Joseph J. Eiden

Improved detection of rotavirus shedding by polymerase chain reaction

To improve identification of children excreting rotavirus a method for the amplification of rotavirus RNA by the polymerase chain reaction (PCR) was developed. The assay was compared with a solid-phase enzyme immunoassay in the detection of rotavirus shedding by infants in hospital during the winter peak of rotavirus infections. Forty children were studied in an intermediate care unit after transfer from intensive care units. Only two were admitted primarily because of diarrhoea; the other thirty-eight were admitted for management of various other disorders. Rotavirus shedding was detected by enzyme immunoassay in twenty of the infants, and nine of these (aged 1 week to 8 months) remained in hospital for more than 5 days after the initial detection of rotavirus and could be studied long term. Of 103 faecal samples from the nine infants, 60 (58%) contained rotavirus RNA detected by reverse-transcriptase (RT)/PCR, whereas only 37 (36%) were positive for rotavirus antigen by the immunoassay (chi 2 = 10.3, p less than 0.002). The geometric mean time of rotavirus shedding was 9.5 (range 1-19) days as detected by RT/PCR and 5.7 (range 1-17) days by the immunoassay (p less than 0.018). In five of the nine children, RT/PCR detected rotavirus shedding for 2-7 days longer than the immunoassay and in four children RT/PCR was positive 1 or more days before rotavirus antigen was detected. Further studies should attempt to find out whether infected infants are capable of spreading wild-type virus during periods when they are not shedding antigen as detectable by enzyme immunoassay.

Rotavirus Immunoglobulin A Responses Stimulated by Each of 3 Doses of a Quadrivalent Human/ Bovine Reassortant Rotavirus Vaccine

A quadrivalent precursor to the pentavalent rotavirus vaccine candidate RotaTeq was evaluated in a 3-dose, 439-subject study. To determine immunogenicity, the quantity of rotavirus immunoglobulin A (IgA) in stool specimens obtained, at 1 of 10 study sites, from 37 placebo and 37 vaccine recipients was measured. None of the placebo recipients showed a clinically important (>/=3-fold) increase in stool rotavirus IgA, whereas 31 vaccine recipients showed an increase after at least 1 dose of vaccine. In total, 16, 19, and 15 vaccine recipients had increases after 1, 2, and 3 doses, respectively, indicating that a 3-dose regimen increased the immune response elicited by this vaccine.

Phylogenetic Relationship of Chlamydia pneumoniae to Chlamydia psittaci and Chlamydia trachomatis as Determined by Analysis of 16S Ribosomal DNA Sequences

The 16S ribosomal DNA sequence of Chlamydia pneumoniae was determined and compared with the corresponding gene sequences of Chlamydia psittaci and Chlamydia trachomatis. C. pneumoniae has been reported to exhibit little chromosomal DNA homology with the other chlamydial species, and its phylogenetic relationships within the genus Chlamydia have not been described. A polymerase chain reaction was employed to determine the 16S rRNA gene sequence of C. pneumoniae. Ten primers from the C. psittaci sequences were used to amplify a C. pneumoniae template in overlapping segments of the gene. Sequence data for 1,554 bases indicated that the levels of homology of C. pneumoniae with C. psittaci and C. trachomatis were 96.19 and 94.07%, respectively. These data support the results of previous biochemical and developmental studies indicating that C. pneumoniae is more closely related to C. psittaci than to C. trachomatis.

EVIDENCE THAT A NOVEL ROTAVIRUS-LIKE AGENT OF RATS CAN CAUSE GASTROENTERITIS IN MAN

A rotavirus-like agent known to cause gastroenteritis in rats was also found to be associated with diarrhoea in a human population. Serological studies indicated that infection with this virus is prevalent in children and adults living in Baltimore, Maryland. The virus is antigenically and genomically distinct from group A rotaviruses and this should be borne in mind in efforts to develop vaccines for protection against infantile gastroenteritis.

Rotavirus rna variation during chronic infection of immunocompromised children

Polyacrylamide gel electrophoresis was performed on RNA from rotaviruses isolated from two immunocompromised patients with prolonged rotavirus diarrhea. Extensive variations were observed in the genomes of rotaviruses isolated from each patient during a 16-week period of their illness. An animal model of rotavirus infection was used to help evaluate the results found in our patients. Individual mouse rotavirus strains were found to be stable during serial passage, but individual animals could be coinfected with more than one viral strain. The changes found in the rotavirus RNA from our patients possibly represented reinfection by different viral strains. Appropriate precautions should be observed to prevent such reinfections in immunocompromised patients.

Comparison of group B rotavirus genes 9 and 11

Group B rotaviruses (GBRs) were recognized recently as causative agents of gastroenteritis. Investigations into the relatedness of various heterologous GBR strains have been hindered by the difficulty of growing these viruses in cell culture. Viral RNA extracted from experimentally infected rats used to prepare cDNA clones. From these, the nucleotide sequences of genes 9 and 11 of the IDIR strain of GBR were determined and compared with the corresponding sequences of the human ADRV strain of GBR. IDIR gene 11 is 643 bp in length with a single open reading frame (ORF) encoding 174 amino acids; IDIR gene 9 is 804 bp in length with a single ORF encoding 246 amino acids. Comparison of the IDIR sequences with those of ADRV showed that nucleotide sequence similarity was 60.6% and 71.9% for genes 9 and 11, respectively. The deduced amino acid sequence similarity was 51.2% for the gene 9 and 66.5% for the gene 11 product. This sequence diversity indicates that GBRs are more distantly related than strains of group A rotavirus.

Molecular cloning, sequence analysis, in vitro expression, and immunoprecipitation of the major inner capsid protein of the IDIR strain of group B rotavirus (GBR)

The sixth genomic segment of the infectious diarrhea of infant rats (IDIR) strain of group B rotavirus (GBR) was cloned from double-stranded RNA purified from infected rat feces. Sequence comparison with group A rotaviruses (GAR) and the human ADRV strain of GBR indicated that IDIR gene 6 encoded the major inner capsid protein. The nucleic acid sequences of the two GBR genes were 72.9% conserved, and 83.4% of the amino acids were identical. Sequence substitutions between IDIR and ADRV were more numerous than reported for heterologous GAR strains, indicating that the two GBR strains may have diverged from one another over a longer period of time. Despite the sequence heterogeneity exhibited by the major inner capsid proteins of ADRV and IDIR, hydrophilicity plots of the two gene products were nearly indistinguishable. The GBR hydrophilicity plots displayed little similarity with those of rotavirus groups A or C, indicating substantial differences in the structures of those major inner capsid proteins. In vitro transcription and translation of IDIR gene 6 yielded a polypeptide product consistent in size with that predicted from the deduced amino acid sequence and the virion major inner capsid protein. The IDIR 6 polypeptide was immunoprecipitated by antisera directed against IDIR as well as antisera directed against ADRV and a heterologous bovine strain of GBR. No immunoprecipitation was observed with control sera or antisera directed against GAR. These results confirmed that group-specific epitopes were displayed by the major inner capsid protein encoded by IDIR gene 6. Reactivity with heterologous GBR antisera also indicated that the IDIR gene 6 product may prove useful as a standard reagent in immunoassays for the detection of GBR.

Infant immune response to human rotavirus serotype G1 vaccine candidate reassortant WI79-9: different dose response patterns to virus surface proteins VP7 and VP4.

Background. Rotavirus is the leading cause of morbidity from gastroenteritis in the developed world and the leading cause of mortality from viral gastroenteritis (estimated 600 000 deaths) worldwide. G1 is the most prevalent human serotype. Reassortant rotavirus between simian rotavirus RRV or bovine rotavirus WC3 and human strain rotaviruses have been extensively tested as candidate vaccines. Rotavirus (RV) reassortant strain WI79-9 consists of a human (strain WI79) G1 serotype VP7 surface protein on a bovine (strain WC3) background. It is a key component of a pentavalent (G1, G2, G3, G4 and P1) WC3 reassortant vaccine candidate, RotaTeq, now being tested in Phase III clinical trials. Methods. We studied 84 infants between the ages of 2 and 8 months who received 3 oral doses of WI79-9. Serum neutralizing antibody was measured to the human (WI79 serotype P1 G1) and bovine (WC3 serotype P7 G6) parent RV after each dose. A significant response was defined as a ≥3-fold rise in antibody titer between the predose and postdose sera. Results. In two separate cohorts of vaccinees given three doses of WI79-9 reassortant rotavirus, 68 to 75% of infants demonstrated a significant response to WC3 (VP4, P7) after Dose 1, fewer (24 to 39%) responses were detected after Dose 2 and rare (0 to 4%) additional responses occurred after Dose 3. The cumulative response rate to WC3 after three doses was 95% in both trials. In contrast 23 to 37% had a significant response to WI79 (VP7, G1) after Dose 1, and 57 to 61% had a significant response after Dose 2. Additional significant responses after Dose 3 led to a cumulative response of 70 to 84%. Conclusion. Two doses of G1 reassortant WI79 were necessary to induce significant antibody responses to human G1 (VP7) antigen in >50% of infants. Three doses were required to achieve significant antibody responses to VP7 in >70% of infants.

Use of ciprofloxacin in an infant with ventriculitis

Ciprofloxacin was used successfully in a neonate with ventriculitis caused by a multiply resistant strain of Enterobacter cloacae. Limited pharmacokinetic data indicated that adequate concentrations of drug could be attained in cerebrospinal fluid.

Engineering the Assembly Pathway of the Baculovirus‐Insect Cell Expression Systema

The synthesis of complex biological structures such as antibodies using recombinant DNA technology is a major biotechnological advance. Active murine antibody (IgG) oligomers, composed of two heavy (H) and two light (L) polypeptide chains, have been expressed and secreted by the baculovirus-insect cell expression system. Unfortunately, expression of the functional antibodies is accompanied by the formation of abnormal protein complexes and aggregates in which the polypeptide chains are bound together into incorrect associations. The formation of these abnormal complexes or protein aggregates in insect cells may be caused by insufficient intracellular levels of two catalytic proteins, immunoglobulin binding protein (BiP or GRP78), and protein disulfide isomerase (PDI). Consequently, we obtained the genes coding for murine BiP and PDI and cloned the genes into the baculovirus vector (Autographa californica nuclear polyhedrosis virus) to obtain AcBB-BiP and AcBB-PDI. Infection of Spodoptera frugiperda (Sf-9) insect cells with these two baculoviruses yielded recombinant proteins of the correct size that were recognized by antibodies to these proteins. Cloning these genes into the baculovirus vector is one approach to engineering the assembly pathway in order to lower aggregation and increase production of functionally active proteins and oligomers.