Splice site requirement for the efficient accumulation of polyoma virus late mRNAs.

Polyoma virus late nuclear primary transcripts are giant and heterogeneous, containing tandem repeats of the late strand of the circular viral genome. Late pre-mRNA processing involves the splicing of noncoding 'leader' exons to each other (removing genome-length introns), with the joining of the last leader to a coding 'body' exon. We have constructed a number of mutants blocked only in leader-leader splicing, or blocked in both leader-leader and leader-body splicing. We examined the accumulation of both nuclear and cytoplasmic late-strand RNAs in NIH3T3 cells. Consistent with our previous results, mutants lacking the 3' splice site of the late leader (leader-leader splicing blocked) showed a 10-20 fold defect in late RNA accumulation. Mutants which lacked the leader 5' splice site (leader-body splicing blocked) had a more profound defect, exhibiting virtually no late-strand cytoplasmic or nuclear RNA. This result was unexpected as a substantial proportion of wild type late cytoplasmic messages are unspliced. A mutant with no intron, but having functional 3' and 5' splice sites bordering the leader exon, is capable of producing large amounts of unspliced late mRNA. This demonstrates that an excisable intron is not a requirement for late mRNA accumulation. The accumulation of polyoma late mRNAs requires the presence of leader exons bordered by functional 3' and 5' splice sites, whether or not these sites are used during pre-mRNA processing.

The sequence and context of the 5' splice site govern the nuclear stability of polyoma virus late RNAs.

We have examined the influence of splicing signals on the stability of polyoma virus late RNAs in the nucleus. Late primary transcripts contain a single 5' splice site and three alternative 3' splice sites. In earlier work we showed that the presence of introns was not required for late RNA accumulation, however, the 5' splice site was essential, as removal of only the 5' splice site was sufficient to destabilize late RNAs up to 100-fold when compared with early RNAs. A complementary clone which retained the 5' splice site but which carried small deletions of all late region 3' splice sites produced wild-type levels of unspliced late RNA. In order to extend this work we have constructed additional types of mutants. Point mutations in the 5' splice site confirmed its importance for RNA stability. Other mutants included constructs in which the spacing between the 5' splice site and the late promoter was altered and 5' splice site insertion mutants where a 58 bp fragment containing the 5' splice site sequence was inserted separately at various restriction sites in the late region. Both types of mutants lacked all of the late 3' splice sites and had only a single 5' splice site. RNase protection analyses of late and early RNAs from these constructs revealed that moving the 5' splice site away from the late promoter (or from its normal context) destabilized late RNAs > 10-fold relative to the wild-type. We conclude that both 5' splice site integrity and its proximity to the late promoter play important roles in the nuclear stability of polyoma virus late RNAs.

Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs.

Polyomavirus late mRNA molecules contain multiple, tandem copies of a noncoding 57-base "late leader" exon at their 5' ends. This exon is encoded only once in the genome. Leader multiplicity arises from leader-leader splicing in giant primary transcripts, which are the result of multiple circuits of the viral genome by RNA polymerase II. We have been interested in learning more about the role of the leader exon in late viral gene expression. We recently showed that an abbreviated-leader mutant virus (ALM) with a 9-base leader exon is nonviable (G. R. Adami and G. G. Carmichael, Nucleic Acids Res. 15:2593-2610, 1987) and has a severe defect in both late pre-mRNA splicing and stability. However, a mutant virus with a different, substituted leader sequence of 51 nucleotides (SLM/MP8) is viable and has no apparent defects. Here we examined further the role of the late leader exon in late pre-mRNA processing. When the leader exon length was gradually reduced from 51 nucleotides to 9 nucleotides in a series of mutants, RNA splicing and stability defects were coupled. In this system there was a minimum exon size of between 33 and 27 nucleotides. Next, a number of mutations were introduced into the 3' splice site which precedes the late leader. Such mutations blocked leader-leader splicing. Surprisingly, they also interfered with leader-mVP1 body splicing and resulted in unstable primary transcripts. Thus, polyomavirus leader-leader splicing appears to be important for the efficient accumulation of late viral mRNA molecules.

Clones from the human gene complex coding for salivary proline-rich proteins.

The salivary protein gene complex consists of a series of loci coding for related but distinct proline-rich proteins (PRPs) found chiefly in saliva. We have screened a library of human genomic DNA fragments in bacteriophage lambda Charon 4A with a PRP cDNA synthesized and cloned from rat parotid gland mRNA. Two phages (PRP1 and PRP2) hybridizing to the rat probe under moderately stringent conditions contain related but not identical DNAs. Preliminary nucleotide sequence data indicate that both DNAs include regions comprised of nearly identical tandemly repeated sequences, each able to code for about 21 amino acids. The decoded consensus repeat sequence is homologous to the repeating amino acid units found by others in human PRPs. This and other features demonstrate that these two clones are members of the PRP gene family. Polymorphic differences between the DNAs of different individuals were observed after probing digests of human genomic DNA with a HinfI fragment from PRP1. These DNA polymorphisms reflect size differences, possibly caused by frequent unequal crossing-over between the repeated units in the PRP genes.

Suspected Brazilian purpuric fever in a toddler with overwhelming Epstein-Barr virus infection.

We describe a toddler from Connecticut who developed purulent conjunctivitis, fever, and a morbilliform rash. Blood cultures were positive for Haemophilus influenzae biogroup aegyptius; further investigation was performed to assess the possibility that the illness was consistent with Brazilian purpuric fever, which, to our knowledge, has not been reported in the United States. This isolate shared morphological and some biochemical characteristics with previously studied H. influenzae biogroup aegyptius strains but differed according to slide agglutination testing, plasmid characterization, and ribotyping. Blood and tissue samples obtained during his hospitalization were also positive for Epstein-Barr virus. The child died 8 days after hospitalization. Fifty other cases of invasive H. influenzae infection were identified by active surveillance studies. Of the 49 viable surveillance isolates, 10 were biotype III (two of which had the same ribotype as the strain from our case.

Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995-1997.

OBJECTIVES: This study examined epidemiologic factors affecting mortality from pneumococcal pneumonia in 1995 through 1997. METHODS: Persons residing in a surveillance area who had community-acquired pneumonia requiring hospitalization and Streptococcus pneumoniae isolated from a sterile site were included in the analysis. Factors affecting mortality were evaluated in univariate and multivariate analyses. The number of deaths from pneumococcal pneumonia requiring hospitalization in the United States in 1996 was estimated. RESULTS: Of 5837 cases, 12% were fatal. Increased mortality was associated with older age, underlying disease. Asian race, and residence in Toronto/Peel, Ontario. When these factors were controlled for, increased mortality was not associated with resistance to penicillin or cefotaxime. However, when deaths during the first 4 hospital days were excluded, mortality was significantly associated with penicillin minimum inhibitory concentrations of 4.0 or higher and cefotaxime minimum inhibitory concentrations of 2.0 or higher. In 1996, about 7000 to 12,500 deaths occurred in the United States from pneumococcal pneumonia requiring hospitalization. CONCLUSIONS: Older age and underlying disease remain the most important factors influencing death from pneumococcal pneumonia. Mortality was not elevated in most infections with beta-lactam-resistant pneumococci.

Hemolytic-Uremic Syndrome and Escherichia coli O121 at a Lake in Connecticut, 1999.

OBJECTIVE: Non-O157 Shiga toxin-producing Escherichia coli (STEC) have emerged as an important public health problem. Outbreaks attributed to non-O157 STEC rarely are reported. In 1999, follow-up of routine surveillance reports of children with hemolytic- uremic syndrome (HUS) identified a small cluster of 3 cases of HUS, all of whom had spent overlapping time in a Connecticut lake community in the week before onset of symptoms. We conducted an investigation to determine the magnitude and source of the outbreak and to determine risk factors associated with the transmission of illness. METHODS: We conducted a cohort study and an environmental investigation. The study population included all people who were at the lake in a defined geographic area during July 16-25, 1999. This time and area were chosen on the basis of interviews with the 3 HUS case-patients. A case was defined as diarrhea (>/=3 loose stools/d for >/=3 days) in a person who was at the lake during July 16-25, 1999. Stool samples were requested from any lake resident with diarrheal illness. Stools were cultured for Salmonella, Shigella, Campylobacter, and E coli O157. Broth cultures of stools were tested for Shiga toxin. Case-patients were asked to submit a serum specimen for antibody testing to lipopolysaccharides of selected STEC. Environmental samples from sediment, drinking water, lake water, and ice were obtained and cultured for E coli and tested for Shiga toxin. An environmental evaluation of the lake was conducted to identify any septic, water supply system, or other environmental condition that could be related to the outbreak. RESULTS: Information was obtained for 436 people from 165 (78%) households. Eleven (2.5%) people had illnesses that met the case definition, including the 3 children with HUS. The attack rate was highest among those who were younger than 10 years and who swam in the lake on July 17 or 18 (12%; relative risk [RR]: 7.3). Illness was associated with swimming (RR = 8.3) and with swallowing water while swimming (RR = 7.0) on these days. No person who swam only after July 18 developed illness. Clinical characteristics of case-patients included fever (27%), bloody diarrhea (27%), and severe abdominal cramping (73%). Only the 3 children with HUS required hospitalization. No bacterial pathogen was isolated from the stool of any case-patient. Among lake residents outside the study area, E coli O121:H19 was obtained from a Shiga toxin-producing isolate from a toddler who swam in the lake. Serum was obtained from 7 of 11 case-patients. Six of 7 case-patients had E coli O121 antibody titers that ranged from 1:320 to >1:20 480. E coli indicative of fecal contamination was identified from sediment and water samples taken from a storm drain that emptied into the beach area and from a stream bed located between 2 houses, but no Shiga toxin-producing strain was identified. CONCLUSIONS: Our findings are consistent with a transient local beach contamination in mid-July, probably with E coli O121:H19, which seems to be able to cause severe illness. Without HUS surveillance, this outbreak may have gone undetected by public health officials. This outbreak might have been detected sooner if Shiga toxin screening had been conducted routinely in HUS cases. Laboratory testing that relies solely on the inability of an isolate to ferment sorbitol will miss non-O157 STEC, such as E coli O121. Serologic testing can be used as an adjunct in the diagnosis of STEC infections. Lake-specific recommendations included education, frequent water sampling, and alternative means for toddlers to use lake facilities.

Epidemiology of invasive Streptococcus pneumoniae infections in the United States, 1995-1998: Opportunities for prevention in the conjugate vaccine era.

CONTEXT: Pneumococcal polysaccharide vaccine is recommended for elderly persons and adults with certain chronic illnesses. Additionally, a recently licensed pneumococcal 7-valent conjugate vaccine has been recommended for use in young children and could dramatically change the epidemiology of pneumococcal disease. OBJECTIVES: To assess pneumococcal disease burden in the United States, estimate the potential impact of new vaccines, and identify gaps in vaccine recommendations. DESIGN AND SETTING: Analysis of data from the Active Bacterial Core Surveillance (ABCs)/Emerging Infections Program Network, an active, population-based system in 9 states. PATIENTS: A total of 15 860 cases of invasive pneumococcal disease occurring between January 1, 1995, and December 31, 1998. MAIN OUTCOME MEASURES: Age- and race-specific pneumoccocal disease incidence rates per 100 000 persons, case-fatality rates, and vaccine preventability. RESULTS: In 1998, overall incidence was 23.2 cases per 100 000, corresponding to an estimated 62 840 cases in the United States. Incidence was highest among children younger than 2 years (166.9) and adults aged 65 years or older (59.7). Incidence among blacks was 2.6 times higher than among whites (95% confidence interval [CI], 2.4-2.8). Overall, 28.6% of case-patients were at least 65 years old and 85.9% of cases in this age group were due to serotypes included in the 23-valent polysaccharide vaccine; 19.3% of case-patients were younger than 2 years and 82.2% of cases in this age group were due to serotypes included in the 7-valent conjugate vaccine. Among patients aged 2 to 64 years, 50.6% had a vaccine indication as defined by the Advisory Committee on Immunization Practices (ACIP). The case-fatality rate among patients aged 18 to 64 years with an ACIP indication was 12.1% compared with 5.4% for those without an indication (relative risk, 2.2; 95% CI, 1.7-2.9). CONCLUSIONS: Young children, elderly persons, and black persons of all ages are disproportionately affected by invasive pneumococcal disease. Current ACIP recommendations do not address a subset of persons aged 18 to 64 years but do include those at highest risk for death from invasive pneumococcal disease.

Macrolide resistance among invasive Streptococcus pneumoniae isolates.

CONTEXT: Macrolide antibiotics, including erythromycin, clarithromycin, and azithromycin, are the mainstays of empirical pneumonia therapy. Macrolide resistance among Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, is increasing in the United States. Whether resistance is a significant problem or whether macrolides remain useful for treatment of most resistant strains is unknown. OBJECTIVE: To examine the epidemiology of macrolide-resistant pneumococci in the United States. DESIGN AND SETTING: Analysis of 15 481 invasive isolates from 1995 to 1999 collected by the Centers for Disease Control and Prevention's Active Bacterial Core surveillance system in 8 states. MAIN OUTCOME MEASURES: Trends in macrolide use (1993-1999) and resistance and factors associated with resistance, including examination of 2 subtypes, the M phenotype, associated with moderate minimum inhibitory concentrations (MICs), and the MLS(B) phenotype, associated with high MICs and clindamycin resistance. RESULTS: From 1993 to 1999, macrolide use increased 13%; macrolide use increased 320% among children younger than 5 years. Macrolide resistance increased from 10.6% in 1995 to 20.4% in 1999. M phenotype isolates increased from 7.4% to 16.5% (P<.001), while the proportion with the MLS(B) phenotype was stable (3%-4%). The median erythromycin MIC (MIC(50)) of M phenotype isolates increased from 4 microg/mL to 8 microg/mL. In 1999, M phenotype strains were more often from children than persons 5 years or older (25.2% vs 12.6%; P<.001) and from whites than blacks (19.3% vs 11.2%; P<.001). CONCLUSIONS: In the setting of increasing macrolide use, pneumococcal resistance has become common. Most resistant strains have MICs in the range in which treatment failures have been reported. Further study and surveillance are critical to understanding the clinical implications of our findings.