Sequence polymorphism in the E3 7.7K ORF of subspecies B1 human adenoviruses.

Sequences corresponding to the 7.7K open-reading frame (ORF) of the E3 region of subspecies B1 adenoviruses (Ads) were compared with prototype strains of Ad3, Ad7, Ad16, Ad21, and Ad50 and field isolates representing a variety of genome restriction types of Ad3 and Ad7 to better assess the extent of genetic variation in this intriguing region of the viral genome encoding a product whose function is still unknown. Alignment of 55 species B1 Ad sequences revealed a marked polymorphism in the 7.7K ORF and allowed the identification of eight distinct sequence profiles (SPs) characterized by (1) deletions that retain or change the reading frame, (2) single-base mutations (SBMs) that change the start codon (ATG to ATT or ATC), and (3) other SBMs. mRNAs of expected size for the observed sequence polymorphisms were identified by RT-PCR from DNAse I-treated total RNA extracts of infected cells. Predicted proteins ranged from 0 to 94 amino acids corresponding to molecular masses of 0-11 K. Together with the hypervariable regions of the hexon gene, the E3 7.7K ORF appears to be another area of the Ad genome in which genetic diversity may be generated by illegitimate recombination.

An evaluation of measles revaccination among school-entry-aged children.

BACKGROUND: A two dose measles vaccination schedule is recommended routinely for all school-entry-aged children. We evaluated this recommendation by determining both measles antibody seroprevalence and the response to revaccination in seronegative children in this age group. METHODS: Children 4 to 6 years of age who had received a single dose of measles vaccine between the ages of 15 to 17 months were tested for measles antibody by using enzyme-linked immunosorbent assay (ELISA) microneutralization technique. Seronegative children were revaccinated and again tested for measles antibody (immunoglobulin M [IgM] and neutralizing). RESULTS: Of 679 children tested, 37 (5.4%) were seronegative. Seronegativity was not significantly associated with age, sex, race, age at initial vaccination, time since vaccination, or maternal year of birth. However, children mothers with a college degree were 12 times more likely to be seronegative than children of mothers who never attended college (P < .01). Of the 37 seronegative children, 36 seroconverted after revaccination--33 producing IgM measles antibody, suggestive of a primary immune response. The cost per seroconversion would have been an estimated $415 if all 679 children had been revaccinated. CONCLUSIONS: Revaccination reduces the pool of children who are susceptible to measles. Although the cost per seroconversion is high, a two-dose schedule should reduce the substantial costs of controlling measles out breaks by reducing the number of outbreaks.

Nosocomial exposure to parvovirus B19: low risk of transmission to healthcare workers.

OBJECTIVE: To evaluate the risk of nosocomial transmission of parvovirus B19 (B19) infection to healthcare workers (HCWs) exposed to patients with transient aplastic crisis (TAC) caused by acute B19 infection. DESIGN: Cohort study. SETTING: 1,000-bed, urban teaching hospital in Atlanta, Georgia. PARTICIPANTS: Eighty-seven exposed HCWs who cared for two patients with TAC prior to the time they were isolated and a comparison group of 88 unexposed HCWs from wards or clinics where the patients did not receive care. INTERVENTION: Self-administered questionnaire on hospital contact with index patients, B19 community risk factors, and signs and symptoms suggestive of B19 disease. Serology for B19-specific IgM and IgG antibodies measured by antibody-capture enzyme-linked immunosorbent assay. RESULTS: 1 (3.1%) of the 32 nonimmune exposed HCWs had serologic evidence of recent B19 infection compared to 3 (8.1%) of the 37 nonimmune HCWs in the comparison group (P = .6). In a subgroup analysis of exposed HCWs who cared for index patients during the time when the virus load was expected to be greatest, a recent infection rate of 5.8% (1/17) was found among nonimmune HCWs. CONCLUSIONS: The finding of similar rates of recent infection in nonimmune exposed and unexposed HCWs suggests that transmission to HCWs did not occur, despite failure to place the patients in isolation at the onset of hospitalization.

Rapid molecular epidemiologic studies of human parainfluenza viruses based on direct sequencing of amplified DNA from a multiplex RT-PCR assay.

Sequencing studies of limited regions of the human parainfluenza viruses (HPIVs) genomes have helped describe patterns of virus circulation and characterize institutional outbreaks of HPIVs-associated respiratory illness. In this study, we sequenced reverse transcription polymerase chain reaction (RT-PCR)-amplified HPIVs RNA obtained from a multiplex RT-PCR assay described previously for simultaneous detection of HPIV-1, 2 and 3. Differences in the nucleotide sequences of limited regions of the HN gene allowed us to distinguish temporally and geographically diverse HPIV isolates (43 HPIV-1, 7 HPIV-2, 12 HPIV-3 isolates from this and previously published studies). In addition, an outbreak of HPIV-3-associated illness among infants on a pediatric ward was investigated by comparing sequences of three ward isolates with three matched community controls. Sequences of all ward isolates were identical and differed from those of the community controls, suggesting a single introduction and nosocomial transmission of the virus. Combining multiplex reverse transcription polymerase chain reaction (RT-PCR) assays with direct sequencing of the PCR products can provide an integrated system for rapid diagnosis and characterization of HPIVs.

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.

Use of mixture models in determining laboratory criterion for identification of seropositive individuals: application to parvovirus B19 serology.

Selecting the cut-off value to identify infected individuals requires a trade-off between sensitivity and specificity. The standard technique for selecting a cut-off uses results of a population either known or assumed uninfected. Using mixture models, however, it is not necessary to identify an uninfected population. This technique also provides methods to estimate the probability that an individual will be accurately classified. We illustrate this technique in determining cut-off values for the serological diagnosis of human parvovirus B19 infection.

Chemiluminescent microwell hybridization assay for direct detection of human parvovirus B19 DNA.

A method for the direct detection of human parvovirus DNA in serum samples that uses a digoxigenin-labeled RNA probe to hybridize with target B19 DNA, followed by capture of the hybrid onto a microtiter plate wells previously coated with a second oligonucleotide probe was developed. The captured hybrid is then detected with anti-digoxigenin-alkaline phosphatase conjugate and chemiluminescent substrate and the reaction read on a scintillation counter. The relative sensitivities of the microwell and standard dot blot hybridization assays were compared. The chemiluminescent microwell hybridization assay was more sensitive than dot-blot hybridization and could be performed in a few hours. This format, therefore, permits rapid and sensitive detection of parvovirus DNA suitable for the clinical setting.

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.

Genetic diversity in the non-structural gene of parvovirus B19 detected by single-stranded conformational polymorphism assay (SSCP) and partial nucleotide sequencing.

A homologous region in the parvovirus B19 non-structural gene (B19 nt 1399-1682) was examined in 50 samples from patients with a wide variety of B19-related disease from various countries by PCR amplification, single-stranded conformational polymorphism (SSCP) assay and nucleotide sequencing. Five SSCP types were confirmed by nucleotide sequence analysis. Of a total of 6 mutations, all were silent. Types 3 and 4 accounted for 92% of strains. There was no correlation between genome type and either clinical illness or patient age. However, there was a correlation between SSCP type and country of origin. Type 3 strains predominated in Japan (18/26) and the UK (6/8), whereas type 4 predominated in the USA (9/12). Notably, type 3 strains also predominated among females (14/18), whereas there were approximately equal numbers of strain types 3 (7/17) and 4 (8/17) among males; an observation which remains unexplained. Within the Japanese group, although type 3 strains predominated overall, strains isolated from 1981 to 1987 consisted of types 1 (2/15), 2 (1/15), 3 (8/15), and 4 (4/15), whereas strains isolated from 1990 to 1994 consisted almost entirely of type 3 (10/11).

Non-enveloped viruses transmitted by blood and blood products.

Since the advent of solvent detergent (S-D) treatment for inactivation of enveloped viruses, there has been no transmission of human immunodeficiency virus, hepatitis B virus, or hepatitis C virus by treated blood products. However, shortly after the introduction of S-D treatment, transmission of hepatitis A with S-D treated factor concentrates was reported in Germany, Italy, Ireland, the United States and South Africa, and this raised awareness of the potential for blood transmission of non-enveloped viruses in general. This report summarizes the physical and epidemiological features of three non-enveloped viruses, hepatitis A virus, parvovirus B19, and the recently identified TT virus, and their transmission by blood and blood products.

Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene.

Human adenoviruses (Ads) are responsible for a substantial disease burden. Type-specific identification of Ads can help guide therapeutic and disease prevention strategies and aid epidemiological investigations. Immunotyping of Ads by serum neutralization (SN) is laborious and time consuming and depends upon type-specific antisera that are in short supply. A rapid molecular typing assay based on polymerase chain reaction (PCR) amplification and sequencing of Ad hexon gene hyper-variable regions 1-6 (HVR(1-6)) known to contain type-specific epitopes was evaluated as an alternative to SN. Deduced amino acid sequences of HVR(1-6) obtained from all 51 currently recognized Ad prototype strains were well resolved, with the exception of types 15 and 29, which were identical. Of 192 temporally and geographically diverse Ad field isolates sequenced in this study, and 111 previously published sequences, all more closely matched their predicted prototype strains. Ads were also detected and correctly identified directly from 24 clinical specimens positive by culture or antigen detection. PCR and sequencing of HVR(1-6) offers a practical alternative to SN for typing most Ads and can be readily adapted for use in laboratories with molecular capabilities.

Clinical comparison of ethyl acetate and diethyl ether in the formalin-ether sedimentation technique.

A substitute for the volatile solvent diethyl ether has been actively sought for the Formalin-ether sedimentation technique. Ethyl acetate has recently been shown to be a comparable substitute. In an effort to verify these findings and evaluate ethyl acetate under clinical conditions, comparison studies with 62 fresh human stool specimens were performed. Parallel concentrates with diethyl ether and ethyl acetate were prepared for each specimen, and the quantity and appearance of recovered parasite species were determined. Ethyl acetate was found comparable to diethyl ether in the quantitative recovery of parasite eggs, cysts, and larvae; no distortion or alteration of parasite morphology was observed with either solvent. More care was required, however, to completely remove the interface plugs of ethyl acetate and prevent their remixing the concentrate sediment. In addition, wet mounts prepared from ethyl acetate concentrates were occasionally obscured by liquid bubbles probably composed of remaining insoluble ethyl acetate. Clinical laboratories considering substituting ethyl acetate for diethyl ether in the Formalin-ether sedimentation technique should be aware of these problems and take the appropriate precautions to avoid them.

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus.

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.

Development and evaluation of capture immunoglobulin G and M hemadherence assays by using human type O erythrocytes and recombinant parvovirus B19 antigen.

The capacity of human parvovirus B19 to agglutinate human type O erythrocytes was used to develop immunoglobulin G and M antibody capture hemadherence assays. When results of these assays were compared with those of corresponding antibody capture enzyme immunoassays using a well-characterized panel of 125 serum specimens, a 96.8% overall agreement was obtained between the two methods.

Type-specific identification of human adenovirus 3, 7, and 21 by a multiplex PCR assay.

Human adenovirus (Ad) serotypes 3, 7, and 21 of DNA cluster B:1 are often associated with severe respiratory illness, particularly in infants and young children and, in addition to Ad4, are among the most important causes of acute respiratory disease syndrome in new military recruits. To address the inherent problems associated with classic typing methods, we developed a multiplex PCR assay for the rapid, specific identification of Ad3, Ad7, and Ad21 field isolates. To design type-specific primers for our assay, we sequenced the Ad21 hexon gene and compared this sequence with previously published sequences of Ad3, Ad7, and Ad16. The overall nucleotide (nt) and amino acid (aa) identities between Ad21 and Ad3, Ad7, and Ad16 were similar (ranges 78.3-80.8% nt; 84.1-86.2% aa), with significantly greater variability in the regions of the hexon that encode surface loops 1 and 2. Type-specific primers designed to the hypervariable regions correctly identified Ad3, Ad7, and Ad21 prototype strains and 53 previously typed Ad field isolates. No cross-reactions with other Ad serotypes were identified. Our multiplex PCR assay for type-specific identification of Ad3, Ad7, and Ad21 isolates will provide a rapid and convenient tool for the epidemiologic investigation of Ad-associated respiratory illness.

Detection of human parvovirus B19 DNA PCR products by RNA probe hybridization enzyme immunoassay.

We have developed an RNA probe hybridization enzyme immunoassay for detection of human parvovirus B19 PCR-amplified DNA. The assay is easy to perform and increases assay sensitivity without the added inconvenience and risk of false-positive results associated with nested PCR.

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.

Species-specific identification of human adenoviruses by a multiplex PCR assay.

A multiplex PCR assay was developed by using primers to the fiber gene that could differentiate human adenovirus (Ad) species A through F in a single amplification reaction. The assay correctly identified the species of all 49 recognized Ad prototype strains as well as 180 geographically and temporally diverse Ad field isolates. Ad serotype 6 (Ad6) (species C), Ad16 (species B), Ad31 (species A), and Ad40 and Ad41 (species F) could also be distinguished by amplicon size within each respective species. In comparison, a previously described Ad species-specific multiplex PCR assay that used primers to the Ad hexon gene gave equivocal results with several serotypes of species B, whereas our multiplex assay amplified all species B serotypes equally well. Our multiplex PCR assay will permit rapid, accurate, and cost-effective classification of Ad isolates.

Prevalence of antibodies to human parvovirus B19 nonstructural protein in persons with various clinical outcomes following B19 infection.

Persistent infections with human parvovirus B19 (B19) associated with debilitating chronic disease have been described, although evidence linking B19 to these more unusual clinical outcomes has been inconclusive. Recent reports have suggested that the development of antibodies to the B19 nonstructural protein (NS1) following B19 infection might be linked to development of severe arthropathy and chronic infection. To confirm these findings, the C-terminal region of the NS1 protein was expressed for use in Western blot assays for detection of anti-NS1 IgG antibodies in human serum. Among 91 persons tested, 0 of 20 not previously infected with B19, 9(36%) of 25 with past B19 infection, and 5 (12.5%) of 40 with recent B19 infection, had detectable anti-NS1 antibodies. Of 6 persons with chronic B19 infection, 2 had detectable antibodies to NS1. The presence of anti-NS1 antibodies did not appear to correlate with unusual clinical outcomes or chronic B19 infection.