Detection of human parvovirus B19 in early spontaneous abortuses using serology, histology, electron microscopy, in situ hybridization, and the polymerase chain reaction.

OBJECTIVE: To determine whether there is an association between parvovirus B19 infection and early spontaneous abortion at less than 20 weeks' gestation. METHODS: Eighty samples of early spontaneous abortions were analyzed. Each sample was examined histologically for the presence of viral inclusions, and selected cases were analyzed for parvovirus using electron microscopy and in situ hybridization. Polymerase chain reaction DNA amplification for the virus was done in each case. Maternal sera were analyzed for immunoglobulin (Ig) M and IgG parvovirus antibodies and compared with temporally matched controls. RESULTS: Five cases in the study group had evidence of seroconversion for parvovirus, compared with two controls. Products of conception from two of these five cases were positive for virus by polymerase chain reaction amplification, and only one of these two had a characteristic inclusion of parvovirus histologically. Conversely, five chorionic vesicles from mothers who had not seroconverted had histologic changes suggesting parvovirus infection, but all of these cases were negative for parvovirus using in situ hybridization, polymerase chain reaction, and electron microscopy. CONCLUSIONS: Parvovirus B19 DNA was found in two of 80 early spontaneous abortuses. Although viral DNA was detected in two cases, there was no clear evidence that the infections caused fetal death. Neither case showed erythroblastosis with large numbers of inclusions, as is seen in hydropic fetuses with parvovirus infection. In addition, in five cases in which parvovirus infection was not documented serologically or by the polymerase chain reaction, there was erythroid nuclear clearing suggestive of parvovirus B19 inclusions. This indicates that histologic evaluation for parvoviral inclusions is not always reliable in early spontaneous abortuses.

Diarrhea in a non-hospitalized rural Salvadoran population: the role of enterotoxigenic Escherichia coli and rotavirus.

To determine the role of rotavirus, enterotoxigenic Escherichia coli and enteropathogenic E. coli in diarrheal disease of non-hospitalized children and adults living in rural El Salvador, stool specimens were collected from 156 persons with diarrhea and 134 age- and sex-matched controls over a 1-year period. Enterotoxigenic Escherichia coli (ETEC) were isolated as frequently from controls (13.4%) as from diarrhea cases (12.2%). Enteropathogenic E. coli were isolated from 13 cases (8.3%) and 10 (7.7%) controls. Rotavirus was demonstrated in only five of the 129 specimens from cases examined; the five persons infected were less than or equal to 3 years of age. No invasive E. coli were found. Serotyping of ETEC revealed serogroups of ETEC previously associated with enterotoxigenicity but was not helpful in separating infection from disease. The etiology of diarrhea in this rural, non-hospitalized population was complex. Isolation of a known pathogen did not prove etiology. The rotaviruses, which have been isolated frequently from hospitalized persons, were rare. Further laboratory and epidemiologic studies in such populations are needed to identify those factors that determine pathogenicity.

Development and evaluation of an IgM capture enzyme immunoassay for diagnosis of recent Norwalk virus infection.

A capture enzyme immunoassay specific for Norwalk IgM class antibody was developed. The assay was moderately sensitive, identifying 33/53 (62%) of patients with naturally acquired Norwalk virus infection and 17/18 (94%) of experimentally infected volunteers. The assay was also specific for IgM class antibody and acute Norwalk virus infection and results were generally reproducible. A specific IgM response correlated with seroconversion by total antibody blocking assay and occurred independently of clinical symptoms. Among 81 symptomatic cases composing seven Norwalk outbreaks, specific IgM was absent from acute phase sera collected less than or equal to three days post onset, and was uncommon in sera collected within one week and after five weeks, with an optimal collection time at about two to three weeks. The Norwalk IgM capture immunoassay may be used to augment paired sera assays in the identification of Norwalk-associated outbreaks of acute gastroenteritis.

Multiple primer pairs for polymerase chain reaction (PCR) amplification of human parvovirus B19 DNA.

Human parvovirus B19 is the etiologic agent of erythema infectiosum and transient aplastic crisis in patients with hemolytic anemias and has been associated with fetal death, arthritis, and chronic anemia. Acute B19 infection is best diagnosed by detection of IgM antibodies, whereas the diagnosis of chronic infection often requires the sensitivity of PCR to demonstrate presence of virus over time. To improve our ability to detect B19 DNA by polymerase chain reaction (PCR), we evaluated 19 primers combined into 16 different primer pairs for their ability to detect temporally and geographically diverse B19 isolates. All 16 pairs reacted with all isolates tested but with different sensitivity. Sequence analysis showed few nucleotide changes compared with published sequences. These changes did not explain observed differences in sensitivity between primer pairs. The most sensitive primer pairs detected 350 to 3500 DNA copies after 35 cycles. A second amplification cycle with nested primers improved the sensitivity 100-fold. These 16 primer pairs provide the diagnostic virologist with multiple options for B19 PCR assays.

A neutralization test survey for Lassa Fever activity in Lassa, Nigeria.

5-8% of 434 sera collected in Lassa village, Nigeria, in August 1970 were positive by neutralization test for Lassa fever (LF) virus. A second survey in March 1971 found 15 of 47 complete compounds tested in Lassa, Dille and Yuba villages had at least one peron with serologically demonstrable experience with LF virus. 67% of positive compounds had more than one infected member. Four of 17 hospital staff sero-converted between August 1970 and March 1971 without symptoms, evidence that subclinical infections of LF occr.

An outbreak of acute infectious nonbacterial gastroenteritis in a high school in Maryland.

An outbreak of acute infectious nonbacterial gastroenteritis (AING) occurred in a high school in Maryland in 1984. Thirty-six percent of students surveyed met the case definition of gastroenteritis, as did 24 percent of school employees. Eating lunch in the cafeteria on January 30 was significantly associated with illness. After controlling for other food items consumed during the January 30 lunch, only the sandwiches were significantly associated with illness, but the source of the contamination was not identified. Four of 17 serum pairs from sick students and none of the 8 serum pairs from exposed controls (a nonsignificant difference) showed at least a 4-fold rise in antibody titre to Norwalk virus between acute- and convalescent-phase specimens. This outbreak of AING is believed to be the first to implicate epidemiologically sandwiches as vehicles of transmission. The outbreak highlights the need for investigators to look for a viral etiology in gastroenteritis outbreaks.

Susceptibility of nonhuman primate species to infection by simian rotavirus SA-11.

Enzyme-linked immunosorbent assay examination of sera from three nonhuman primate species demonstrated the presence of antibody reacting with simian rotavirus SA-11 and calf rotavirus C486. The occurrence of this antibody in sera from adult, wild-caught animals suggests natural rotovirus infection. The occurrence of antibody was highest in the chimpanzee and declined, respectively, in the rhesus macaque and in the squirrel monkey. Inoculation of three infant rhesus macaques, a nursery-reared chimpanzee, and a cesarian-derived nursery-reared baboon with SA-11 virus resulted in enteric infection, with virus excretion beginning 48 to 72 hours after oral administration of the virus. Clinical disease, as manifested by diarrhea, was observed only in the chimpanzee. Inoculation of ten squirrel monkeys, from 30 to 191 days old, induced infection only in the monkey inoculated at 30 days of age. This monkey became ill within 48 hours after viral administration and was euthanatized. Necropsy demonstrated a generalized infection, with virus recovered from lung, liver, kidney, spleen, and intestine. The remaining nine inoculated squirrel monkeys failed to develop enteric infection and did not respond with antibody to SA-11 virus.

Severe anemia caused by human parvovirus in a leukemia patient on maintenance chemotherapy.

A 6-year-old boy on maintenance chemotherapy for acute lymphocytic leukemia developed severe hypoplastic anemia during chemotherapy previously well tolerated. The hypoplastic episode persisted for approximately 30 days. Human parvovirus (B19), the etiologic agent of aplastic crisis in persons with underlying hemolytic syndromes, was detected in the patient's serum 25-30 days after onset of hemoglobin decrease, and B19 IgM seroconversion occurred 1 week later. The patient's hypoplastic anemia was presumably caused by prolonged B19 infection resulting from a blunted immune response. An immune response to the B19 infection and resolution of the illness were temporally associated with brief cessation of chemotherapy.

Comparative complement fixation and serum neutralization antibody titers to herpes simplex virus type 1 and Herpesvirus simiae in Macaca mulatta and humans.

The serological relationship of herpes simplex type 1 virus and Herpesvirus simiae was studied. Antibody titers to these viruses were determined in 163 Macaca mulatta sera and 67 human sera by serum neutralization (SN) and complement fixation (CF) tests. Both groups of sera were also tested by CF with envelope and capsid antigens of herpes simplex type 1. By SN, the majority of the monkeys and all of the humans had a higher titer to herpes simplex type 1 than to H. simiae. By CF, with crude antigens the titers in the monkey sera were greater to H. simiae than to herpes simplex type 1, although four sera had equal titers to both antigens; the titers in the human sera were conversely higher with the herpes simplex type 1 antigen, except for four sera which had equal titers to both antigens. The capsid CF antigen of herpes simplex type 1 was reactive with the human sera but virtually nonreactive with the monkey sera; the envelope CF antigen of herpes simplex type 1 was reactive with both monkey and human sera but was somewhat less reactive than the crude herpes simplex type 1 CF antigen. In addition, serum samples from a patient recently infected with H. simiae were examined by CF and SN for antibody to both herpes simplex type 1 and H. simiae viruses. The serological profile indicated a positive correlation with the infecting virus. Although the SN titers did not conclusively reflect an infection with H. simiae, the CF titers were higher to H. simiae than to herpes simplex in later sera and thus appeared to be compatible with H. simiae infection.

Norwalk-related viral gastroenteritis due to contaminated drinking water.

An explosive outbreak of gastrointestinal illness clinically compatible with infection by an agent serologically related to Norwalk virus agent occurred in an elementary school in May 1978. Seroconversion by radioimmunoassay to the Norwalk antigen was noted in two of three ill persons, but no viral particles were identified in stool. Illness developed in 72% of students and teachers at the school, and 32% of household contacts of these ill persons. Of household contacts of persons exposed at school but not clinically ill, 11% developed illness. This value, however, was not statistically different from the level of illness observed concurrently in household contacts of students at an unaffected school nearby. Epidemiologic investigation implicated water as the mode of transmission. Average consumption of one or more glasses per day was strongly associated with illness (p less than 0.00000001). Among soccer team members with limited school contact, water consumption at the school was associated with a 14-fold greater risk of illness (p less than 0.000001). Drinking water was most likely contaminated by back-siphonage through a cross-connection between the school's well and septic tank. This contamination occurred approximately 24 to 36 hours before the outbreak developed.

Characteristics of noncultivable adenoviruses associated with diarrhea in infants: a new subgroup of human adenoviruses.

Virus particles morphologically resembling adenovirus were found in fecal specimens from infants and were examined for cultivability with standard cell culture techniques and for characteristics of human adenoviruses. Specimens from 13 of 15 infants could not be cultivated in cell cultures. The two adenoviruses that were cultivated, types 1 and 31, reacted in the expected manner in all tests. Counterimmunoelectrophoresis with group-specific anti-hexon serum confirmed that the observed particles in the 15 specimens were human adenoviruses. The buoyant density in sucrose of five of the noncultivable adenoviruses in original stool suspensions averaged 1.335 g/cm(3) and that of the two cultivable ones averaged 1.332 g/cm(3); both groups had typical adenovirus morphology by electron microscopy. Treatment of the specimens and of a variety of tissue culture cells with proteolytic and other enzymes did not improve cultivability. Examination of partially purified virus by immunoelectron microscopy did not reveal evidence of immunoglobulin A, G, or M coating on the particles, an indication that coproantibody inhibition was not the cause of noncultivability. Fluorescent-antibody studies with an antihexon conjugate and counterimmunoelectrophoresis studies of serially passaged noncultivable viruses indicated that the viruses are infecting cells but are not undergoing effective replication. Antisera to three of the noncultivable viruses demonstrated homologous reactions in counterimmunoelectrophoresis with the respective immunizing antigens but showed only low levels of hemagglutination-inhibiting and neutralizing activity to a few of the known human adenoviruses. We concluded that the noncultivable viruses in these infant diarrhea cases were indeed human adenoviruses, were not defective particles, were not bound to coproantibody, were infectious but incapable of effective relication in conventional cell cultures, were serologically related to types 11, 17, 32, and 33, and should be considered a new, distinct subgroup.

Comparison of hemagglutination-inhibition, complement-fixation and enzyme-linked immunosorbent assay for quantitation of human rotavirus antibodies.

The hemagglutination-inhibition test for detecting rotavirus antibody was evaluated by using simian rotavirus SA-11 as hemagglutinating antigen. Results show that the test is as sensitive as either complement fixation or the enzyme-linked immunosorbent assay for detecting antibody to rotavirus in human sera.

Serum immunoglobulin A response to Norwalk virus infection.

We describe the serum immunoglobulin A (IgA) antibody response to Norwalk virus infection in human volunteers and compare it with previously described IgM and total antibody responses. Whereas specific IgA and IgM peak within 2 weeks after onset of symptoms, titers of total blocking antibody continue to rise, implying mediation by IgG antibody.

Monoclonal IgG to the inner capsid of human rotavirus.

Monoclonal IgG to the inner capsid of human rotavirus was produced by mice immunized with hybrid cells derived from mouse myeloma and the spleens of mice immunized with rotavirus. This IgG reacted with an antigenic determinant of mammalian rotaviruses that was not present on that of avian rotaviruses.

Adaptation of two avian rotaviruses to mammalian cells and characterization by haemagglutination and RNA electrophoresis.

Turkey and chicken rotaviruses were successfully adapted to replicate in a rhesus monkey embryo kidney (MA104) cell line. Trypsin treatment of virus and cells was essential for serial passage of the viruses. Polyacrylamide gel electrophoresis separated the viral RNA into 11 segments with clear resolution of segments 10 and 11. Segment 5 migrated with size class I, and this characteristic appeared to be a unique feature of the avian rotavirus genome. The ability of the avian rotaviruses to haemagglutinate a variety of erythrocytes was demonstrated. A type-specific antigen was detected by haemagglutination-inhibition assays and a group or subgroup antigen by enzyme immunoassays. Treatment of serum with heparin-MnCl2 was shown to be the best method for removing non-specific inhibitors of haemagglutination.

Chronic parvovirus infection in a presumably immunologically healthy woman.

Infection due to parvovirus B19 is common and usually resolves over several weeks. Prolonged infection has been reported primarily in immunodeficient hosts. The present report describes a chronic infection in an apparently immunologically healthy woman. The illness was characterized by recurrent episodes of paresthesia without anemia. Laboratory studies demonstrated persistence of parvovirus-specific DNA for nearly 4 years.

Comparison of agar and agarose preparations for mengovirus plaque formation.

An agarose overlay yielded mengovirus plaques earlier and in greater size and number than overlays of chemically undefined agars with or without enhancers. Marked variability in plaque-forming efficacy of commercial agarose preparations was noted.