Pattern of shedding of the Norwalk particle in stools during experimentally induced gastroenteritis in volunteers as determined by immune electron microscopy.

In 11 of 23 volunteers the Norwalk virus-like particle was visualized by immune electron microscopy in at least one stool specimen obtained during the acute phase of experimentally induced nonbacterial gastroenteritis. Examination of multiple stool specimens obtained during the course of illness in these 11 volunteers revealed maximal concentration of Norwalk virus-like particle at the onset of illness and shortly thereafter; in no case was the Norwalk particle visualized in stools obtained before the onset of illness. This finding further suggests that the Norwalk particle was the etiological agent of the Norwalk gastroenteritis outbreak. The limit of reliability of our immune electron microscopy assay system for particle counting was examined.     

Norwalk virus in Norway: an outbreak of gastroenteritis studied by electron microscopy and radioimmunoassay

An outbreak of acute gastroenteritis affected approximately half of 40 children staying at a holiday centre in Southern Norway. By direct electron microscopy Norwalk-like viruses were demonstrated in 4/8 available stool specimens. No other pathogens were detected. Antibody against these viruses was demonstrated by immune electron microscopy in all of 7 convalescent phase sera but in none of 11 acute phase sera collected. Radioimmunoassay examination showed a rise in titre of Norwalk virus antibody in 6 available paired sera. This outbreak of Norwalk virus gastroenteritis in Norway was thus documented by a combined use of direct and immune electron microscopy and radioimmunoassay.     

The discovery of the 27-nm Norwalk virus: an historic perspective

In 1972, a 27-nm virus-like particle was discovered by use of immune electron microscopy (IEM) in an infectious stool filtrate derived from an outbreak of gastroenteritis in an elementary school in Norwalk, Ohio. IEM enabled the direct visualization of antigen-antibody interaction, as the particles were aggregated and coated by specific antibodies. This allowed the recognition and identification of a 27-nm virus-like particle that did not have a distinctive morphology, was low-titered, and was among the smallest viruses known. Serum antibody responses to the 27-nm particle were demonstrated in key individuals infected under natural or experimental conditions; this and other evidence suggested that this virus-like particle was the etiologic agent of the Norwalk gastroenteritis outbreak. The fastidious 27-nm Norwalk virus is now considered to be the prototype strain of a group of noncultivatable viruses that are important etiologic agents of epidemic gastroenteritis in adults and older children.     

Viral gastroenteritis induced by the Hawaii agent: Jejunal histopathology and serologic response

Peroral jejunal biopsies were performed in seven normal volunteer subjects prior to, 48 hours after and two weeks after the administration of the Hawaii agent of viral gastroenteritis. Light and electron microscopic examination revealed an intact mucosa with blunted villi, shortened and distorted microvilli, swollen mitochondria and intercellular edema. These histologic changes were seen only in acutely ill volunteer subjects and were absent two weeks after illness in three of four who were previously ill. This reversible lesion was similar to, but not identical with, that previously described in viral gastroenteritis induced by the Norwalk agent.Serum antibody increases in response to the Hawaii agent as measured by immune electron microscopy were present in three of four ill volunteer subjects and in none of three who remained well.     

Hepatitis A antigen particles in liver, bile, and stool of chimpanzees.

Virus-like hepatitis A antigen (HA Ag) particles, presumably hepatitis A virus, were isolated from the liver, bile, and stool of three chimpanzees that had been infected with stool filtrates containing HA Ag particles. Specimens of serum, stool, liver biopsy material, and bile were obtained at selected intervals during the experiment. The animals developed mild hepatitis 19-21 days after inoculation, and antibody to HA Ag appeared de novo in their convalescent-phase serum. During acute illness, virus-like particles similar to the HA Ag particle were seen in liver cell cytoplasm by electron microscopy. HA Ag particles were detected by immune electron microscopy and a new radioimmunoassay in isopycnically banded samples of liver, bile, and stool. HA Ag particles were found at densities of 1.29-1.39 g/cm3, but the major peak density for antigen particles in samples of liver, bile, and stool was approximately 1.34 g/cm3. The fact that HA Ag particles can be recovered from chimpanzee liver, bile, and stool makes these potentially important sources of infectious and antigenic materials.     

A 27-nm virus isolated during an outbreak of acute infectious nonbacterial gastroenteritis in a convalescent hospital: a possible new serotype

An outbreak of acute infectious nonbacterial gastroenteritis began among elderly patients in a convalescent hospital in Marin County in northern California in March 1978 and persisted through May 1978. The overall clinical attack rate was 51% of 187 residents and 12% of 180 employees. A 27-nm viruslike particle was observed by immune electron microscopy in stools obtained at or near the onset of illness from four of 32 patients. Seroresponses to the 27-nm particles were found by immune electron microscopy in 16 of 18 patients. In addition, serologic evidence of infection with this or a related agent was demonstrated in persons who developed illness in another large outbreak of acute infectious nonbacterial gastroenteritis which occurred in a nearby county. This agent is morphologically similar to but serologically unrelated to the Norwalk and Hawaii gastroenteritis agents and has been designated the Marin agent pending further classification.     

Antigenic relationships between human caliciviruses and Norwalk virus

We investigated antigenic relationships between human calicivirus (HCV) strains and Norwalk virus by using immune electron microscopy (IEM) and radioimmunoassay (RIA). Three serologically distinct HCV strains, UK1, UK2, and Japan, were demonstrated by IEM, as was evidence for two additional strains, UK3 and UK4. Although HCV strains and Norwalk virus were distinct by IEM, 12 of 20 patients with gastroenteritis due to HCV UK4 and two of eight with gastroenteritis due to UK2 showed seroconversions to Norwalk virus by RIA. These naturally occurring antibody responses in humans, as detected by RIA, support the concept that Norwalk virus belongs to the family Caliciviridae. An RIA for HCV Japan antigen also detected HCV UK1, UK2, and UK4 and thus appears to identify a group-specific antigen for these viruses. An RIA for antibody to HCV Japan failed to identify seroconversions in 45 of 47 patients with gastroenteritis due to HCV UK. These results may reflect different reactivities of various immunologic tests for the identification of infections due to small gastroenteritis viruses.     

Serological responses among teenagers after natural exposure to Norwalk virus

Twenty-one teenagers exposed to a contaminated water supply during an outbreak of gastroenteritis were tested for seroconversion to Norwalk virus. Serum specimens were collected within 72 hr of exposure and four weeks later. Each of the 11 individuals who developed symptoms and five of the 10 who remained well had a whole-antibody response in serum. None of the remaining five teenagers became ill or seroconverted. Neither seroconversion nor susceptibility to illness was associated with an absence of detectable antibody from acute-phase serum specimens. These findings support the view that immunity to Norwalk virus is not determined by serum antibody. Furthermore, the results are consistent with the possibility, suggested by previous studies in volunteers, that susceptibility is determined by Norwalk virus-specific intestinal receptor sites. IgM responses to the Norwalk virus were detected in only seven persons who became ill (64%) and nine who seroconverted (56%). The seroassay for the Norwalk IgM component might have proved a more sensitive diagnostic tool in this outbreak if convalescent-phase specimens had been collected sooner than four weeks after the onset of illness.     

Prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea

Diarrhea due to enteric pathogens is an important complication of advanced human immunodeficiency virus infection. Whereas numerous bacterial and parasitic agents have been implicated, the role of pathogenic enteric viruses is less clear. Stools from 153 human immunodeficiency virus seropositive men were tested by electrophoresis, enzyme-linked immunosorbent assay, and immune electron microscopy for the presence of rotaviruses (group A and non-group A), adenoviruses, and Norwalk agent. Virus was detected in 9% of the patients with acquired immunodeficiency syndrome, 3% of the patients with acquired immunodeficiency syndrome-related complex, and none of the seropositive men without these diagnoses. Virus detection was not more likely in stool from patients with diarrhea.     

Diagnosis of astrovirus gastroenteritis by antigen detection with monoclonal antibodies

An enzyme-linked immunosorbent assay (ELISA), based on monoclonal antibodies to the astrovirus group antigen, was designed for the detection of astroviruses in stools of patients with gastroenteritis. Compared to immune electron microscopy used as the standard test, the sensitivity of the astrovirus ELISA was 91% (31/34) and the specificity was 96% (54/56). All five of the known astrovirus serotypes could be detected in 16 samples on which serotyping was done. In tests on 155 stools containing other enteric viruses, including adenoviruses, rotaviruses, caliciviruses, Hawaii virus, Snow Mountain virus, and Norwalk virus (30, 20, 70, 24, 4, and 7 samples, respectively), only 3 were positive in the astrovirus ELISA. The combined specificity for all astrovirus immune electron microscopy-negative samples was 98% (206/211). The results demonstrate that the new ELISA provides a sensitive and specific means for the diagnosis of astrovirus gastroenteritis.     

Experimental infection of chimpanzees with hepatitis A virus.

The susceptibility of chimpanzees to viral hepatitis type A was examined with immine electron microscopy. Of four seronegative infant chimpanzees, two were inoculated with a hepatitis A acute-phase stool filtrate rich in 27 nm virus-like hepatitis A antigen (HA Ag) particles, and two were inoculated with an HA Ag-negative preinfection stool filtrate. One of each pair of chimpanzees was inoculated intravenously, the other orally. One month later both chimpanzees that had received the HA Ag-positive filtrate developed biochemical, histologic, and clinical evidence of acute viral hepatitis. HA Ag particle (27 nm) were detected in their stools by immune electron microscopy; particle shedding followed a pattern similar to that in human volunteers. Immune electron microscopy also showed that antibody HA Ag had developed in the convalescent-phase sera of the infected chimpanzees. Control animals remained free of illness at this time but did develop hepatitis three to five weeks after exposure to the two infected chimpanzee-. The infectious inoculum was titrated in two additional seronegative chimpanzees. It was concluded that hepatitis a can be successfully transmitted to seronegative chimpanzees. Moreover, these studies provide further evidence that the 27-nm virus-like HA Ag particle is the etiologic agent of viral hepatitis type A.     

Role of Norwalk virus in outbreaks of nonbacterial gastroenteritis.

Twenty-five separate outbreaks of nonbacterial gastrointestinal illnesses were studied serologically for evidence of infection with the Norwalk virus and the rotaviruses that affect humans. Eight of 25 outbreaks appeared to be related to the Norwalk virus. In one of the 25 outbreaks, there was evidence of rotavirus infection. These observations suggest that the Norwalk virus or serologically related agents play an important role in epidemic nonbacterial gastroenteritis in adults and older children.     

Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.     

A prospective study of rotavirus infection in infants and young children

Diarrhea in neonates, followed as a cohort, and their families was studied prospectively. The families were followed for an average of 16.3 months. Stool and serum specimens were obtained at least every three months. Stool specimens were examined for viruses by electron microscopy and cultured for enteropathogens, and serum specimens were tested for antibodies to rotavirus and Norwalk virus. During the study, 237 episodes of gastroenteritis were observed in 104 infants and their 62 siblings. Rotavirus, detected 82 times in 72 children, was by far the most common enteropathogen. It was associated with gastrointestinal symptoms in 72% (with diarrhea in 65%). Rotavirus diarrhea occurred mostly in winter months and was significantly more frequently associated with respiratory symptoms than were diarrheas with other etiologies. Rotavirus infection was uncommon in the first six months of life, but by two years of age, 62% of the infants had had at least one infection. Neither breast feeding nor the presence of antibody to rotavirus in cord blood appeared to be protective.     

Investigation of an outbreak of adult diarrhea rotavirus in China

In 1987 an epidemic of diarrhea associated with adult diarrhea rotavirus (ADRV) occurred in Qinhuangdao City, China, affecting more than 200 persons and causing 2 deaths. The outbreak was introduced by a person returning from an epidemic area and was spread initially to his family members and subsequently to the entire community. Adults were at greater risk of diarrhea than children 0-4 y of age and, the duration of illness increased significantly with increasing age. ADRV was identified by ELISA and electron microscopy. The electropherotypes of all positive specimens were identical, consistent with the single point-source introduction of the virus. Seroconversion was detected in 6 of 7 ill persons with a blocking ELISA. Both asymptomatic infection and person-to-person spread identified in this epidemic suggest that current emphasis on preventing waterborne transmission may not control the introduction of ADRV into new areas. The predisposition of adults for more severe disease with ADRV is similar to the pattern observed with other enteric viruses such as the Norwalk agent and hepatitis A.     

Multiple-challenge study of host susceptibility to Norwalk gastroenteritis in US adults

In a multiple-challenge study of US adult volunteers with low or high levels of serum antibody to Norwalk virus, Norwalk inoculum 8FIIa was administered to 42 subjects, 22 were challenged 6 months later, and 19 received a third challenge after 6 more months. All 12 with high (greater than or equal to 1:200) but only 19 of 30 with low (less than 1:100) prechallenge titers experienced illness or a fourfold increase in titer after the first challenge (P less than .025). Only 4 of those challenged twice became ill; all had low initial titers but 3 had high titers before the second challenge. None became ill after a third challenge. Nine (47%) had high titers immediately before the third challenge; 3 had low titers before and after each challenge and remained asymptomatic. Thus, preexisting serum antibody to Norwalk virus does not seem to be associated with protective immunity, but antibody levels become associated with protection after repetitive exposure. Short-term resistance lasts greater than or equal to 6 months after challenge, and a small percentage of resistant individuals maintain low antibody titers even after multiple challenges.     

A large nontypical outbreak of Norwalk virus. Gastroenteritis associated with exposing celery to nonpotable water and with Citrobacter freundii.

The US Air Force Academy experienced a point-source outbreak of gastroenteritis originally believed to be caused by Salmonella. The overall attack rate was 48% among approximately 3000 cadets and staff. Food-specific attack rates implicated chicken salad. The odds ratio for chicken salad consumption in ill cadets was 10.7 (95% confidence interval: 8.2; 13.8). The celery component had been exposed to nonpotable water. Citrobacter freundii were statistically associated with consumption of the suspected vehicle and subsequent illness. Most aspects were consistent with the epidemiology of Norwalk gastroenteritis. However, the clinical presentation was not typical of reported outbreaks. One hundred five cadets required intravenous rehydration. Serum samples implicated Norwalk virus as the most probable cause of this outbreak. The Centers for Disease Control (Atlanta, Ga) recently began national surveillance for viral gastroenteritis. All outbreaks of gastroenteritis associated with nonpotable water should be investigated for evidence of viral cause.     

Characterization of serotypes of human rotavirus strains by solid-phase immune electron microscopy

Serotyping of human rotaviruses (HRVs) by neutralization requires the previous adaptation of strains to growth in cell cultures, which is often unsuccessful. By using the solid-phase immune electron microscopy (SPIEM) technique with protein A and type-specific, cross-adsorbed, polyclonal immune sera, we divided 40 previously typed culture-adapted strains into the same four serotypes distinguished by neutralization, but HRV strains could also be typed directly on stool extracts. Of 171 HRV strains tested by SPIEM, 163 were typed as a single serotype, two were shown to be mixed serotypes, three could not be typed since they were partially antibody-coated, and three were lacking the common group antigen (atypical rotaviruses or pararotaviruses). In addition, strains of serotype 4 could be classified by SPIEM into two subtypes, 4A and 4B. Overall, strains of serotype 1 were detected in nearly 50% of the 171 HRV-positive stools examined by SPIEM, strains of either serotype 2 or 4 in about 20%, and strains of serotype 3 in nearly 10%. Although the data were too scarce to allow for definite conclusions, the prevalence of the four serotypes appeared to change with the geographic area and the year.     

Infection of owl monkeys (Aotus trivirgatus) and cynomolgus monkeys (Macaca fascicularis) with hepatitis E virus from Mexico.

Owl and cynomolgus monkeys were inoculated with hepatitis E virus (HEV) to compare disease models and produce antibody and virus. By immune electron microscopy (IEM), all six owl monkeys were shown to have serologic responses manifested by unusually high levels of anti-HEV at 6 months, but only three developed hepatitis. Virus-related antigen in liver (HEV Ag) was detected by immunofluorescence microscopy of biopsies from two of four owl monkeys; one with HEV Ag also had HEV in acute-phase bile (detected by IEM) and feces (detected by infecting another owl monkey). In contrast, cynomolgus monkeys propagated HEV to higher levels and all five had hepatitis. Moderate-to-high levels of HEV Ag correlated with detectable HEV in bile for both species. Thus, the value of using HEV-infected cynomolgus was confirmed. Owl monkeys were shown to be HEV-susceptible and sources of high-level anti-HEV; Sustained anti-HEV in these monkeys may also be useful for understanding immune responses.     

Food-borne outbreaks of gastroenteritis caused by small round structured viruses. 1. Four outbreaks of gastroenteritis associated with oyster consumption]

Between December 9, 1988 and January 28, 1989, there were four outbreaks of acute gastroenteritis in Saitama prefecture. Eighty-two of 123 persons (67%) attending four banquets in restaurants became ill: 44 cases attending three banquets were related to eating raw oysters, and 38 attending one banquet to eating sashimi. The most common symptoms were nausea, diarrhea, abdominal cramps, and vomiting. Average incubation periods were 29 to 32 hours long. Bacteriologic analysis of stool specimens did not reveal causative agents. Small round structured viruses were detected in fecal specimens from 19 of 39 ill persons (49%) by electron microscopy. In one of four outbreaks, the formation of antibody to small round structured virus in paired serum samples was detected by western blot test. Small round structured viruses were implicated as the etiologic agents in four outbreaks of acute gastroenteritis.