presence of viral antigen in the cytoplasm in 2 to 30% of enterocytes isolated from suckling mice but not in any of the enterocytes from adult mice. The peak age of mice with MRV-positive enterocytes was 6-11 days. In all instances enterocytes were isolated 24 hr following oral inoculation of mice with control media or MRV. Isolated dispersed enterocytes from uninfected adult or suckling mice were incubated with purified MRV-coated red blood cells. Specific binding of virus coated SRBC to enterocytes, as evidenced by formation of rosettes, was most pronounced in enterocytes obtained from suckling mice under 11 days of age while only low levels of rotavirus binding activity persisted in the enterocytes from mice older than 75 days of age. These data suggest that the degree of replication of MRV in the intestine may be determined by the availability of virus-specific receptors on enterocytes. The differences in the relative proportion of such receptors between suckling and adult mice may explain the unique predilection of infants to rotavirus infection. Although the pathogenesis of mouse rotavirus closely resembles human rotavirus infection, the identification of similar receptors on human enterocytes remains to be established.

Effect of Nutritional Deprivation on mucosal viral Infections

Suckling BALB-c mice, subjected to nutritional deprivation in artificially expanded litters (18 to 20 pups), were compared to normally nourished pups (7-9 per litter) in a series of experiments designed to provide data on morphologic and functional alterations of the small intestine during malnutrition and infection. The effects of protein calorie malnutrition (PCM) on the viral replication pattern and severity of clinical disease were examined in suckling mice infected with murine rotavirus (MRV). The infection in nutritionally deprived animals was characterized by a significant decrease in the minimal infectious dose and in the incubation period for the onset of diarrhea as well as increased severity of disease when compared to well nourished controls. Rotavirus-specific antibody, administered orally prior to virus inoculation, significantly reduced MRV replication in both groups but most strikingly in malnourished animals. Additional studies of the uptake of a macromolecule [ovalbumin (OVA)] following oral administration to experimental and control groups showed more rapid and complete absorption in the malnourished animals. Infection further enhanced the uptake of OVA, suggesting that both PCM and rotavirus infection alter the permeability of the small intestine. An unexpected observation of rotavirus-associated hepatitis in CB-17scid mice was also made in nearly 40% of malnourished mice inoculated with 10(6) PFU of Rhesus rotavirus (RRV). Mice with PCM exhibited a susceptibility to hepatitis between SCID mice (80%) and immunologically normal mice (18%). While these data are not sufficient to confirm a nutritionally-mediated immunoincompetence, they do suggest that either loss of immune competence and/or increased gut permeability in malnourished animals may allow a more severe homologous rotavirus infection as well as extraintestinal spread of heterologous rotavirus.

Negative Staining and Immune Electron Microscopy as Techniques for Rapid Diagnosis of Viral Agents

In 1973, Bishop and her colleagues visualized reovirus-like particles in thin sections of duodenum biopsied from a young child with diarrhea. Shortly afterward, in England’ and Canada,” similar particles were seen in feces using direct visualization with negative-stain electron microscopy. Now, nearly ten years later, the impact of rotaviruses on human disease is becoming apparent. In 1972, an outbreak of gastroenteritis affected 50% of students and teachers in an elementary school in Norwalk, Ohio. Particles 27 nm in diameter were visualized in the infectious stool filtrate. This filtrate was mixed with convalescent serum from a patient who had had the disease. The resultant particles were coated with antibody and clumped together. This technique was termed immune electron microscopy and the virus-like particles were called Norwalk agent.* It is estimated that these two agents account for 50% of hitherto undiagnosed gastroenteritis around the world. It is amazing that in this era of tissue culture, virology pathogens were diagnosed initially by electron microscopic techniques and that these techniques are still vital. Even in 1982 cell culture isolation is not an adequate diagnostic tool for rotaviruses. Norwalk agent and other antigenically related virus-like particles’.20 are still not grown in cell culture systems. Even more recently electron microscopy and immune electron microscopy have been shown to be critical techniques for detecting new virus-like particles associated with gastroenteritis in humans. These particles have two things in common. They are smaller than rotavirus ( I 5-40 nm) and are round. They have logically been referred to by some investigators as small round virus-like particles (SRV). They are not antigenically related to Norwalk agent. Because these SRV have been detected by electron microscopy, their morphology is a distinguishing feature for some particles. Astrovirus, a 28 nm particle with a star-like surface was described by Madely and Cosgrove in 1975. Astrovirus has been detected in outbreaks of diarrhea in Scotland, England, and Canada.2.’ 1 ~ 1 3 ~ 1 6 Caliciviruses, also approximately 28 to 30 nm in size, have been associated with diarrhea in human infants according to several

Festschrift Immunological aspects of interaction between rotavirus and the intestine in infancy

Rotaviruses are important pathogens causing severe diarrhoea in human infants and young animals. Available information on the pathogenic mechanisms of and the immune response to rotaviruses is reviewed here. Studies in our laboratory using the suckling mouse model have focused on elucidating the nature of interaction between the virus and the gut, and on the importance of T and B cell mediated immunity in protection and recovery from the disease. Our data suggest that the age dependence of mouse rotavirus (MRV) infection is related to the presence of virus‐specific receptors on the enterocytes. The uptake of rotavirus antigens appears to be limited to the epithelium associated with Peyers patches. The antigen is transported to local and regional lymph nodes. Recent studies have indicated that rotavirus infection also increases the uptake of other macromolecules in the intestine. Rotavirus‐specific mucosal IgA response seems to be related to the location and magnitude of MRV antigen in the lymph follicles in different segments of the small intestine. Studies in mice with different types of immunodeficiency suggest that a specific immune response is required for complete resolution of virus infection. Several parameters of immunity to rotavirus infection have been examined and, similar to other reports, local immunity in the intestine appears to have the most important role in protection. It also has been observed that nutritional factors may be important in modifying disease. However, there are still many questions to be answered concerning the role of immunity in mediating the pathogenesis of rotavirus infection.

Polymerase Chain Reaction Assays for the Detection of Cytomegalovirus in Organ and Bone Marrow Transplant Recipients

Cytomegalovirus (CMV) infection is ubiquitous and results in a wide spectrum of clinical manifestations ranging from asymptomatic infection to severe life threatening disease. Infection in normal children and adults usually causes no symptoms but in the immunocompromised host, CMV may result in severe opportunistic infections with high morbidity and mortality. Historically, virus detection was dependent on culture of the virus or on a centrifugation culture system referred to as a shell vial assay. The shell vial assay frequently lacked sensitivity and was unable to detect infection in its early phase. Also, as with culture assays, the results were affected by antiviral therapy. The CMV antigenemia assay was developed to provide more rapid results and has gained wide usage. This assay is limited to detection of the virus in white blood cells and is more sensitive than culture or the shell vial assay. Application of the polymerase chain reaction (PCR) to these problems has resulted in the development of assays for CMV which are more sensitive than previously available methods. This method employs liquid hybridization with 32P labeled probes and gel retardation analysis for detection of amplified DNA specific for each virus. A comparison of the detection of CMV by an antigenemia assay or the PCR method in the leukocytes of renal transplant patients revealed that the PCR assay detects cytomegalovirus earlier and more consistently than the antigenemia assay. Finally, the application of a fluorescent dye detection system and image analysis of the acrylamide gel with a laser scanner provides additional sensitivity to the detection of cytomegalovirus, as well as avoiding the use of radioactivity, making the assay more adaptable to the clinical laboratory.

Rapid, non-radioactive detection of virus infection by polymerase chain reaction

BACKGROUND Polymerase chain reaction (PCR) diagnosis of infectious diseases, especially virus diseases, offers a very sensitive and specific technique for clinical diagnosis. However, detection systems for amplified DNA requiring radioactive probe hybridization or signal development using blot transfer or nucleotide capture require overnight incubation or specially labeled probe molecules for analysis of amplified DNA. OBJECTIVES To place this technology in the clinical laboratory, rapid and sensitive methods are needed for the detection of amplified DNA which are applicable to the assay of multiple specimens representing many different organisms and requiring a minimum of manipulation. STUDY DESIGN Electrophoretic separation of amplified DNA fragments, stained with the fluorescent dye SYBR Green I, and laser scanning of the gels for detection of virus-specific PCR products was compared with detection of amplified DNA by liquid hybridization with radioactive probes and gel retardation analysis of labeled probe molecules. RESULTS Fluorescent scanning methodology was applied to the detection of cytomegalovirus (CMV), herpes simplex virus (HSV) and the human immunodeficiency virus (HIV). This method was at least 10 times more sensitive than radioactive probe hybridization in the detection of CMV-specific PCR products. This method also required less time and avoided the use of radioactivity. CONCLUSIONS Clinical diagnosis of virus infections can be conveniently and rapidly accomplished, while avoiding the dangers of radioactive probe handling, by fluorescence staining and laser scanning of specifically amplified gene fragments. This technology is applicable to the detection of genes from many different organisms, without specially synthesized and/or labeled oligonucleotide primer or probe sequences.

Effect of malnutrition on extraintestinal spread of rotavirus and development of hepatitis in mice

Abstract The effects of malnutrition on the extramucosal spread and severity of the clinical disease were examined in suckling BALB/c mice infected with heterologous rhesus rotavirus (RRV). Control animals were inoculated with homologous murine rotavirus (MRV). Following oral infection with RRV, 100% of malnourished and 90% of normally nourished mice exhibited watery diarrhea within 48 hours. The clinical symptoms appeared more severe and of longer duration in the malnourished group. At 7 to 14 days post-inoculation, 30 of 79 (38%) malnourished and 19 of 92 (21%) well nourished mice developed icterus, clay-colored feces and dark yellow urine. The mortality rates were 9% and 0%, respectively. Light microscopy revealed an acute hepatitis with foci of necrosis. Rotavirus was detected in liver tissue by cell culture infectivity, immunofluorescence assay and electron microscopy. Homologous MRV infection resulted in diarrhea in all animals and with increased severity in the malnourished group. Significantly, however, none of the control animals developed hepatitis and no virus could be detected in the liver. These results suggest that malnutrition may exert significant influence on the severity of rotavirus infection and on the extent of extramucosal spread of RRV. Since several heterologous rotavirus strains are currently being tested in human vaccine trials, the implications of our observations suggest that special attention should be paid to malnourished infants and that screening of hepatic function should be included in vaccine protocols.

Effect of intestinal mucosal environment on the hepatopathogenic potential of rotavirus in normal and immunocompromised hosts

The worldwide impact of rotaviruses (RV) as the leading cause of diarrhea in children has generated much interest in disease prevention by a specific vaccine. The Jennerian approach to RV vaccination of humans using orally administered heterologous (animal) strains has been the most promising [1]. The suckling mouse model has recently been shown to be applicable to study the disease induced by heterologous RVs [2]. Mice with severe combined immunodeficiency (SCID), which are known to suffer persistent murine RV infection, constitute a good model for the immunocompromised host. Recent studies in our laboratory have suggested that mucosal immunocompetence may play an important role in influencing heterologous RV infection in mice [3]. This was evidenced by extramucosal spread and hepatic virus replication of rhesus RV (RRV) in SCID mice. The aim of this study was to examine the effect of different routes of administration on the hepatopathogenic potential of RRV in SCID and normal mice.