Comprehensive analysis of RHD splicing transcripts reveals the molecular basis for the weak anti-D reactivity of Del -red blood cells.

BACKGROUND: Of the Rh blood type, Del is a rare variant that elicits the weakest anti-D reactivity. In this study, we revisit the genetic changes of Del allele and characterise the RHD splicing transcripts to realise the molecular basis of Del formation in the Taiwanese population. STUDY DESIGN AND METHODS: The RHD exons from Del and D-positive individuals were amplified by polymerase chain reaction (PCR) using different primer pairs followed by sequencing analyses. In addition, full-length RHD transcripts were reversed transcribed and amplified by nested-PCR. The type and frequency of the RHD splicing transcripts were analysed after sequencing the PCR products that were subcloned into a cloning vector. RESULTS: All Del individuals had a characteristic 1227G>A mutation. No deletion of the exon sequences was found. At least nine types of RHD splicing transcripts including exons 7/8/9 deletion, 7/9 deletion, 8/9 deletion, 9 deletion, 2/3/7/9 deletion, 2/3/7/8/9 deletion, exons 7/8/9 deletion with replacement of exon 3 with RHCE exon 3, exon 9 deletion with cryptic insertion of 170 bp of intron 7 and exons 7/8/9 deletion with cryptic insertion of 117 bp of intron 3 were identified in the Del -RBC. These aberrant splicing transcripts led to production of frame shift or truncated D antigen. Notably, no full-length RHD transcript was identified in the Del -RBC. CONCLUSION: The RHD 1227G>A mutation contributes to the molecular basis of Del phenotype in the Taiwanese population. The point mutation results in aberrant frame shift or exon deletion transcripts and generates D protein with weak antigen presenting function.

Genetic and mechanistic evaluation for the weak A phenotype in Ael blood type with IVS6 + 5G>A ABO gene mutation.

BACKGROUND AND OBJECTIVES: Ael is a rare blood type that is characterized by weak agglutination of RBCs when reacts with anti-A antibody in adsorption-elution test. Although IVS6 + 5G→A mutation is known to associate with the Ael blood type, genetic and mechanistic evaluation for the weak agglutination of Ael with IVS6 + 5G→A mutation has not yet been completely addressed. MATERIALS AND METHODS: In this study, five cases of confirmed Ael individuals were analysed. The cDNAs for the A(el) alleles were obtained by cloning method for sequence analyses. The erythroleukemia K562 cells were used as the cell study model and were transfected with the A(el) expression construct. Flow cytometry analysis was then performed to determine the levels of surface antigen expression. RESULTS: The results indicated that IVS6 + 5G→A attributes to all cases of Ael . RT-PCR analyses revealed the presence of at least 10 types of aberrant A(el) splicing transcripts. Most of the transcripts caused early termination and produced non-functional protein during translation. Nevertheless, the transcript without exons 5-6 was predicted to generate functional Ael glycosyltransferase lacking 57 amino acids at the N-terminal segment. When the exons 5-6 deletion transcript was stably expressed in the K562 cells, weak agglutination of the cells can be induced by adding anti-A antibody followed by adsorption-elution test. CONCLUSION: This study demonstrates that aberrant splicing of A transcripts contributes to weak A expression and the weak agglutination of Ael -RBCs, adding to the complexity for the regulatory mechanisms of ABO gene expression.

Duration of enterovirus shedding in stool.

Excretion of enterovirus (EV) may persist for months after an EV infection; the exact duration of excretion, however, is not yet known. Twelve children who were infected with EV between September 1998 and June 1999 were enrolled into this study. The patients included 4 boys and 8 girls, aged from 1 month to 5 years. Six patients were asked to join this virus isolation program, and the other 6 were followed-up regularly. Only 2 of the patients were infected with EV 71. To delineate the duration of EV shedding in each case, throat swabs for virus isolation were performed every 1 or 2 weeks for at least 1 month, and stools were analyzed for at least 2 months following the same schedule. After the infection, EV was identifiable in the throat in 4 patients for 1 to 2 weeks. Excretion of EV through stool was evidenced for up to 7 weeks in 6 patients, 8 weeks in 3, and 11 weeks in 1. In the 2 patients who failed to show up for follow-up visits from the 7th week, excretion of EV through stool was recorded for at least 7 weeks. Different serotypes of EV could be isolated from the same patient who was not experiencing febrile illness in 2 instances in a series of virus cultures. Coexistence of vaccine poliovirus and non-polio EV, both isolated from stool, was evidenced in 2 patients. Results from this study suggest that EV may not be identified from the throat 2 weeks after the infection, but its excretion through stool can persist for up to 11 weeks. This study also demonstrated that subclinical EV coinfection could occur, and that live vaccine poliovirus did not interfere with the invasion of other non-polio EV.

Acute encephalomyelitis during an outbreak of enterovirus type 71 infection in Taiwan: report of an autopsy case with pathologic, immunofluorescence, and molecular studies.

We report a fatal case of enterovirus type 71 (EV 71) infection in an 8-year-old girl during a summer outbreak of hand, foot, and mouth disease in 1998 in Taiwan. The clinical course was rapidly progressive, with manifestations of hand, foot, and mouth disease, aseptic meningitis, encephalomyelitis, and pulmonary edema. The patient died 24 hours after admission. Postmortem study revealed extensive inflammation in the meninges and central nervous system and marked pulmonary edema with focal hemorrhage. Brain stem and spinal cord were most severely involved. The inflammatory infiltrates consisted largely of neutrophils involving primarily the gray matter with perivascular lymphocytic cuffing, and neuronophagia. The lungs and heart showed no evidence of inflammation. EV 71 was isolated from the fresh brain tissues and identified by immunofluorescence method with type-specific EV 71 monoclonal antibody. It was also confirmed by neutralization test and reverse-transcriptase polymerase chain reaction with sequence analysis. The present case was the first example in which EV 71 was demonstrated to be the causative agent of fatal encephalomyelitis during its epidemic in Taiwan.

Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998.

A large scale outbreak of hand-foot-and-mouth disease (HFMD) occurred in Taiwan in 1998, in which more than 80 children died of shock syndrome with pulmonary edema/hemorrhage. Enterovirus 71 was implicated as the cause of this outbreak. In order to understand the virological basis responsible for mortality on this scale, nucleotide sequences of VP1 that is important for serotypic specificity, and the 5'-non-coding region (5'-NCR) that is important for replication efficiency, were analyzed comparatively. Phylogenetic analysis of both VP1 and 5'-NCR of nine EV71 isolates derived from specimens of fatal patients and seven isolates derived from uncomplicated HFMD patients showed that all but one isolate fell into genotype B. The one distinct isolate from a case of uncomplicated HFMD belonged to genotype C that was clustered along with one isolate from Taiwan in 1986. Complete sequence analysis of two selected isolates, one from the spinal cord of a fatal case and one from the vesicle fluid of a patient with mild HFMD, confirmed a high degree (97-100%) of identity in nucleotide sequence throughout the entire genome, except focal regions of 3C and 3'-NCR where the nucleotide homology was 90-91%. The identity of the deduced amino acid sequence in the 3C region that encodes viral proteinase dropped further to 86%, a result of missense mutations at the first nucleotide position of many codons.

Expression of capsid [correction of caspid] protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection.

To produce enterovirus 71 antigen for diagnostic purposes, the gene encoding the entire capsid protein VP1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and expressed in Escherichia coli as a poly-histidine fusion protein. Western blotting experiments with sera from patients with enterovirus 71 infection indicated that immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide VP1. According to these results, IgM anti-VP1 appeared in sera of patients with a symptomatic enterovirus 71 acute infection, whereas IgG anti-VP1 was present in sera of past infection. This finding suggests that detecting IgG and IgM immune responses against linear epitopes of recombinant VP1 is an effective means of determining the different phases of enterovirus 71 infection. In addition, sera containing coxsackie virus 16 (CA16) antibodies did not cross-react with the recombinant VP1 of enterovirus 71, despite the homology between VP1 proteins of both viruses. Comparison with reference PCR and neutralization assays showed these antibody tests to be appropriate for the serodiagnosis of enterovirus 71 infection.

Comparison of enterovirus 71 and coxsackie-virus A16 clinical illnesses during the Taiwan enterovirus epidemic, 1998.

OBJECTIVES: To compare enterovirus 71 (EV 71) with coxsackievirus A16 (Cox A16) clinical illness in patients at Chang Gung Children's Hospital during Taiwan's enterovirus epidemic of 1998. METHODS: With the use of the immunofluorescence assay and neutralization test, 177 cases of EV 71 and 64 cases of Cox A16 illness were confirmed from April to September, 1998. The clinical signs and symptoms, complications and case fatality rates were compared. RESULTS: Three-fourths of the cases were younger than 3 years of age, and the ratio of males to females was 1.3 in the EV 71 group and 1.2 in the Cox A16 group. In the EV 71 group 120 (68%) cases were uncomplicated, including 94 cases of hand, foot and mouth disease and 15 cases of herpangina, and 57 (32%) cases had complications, including 13 (7.3%) cases of aseptic meningitis, 18 (10%) cases of encephalitis, 4 (2.3%) cases of polio-like syndrome, 8 (4.5%) cases of encephalomyelitis and 12 (6.8%) cases of fatal pulmonary edema. Fourteen (7.9%) patients died, including 12 cases of pulmonary edema and 2 cases of encephalitis; seven (4%) patients had sequelae. By contrast, 60 (94%) of the 64 cases of Cox A16 infection were uncomplicated and only 4 (6.3%) cases were complicated by aseptic meningitis; no fatalities or sequelae were observed. By multivariate analysis vomiting (P = 0.01) and fever higher than 39 degrees C plus lasting longer than 3 days (P = 0.02) were significantly more frequent in the EV 71 group. CONCLUSION: EV 71 illness is more severe with significantly greater frequency of serious complications and fatality than is illness caused by Cox A16.

Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease.

BACKGROUND: In Taiwan, from April to July, 1998, an epidemic of hand, foot, and mouth disease associated with enterovirus 71 (EV71) occurred with fatal complications. We did a clinical study of EV71-related diseases in Taiwan. METHODS: We studied 154 children with virus-culture confirmed EV71 infection. Children were divided into three groups: 11 patients with pulmonary oedema; 38 patients with central nervous system (CNS) involvement and no pulmonary oedema; and 105 children without complications. We compared the clinical features, laboratory findings, risk factors, and outcome among these three groups. FINDINGS: Nine children with pulmonary oedema had hand, foot, and mouth disease, one had herpangina, and one had febrile illness with eight children with limb weakness and one with limb hypesthesia. All children had had sudden onset of tachycardia, tachypnoea, and cyanosis 1-3 days after onset of the disease. Nine of 11 children died within 12 h of intubation; one child was braindead within 15 h and died 17 days after intubation; one child was in deep coma and died 3 months later. In children with CNS complication and no pulmonary oedema, one child died of pneumonia after 4 months of ventilator support and four children had sequelae. All 105 children without complications recovered. There was a significant association between CNS involvement and pulmonary oedema (odds ratio 12.4 [95% CI 2.6-60.1], p=0.001). Risk factors for pulmonary oedema after CNS involvement were hyperglycaemia, leucocytosis, and limb weakness. Hyperglycaemia was the most significant prognostic factor for pulmonary oedema (odds ratio 21.5 [3-159], p=0.003). INTERPRETATION: EV71 can cause hand, foot, and mouth disease, CNS involvement with severe sequelae, and fatal pulmonary oedema. Hyperglycaemia is the most important prognostic factor.

Diagnosis of respiratory tract viruses in 24 h by immunofluorescent staining of shell vial cultures containing Madin-Darby Canine Kidney (MDCK) cells.

Nine hundred and seventy-eight clinical specimens were examined taken from patients with respiratory tract viruses (RV)-like syndrome between November 1996 and July 1998. The study was undertaken to evaluate the effectiveness of centrifuge-enhanced shell vial cultures (SVC) containing Madin-Darby Canine Kidney (MDCK) cells, combined with immunofluorescent (IF) staining in 24 h. This technique rapidly detects and identifies respiratory tract viruses. The conventional tube culture system with multiple cell lines would ordinarily detect RV within 3-30 days. The SVC/IF method using single cell line (MDCK cells) allowed detection of 81.5% of influenza A virus, 72% of parainfluenza virus, 82.6% of respiratory syncytial virus (RSV) and 79.6% of adenovirus in 24 h.

Identification of VP7 serotypes of human rotaviruses by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction.

Serotype specificity of human rotaviruses in Taiwan remained unclear. This study has established three methods for identification of G serotypes, fluorescent focus neutralization test (FFN), enzyme-linked immunosorbent assay by incorporating VP7 (G) serotype-specific monoclonal antibodies (ELISA-MAb) and reverse transcription-polymerase chain reaction (RT-PCR). FFN is a reference standard test for determination of G serotype. RT-PCR and ELISA-MAb were used for serotyping of rotavirus isolates and appeared to be accurate and more sensitive than FFN. Nevertheless, for serotyping the rotaviruses present in clinical stool samples, ELISA-MAb was not satisfactory, only 65.5% of the 264 specimens could be serotyped. The sensitivity of serotype prediction by RT-PCR was 89.4%. When both ELISA-MAb and RT-PCR were taken into account, G serotype could be predicted for 93.2% of the specimens. Serotyping results were comparable with the RNA profiles. Rotaviruses with similar RNA electrophoretic pattern had the same serotype specificity. RNA patterns could be used as a reference for prediction of serotypes. Because ELISA is easy to perform and less expensive, it would be suggested as a screening test. RT-PCR could be used for samples which could not be serotyped by ELISA-MAb. Furthermore, RT-PCR is especially valuable when serotype-specific monoclonal antibodies are not available.

Oral acyclovir prophylaxis of varicella after intimate contact.

OBJECTIVES: Whether oral acyclovir (ACV) given in late incubation can prevent clinical varicella or not. MATERIALS AND METHODS: Twenty-seven healthy infants and children susceptible to varicella received oral ACV (40 mg/kg daily in four divided doses) for 5 days, starting 9 or 11 days after exposure from the index case in the family (2 in the classroom). The clinical features were compared with 13 control children who did not receive ACV. Enzyme-linked immunoassay was used to detect varicella-zoster virus (VZV) antibody and, in follow-up immunologic studies, lymphocyte proliferative response was added. In some cases, blood culture and polymerase chain reaction with Southern hybridization were used for detection of viremia. RESULTS: Among the 27 children in the treatment group, two (7.4%) developed the disease and seroconversion was observed in 17 subjects (63%). Follow-up immunologic studies in 12 of these 17 seroconverted subjects 30 months later showed persistent cellular and/or humoral immunity to VZV. Only one subject, bled 11 days after exposure, had positive VZV DNA and blood culture for VZV. On the other hand 10 of 13 (77%) control subjects developed clinical varicella. CONCLUSIONS: Oral ACV administration to healthy susceptible subjects at the beginning of secondary viremia in the late incubation period (9 days after exposure) can effectively prevent or modify clinical varicella.