MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway.

Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma-infected mice 5days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin (5-HT) and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and 5-HT metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.

Elevated levels of human endogenous retrovirus-W transcripts in blood cells from patients with first episode schizophrenia.

We previously reported on the differential presence of transcripts related to the human endogenous retrovirus (HERV)-W family in cerebrospinal fluid and plasma from patients with first-episode schizophrenia compared with control individuals. Whether this is a consequence of qualitative or quantitative differences in transcription of genomic regions harboring HERV-W elements is not known. The purpose of the present study was therefore to characterize the transcribed HERV-W elements in mononuclear cells obtained from 30 patients first hospitalized for schizophrenia-related psychosis and from 26 healthy control individuals. We observed elevated total levels of HERV-W gag (2.1-fold, P < 0.01) but not env transcripts in the cells of patients compared with controls. By using the melting temperatures of the amplicons as a proxy marker for sequence identity, no absolute qualitative differences was detected between the two groups. Mapping of the detected transcripts identified several intronic and intergenic HERV-W elements transcribed in the cells, including elements previously considered transcriptionally silent. Element-specific assays revealed elevated levels of intronic transcripts containing HERV-W gag sequence from the putative gene PTD015 on chromosome 11q13.5 (1.6-fold, P < 0.05) in the patients compared with the controls. Thus, studies aiming to further understanding of complex human disease such as schizophrenia may need to be extended beyond the strictly protein-coding fraction of the transcriptome.

Maternal infections and subsequent psychosis among offspring.

BACKGROUND: We tested the hypothesis that maternal infections during pregnancy are associated with the subsequent development of schizophrenia and other psychoses in adulthood. METHODS: We conducted a nested case-control study of 27 adults with schizophrenia and other psychotic illnesses and 54 matched unaffected control subjects (matched for sex, ethnicity, and date of birth) from the Providence, RI, cohort of the Collaborative Perinatal Project. We retrieved stored blood samples that had been obtained from these mothers at the end of pregnancy. These samples were analyzed for total class-specific immunoglobulins and for specific antibodies directed at recognized perinatal pathogens capable of affecting brain development. RESULTS: Maternal levels of IgG and IgM class immunoglobulins before the mothers were delivered of their neonates were significantly elevated among the case series (t = 3.06, P =.003; t = 2.93, P =.004, respectively, for IgG and IgM immunoglobulin-albumin ratios). Secondary analyses indicated a significant association between maternal antibodies to herpes simplex virus type 2 glycoprotein gG2 and subsequent psychotic illness (matched t test = 2.43, P =.02). We did not find significant differences between case and control mothers in the serum levels of IgA class immunoglobulins, or in specific IgG antibodies to herpes simplex virus type 1, cytomegalovirus, Toxoplasma gondii, rubella virus, human parvovirus B19, Chlamydia trachomatis, or human papillomavirus type 16. CONCLUSIONS: The offspring of mothers with elevated levels of total IgG and IgM immunoglobulins and antibodies to herpes simplex virus type 2 are at increased risk for the development of schizophrenia and other psychotic illnesses in adulthood.

Retroviral RNA identified in the cerebrospinal fluids and brains of individuals with schizophrenia.

Schizophrenia is a serious brain disease of uncertain etiology. A role for retroviruses in the etiopathogenesis of some cases of schizophrenia has been postulated on the basis of clinical and epidemiological observations. We found sequences homologous to retroviral pol genes in the cell-free cerebrospinal fluids (CSFs) of 10 of 35 (29%) individuals with recent-onset schizophrenia or schizoaffective disorder. Retroviral sequences also were identified in the CSFs of 1 of 20 individuals with chronic schizophrenia. However, retroviral sequences were not identified in any of the CSFs obtained from 22 individuals with noninflammatory neurological diseases or from 30 individuals without evidence of neurological or psychiatric diseases (chi(2) = 19.25, P < 0.001). The nucleotide sequences identified in the CSFs of the individuals with schizophrenia or schizoaffective disorder were related to those of the human endogenous retroviral (HERV)-W family of endogenous retroviruses and to other retroviruses in the murine leukemia virus genus. Transcription of RNA homologous to members of the HERV-W family of retroviruses also was found to be up-regulated differentially in the frontal cortex regions of brains obtained postmortem from individuals with schizophrenia, as compared with corresponding tissue from individuals without psychiatric diseases. The transcriptional activation of certain retroviral elements within the central nervous system may be associated with the development of schizophrenia in at least some individuals. The further characterization of retroviral elements within the central nervous system of individuals with schizophrenia might lead to improved methods for the diagnosis and management of this disorder.

Maternal cytokine levels during pregnancy and adult psychosis.

We investigated levels of maternal cytokines in late pregnancy in relation to the subsequent development of adult schizophrenia and other psychoses in their offspring. The sample included the mothers of 27 adults with schizophrenia and other psychotic illnesses and 50 matched unaffected controls from the Providence cohort of the Collaborative Perinatal Project. Serum samples were analyzed for interleukin 1 beta (IL-1-beta), interleukin 2 (IL-2), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-alpha) by enzyme immunoassay. Maternal levels of TNF-alpha were significantly elevated among the case series (t = 2.22, p =.04), with evidence of increasing odds of psychosis in relation to higher cytokine levels. We did not find significant differences between case and control mothers in the serum levels of IL-1, IL-2, IL-6, or IL-8. These data support previous clinical investigations reporting maternal infections during pregnancy as a potential risk factor for psychotic illness among offspring.

Serum antibodies reactive with non-human primate retroviruses identified in acute onset schizophrenia.

Schizophrenia is a pervasive neuropsychiatric disease of uncertain etiology. Previous studies have postulated that retroviruses may contribute to the etiology of some cases of schizophrenia. We examined the possible relationship between retroviral infection and schizophrenia by measuring antibodies to a number of different primate retroviruses in the sera of individuals undergoing their first hospitalization for this disease. Sera from patients with first onset schizophrenia and matched healthy controls were analyzed by immunoblot and enzyme linked immunosorbent assays using purified retrovirus antigens to identify and quantify antibodies reactive with retrovirus proteins. A significantly increased incidence of antibodies reactive to gag encoded proteins of Mason-Pfizer monkey virus (MPMV), baboon endogenous virus (BaEV) and simian retrovirus type 5 (SRV-5) was observed in the sera of schizophrenia patients compared to controls. The reactivity of the cases and controls displayed the greatest differences in terms of antibodies to the proteins of Mason-Pfizer monkey virus. Employing an algorithm of enzyme linked immunosorbent assay reactivity followed by immunoblot confirmation, we found that MPMV antibodies in 28.9% of the individuals with first episode schizophrenia patients as compared to 3.7% of the unaffected controls (P<0.009, Fisher's Exact Test). These studies are consistent with the occurrence of retrovirus replication in some individuals who are undergoing their first episode of schizophrenia.

PCR-Enzyme immunoassay for detection of Streptococcus pneumoniae DNA in cerebrospinal fluid samples from patients with culture-negative meningitis.

A PCR-based assay was developed to amplify a conserved region of the pneumococcal autolysin gene. The amplified product was labelled with digoxigenin-labelled dUTP and was detected with a biotin-labelled probe in an enzyme immunoassay (EIA). The assay was initially tested with suspensions of various serotypes of Streptococcus pneumoniae and other gram-positive and gram-negative bacteria and was then applied to cerebrospinal fluid (CSF) specimens from patients with meningitis and those with other neurological disorders. The assay detected all the serotypes of S. pneumoniae tested, whereas all the other bacterial strains tested were negative. Seven of the 8 CSF specimens positive for pneumococcus by culture or latex agglutination (LA) were positive by PCR-EIA, whereas all 10 specimens positive for other organisms were negative. Among 11 patients with clinically diagnosed meningitis but with negative culture and LA results, 5 were positive by PCR-EIA. The assay was negative for all but one patient without meningitis; it was positive with the CSF from a child with immunodeficiency and pneumococcal abscesses on the scalp. PCR-EIA is a useful tool for the diagnosis of meningitis, especially when culture and LA are negative because of prior antibiotic treatment.

Enteric pathogens associated with gastrointestinal dysfunction in children with HIV infection.

Infants and young children with HIV infection commonly suffer from gastrointestinal manifestations of their disease. Many HIV infected children have evidence of persistent diarrhoea, malabsorption, malnutrition or growth failure. The aetiology and pathogenesis of gastrointestinal dysfunction in HIV infected children have not been well defined. We performed immunocytochemical analyses on intestinal tissue from 19 HIV-infected children with gastrointestinal dysfunction or growth failure. None of these 19 children had microbial pathogens identified in faecal samples using standard microbiological methods. Intestinal tissues were obtained from the children by biopsy and were examined for antigens from Pneumocystis carinii, cytomegalovirus (CMV) and herpes simplex virus (HSV) using the avidin-biotin-complex immunohistochemical technique and monoclonal or monospecific antibodies. We detected at least one of these pathogens in samples from eight (42%) of 19 HIV infected children. P. carinii was the most prevalent pathogen, found in five of the eight HIV infected children. All of the children with intestinal pneumocystis infection were receiving prophylaxis directed at the prevention of pulmonary disease with this organism and none of them were undergoing active pulmonary infection. We also identified CMV antigens in intestinal tissues from four children and HSV antigens in intestinal tissues from one child. Two children were infected with more than one pathogen. On the other hand, none of these pathogens were found in the tissues obtained from 10 HIV-uninfected patients who had intestinal tissues obtained for chronic non-infectious diarrheal and inflammatory diseases (P < 0.01, Fisher's exact test). Our findings indicate that some children with HIV infection and gastrointestinal dysfunction may be infected with opportunistic pathogens despite negative analyses employing standard microbiological methods. Our study also indicates that HIV infected children can undergo intestinal infection with P. carinii despite the administration of standard immunoprophylactic regimens directed at the prevention of infection with this organism.

Polymerase chain reaction (PCR) search for viral nucleic acid sequences in schizophrenia.

BACKGROUND: Previous studies looking for evidence of viral infection in schizophrenics have yielded conflicting results. We searched for viral nucleic acids to test the hypothesis of the viral aetiology of schizophrenia. METHOD: We used the polymerase chain reaction (PCR) to search for cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza A, Borna disease virus (BDV), and bovine viral diarrhoea virus (BVDV) in: hippocampus from three schizophrenic and three non-schizophrenic subjects; cerebrospinal fluid (CSF) from 48 schizophrenic patients; CSF and peripheral blood mononuclear cells (PBMC) from nine sets of identical twins discordant for schizophrenia; and SK-N-SHEP cells co-cultured with schizophrenic and non-schizophrenic brain homogenates. All patients met DSM-III-R criteria. RESULTS: Virus-specific nucleic acids were not found in any of the samples tested. CONCLUSIONS: The absence of viral nucleic acids in the samples tested suggest that, in these patients, schizophrenia is not associated with a persistent or latent infection due to these viruses.

Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus.

Influenza A virus infections are a major cause of morbidity and mortality worldwide. Standard diagnostic methods either are not efficient in identifying infected individuals in a timely manner or lack sensitivity. We developed a PCR-enzyme immunoassay (PCR-EIA) for the detection of influenza A virus RNA in respiratory secretions. A reverse transcription PCR was performed with oligonucleotide primers directed at a highly conserved area of the influenza A matrix gene. Amplified DNA was identified by hybridization in solution to a nested biotinylated RNA probe and quantitated in an EIA. PCR-EIA detected small quantities of RNA from the three prevalent subtypes of human influenza A virus. Influenza B and C, parainfluenza, measles, mumps, and respiratory syncytial viruses tested negative. The potential efficiency of PCR-EIA for use in clinical diagnosis was determined by testing 90 nasal wash specimens obtained daily over a 10-day period from nine human volunteers infected with influenza A virus. Thirty-seven of the postinfection samples had detectable influenza A virus RNA by PCR-EIA, whereas only 26 postinfection samples were positive by culture. PCR-EIA was particularly efficient for the identification of influenza A virus in samples obtained more than 4 days after infection. Seventeen of 45 such samples were positive, whereas virus was cultivated from 4 samples (P < 0.00005). All preinfection samples from volunteers subsequently infected with influenza A virus were negative by PCR-EIA, as were samples from a volunteer infected with parainfluenza virus type 3. Nucleic acid amplification techniques represent important tools for the timely and sensitive diagnosis of influenza A virus infections and, therefore, their management and control.

Rapid detection of parainfluenza virus type 3 RNA in respiratory specimens: use of reverse transcription-PCR-enzyme immunoassay.

Parainfluenza virus type 3 (PIV-3), an important lower respiratory tract pathogen in young children and immunocompromised individuals, may be underdiagnosed because of the insensitivity of available culturing systems and delay in identification of virus in cell culture. We developed a reverse transcription-PCR-enzyme immunoassay (RT-PCR-EIA) for PIV-3, using primers specific for a highly conserved region of the hemagglutinin-neuraminidase gene. Testing of nasal washes spiked with PIV-3 or other respiratory viruses showed that this assay detected seven strains of PIV-3 but not other respiratory viruses. Of 103 respiratory tract samples obtained from children experimentally infected with a liver PIV-3 vaccine or naturally infected with wild-type PIV-3, 51 were positive by culture and 48 were positive by RT-PCR-EIA. Eleven of the culture-positive samples were negative by RT-PCR-EIA; however, none of these grew virus upon reinoculation into cell culture, indicating that virus was lost or was present at a very low titer. Eight of the culture-negative samples were positive by RT-PCR-EIA: two were obtained from a subject who was culture negative but had a serologic response to PIV-3, four were obtained 7 to 9 days after the first positive culture, and two were obtained 1 day prior to the first positive culture. Thus, this RT-PCR-EIA for PIV-3 is sensitive and specific and can detect viral RNA in samples from which virus cannot be cultivated. This assay could be used for diagnosis late in the course of PIV-3 infection and for accurate detection of disease outbreaks.

Borna disease virus in peripheral blood mononuclear and bone marrow cells of neonatally and chronically infected rats.

Borna disease virus (BDV) establishes a persistent infection in cells of the nervous system in rats. The response, or lack thereof, of the immune system to BDV infection of neurons is responsible for the presence or absence, respectively, of Borna disease. We recently demonstrated transmission of BDV by bone marrow cells from neonatally infected rats. Our findings suggested the possibility of a heretofore unsuspected interaction between BDV and the immune system, that of direct effects of BDV infection on the cells of the immune system. This report enlarges upon the previous findings and confirms the presence of BDV RNA in bone marrow cells of neonatally infected rats, using a reverse transcription-polymerization chain reaction-enzyme immunosorbent assay (RT-PCR-EIA). In addition, we detected BDV RNA in peripheral blood mononuclear cells of neonatally infected rats, and in rats inoculated as adults in the chronic, but not the acute, stage of infection. In addition, the RT-PCR-EIA technique identified BDV RNA in cerebrospinal fluid, nasal secretions, saliva, urine and stool. BDV-sequences were not detected in the plasma of infected animals nor in the body fluids and tissues of normal rats.

Baculovirus expression of pestivirus non-structural proteins.

Bovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3'-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.

A human milk factor inhibits binding of human immunodeficiency virus to the CD4 receptor.

Perinatal transmission of human immunodeficiency virus (HIV) from infected mothers to their children occurs at rates reported as 20-50%. The role of breast feeding in perinatal transmission of viral infections has not been well established. We studied 34 milk and colostral samples obtained from HIV-seropositive and HIV-seronegative women to determine if they contained anti-HIV activity. We found that all the samples contained a factor that inhibited the binding of HIV epitope-specific MAb to recombinant CD4 receptor molecules. The titers of inhibitory activity ranged from 1:200 to 1:10,000 and did not differ between HIV-seropositive and HIV-seronegative mothers. This milk factor also inhibited the binding of gp120 to CD4. Neither human sera nor bovine milk exhibited appreciable inhibitory activity. Fractionation of human milk indicated that the inhibitory activity was confined to the macromolecular fraction; little activity was found in isolated milk lipids or oligosaccharides. Chromatographic procedures indicated that the active macromolecule has an isoelectric point of 9.3-9.6. The active material did not bind to concanavalin A; however, the activity was partially destroyed by chemical and enzymatic treatments that removed sulfated residues. The active material may thus be a sulfated protein, glycoprotein, mucin, or glycosaminoglycan that inhibits the binding of CD4 to HIV envelope glycoproteins. The role of this factor in the natural history of HIV infection in infants and children should be the subject of additional investigations.

Persistent diarrhea and fecal shedding of retroviral nucleic acids in children infected with human immunodeficiency virus.

Gastrointestinal dysfunction is a serious problem in many children infected with human immunodeficiency virus (HIV), the etiology of which has not been clearly defined. Quantitative nucleic acid amplification was used to study the correlation between shedding of HIV nucleic acids and gastrointestinal symptoms in HIV-infected infants and children. Many with HIV infection and persistent diarrheal disease shed HIV nucleic acids in their feces, as did an HIV-infected patient without apparent diarrheal disease. HIV nucleic acids were not found in feces of non-HIV-infected individuals. Intestinal infection with HIV appears to be important in the pathophysiology of gastrointestinal dysfunction in infants and children with HIV infection. Furthermore, the fecal shedding of HIV may play a role in HIV transmission in environments prone to high levels of fecal-oral contamination.

Solid phase capture method for the specific amplification of microbial nucleic acids--avoidance of false-positive and false-negative reactions.

DNA amplification assays such as the polymerase chain reaction are being developed for the amplification of small quantities of microbial nucleic acids. These assays offer the potential for a great deal of sensitivity. However, the high level of sensitivity increases the likelihood of cross-contamination of amplified products and the generation of false-positive reactions. In addition, substances in body fluids can inhibit the efficient performance of the amplification reactions. We have developed an assay format in which microbial nucleic acids are specifically bound to a solid phase surface. Contaminating DNA and enzyme inhibitors present in the sample are removed by washing prior to the performance of the amplification reaction. We could use this system to amplify and detect small amounts of HIV DNA diluted in whole blood. The assay system could distinguish target DNA from contaminating DNA fragments generated by prior amplification reactions.

Humoral immune responses to gag and env proteins from human immunodeficiency virus type 1 in hemophiliac patients.

Solid-phase enzyme immunoassays using recombinant gag and env proteins were developed to study humoral immune responses to HIV infection in a cohort of 105 hemophiliac patients. Thirteen patients with ARC or AIDS and 92 asymptomatic patients were studied. A cross-sectional study showed a wide range of antibody responses to gag and env proteins; however, the differences between the ARC/AIDS and asymptomatic patients were statistically significant for both antigens (P less than .0004). In a longitudinal study, antibody levels in sera from 11 asymptomatic patients with gag antibody log units less than or equal to 1.5 were compared to levels in sera from 10 ARC/AIDS patients and 8 asymptomatic patients with gag antibody greater than 1.5. These patient groups were followed for comparable periods of time (67.1-71.7 mo). The asymptomatic patients with low gag antibody and the ARC/AIDS patients showed a similar pattern of antibody response to gag protein overtime. In hemophiliac patients with HIV-1 infection a low titer of antibody to gag protein is not invariably associated with clinical deterioration and is not a useful serologic marker of impending progression to AIDS.

Growth of group A rotaviruses in a human liver cell line.

Recent observations in children with rotavirus gastroenteritis and in infant mice given rotavirus vaccine by oral administration suggest that this well-known gastrointestinal pathogen may infect the liver. To examine this possibility, the susceptibility of Hep G2 cells to infection with a variety of rotavirus strains was tested. These cells were used because they are considered to be well differentiated and exhibit many liver-specific functions. The Hep G2 cells supported the growth of the simian strain rhesus rotavirus (MMU 18006), a strain currently being used in vaccine trails, but did not support the growth of any human strain (D, DS1, Price or ST3). The rhesus rotavirus infection was cytopathic and resulted in release of lactate dehydrogenase. Rhesus rotavirus growth in Hep G2 cells displayed trypsin-enhanced infectivity and was inhibited by pretreatment of cells with Arthrobacter ureafaciens neuraminidase but not with neuraminidase from Clostridium perfringens. Hep G2 cells were also permissive for another simian strain (SA11), a bovine strain (UK) and single gene substitution reassortants containing VP7 (the major outer capsid neutralization protein) from a human rotavirus strain and the remaining 10 genes from either rhesus rotavirus or UK. In general, UK and its reassortants produced lower levels of antigen than did rhesus rotavirus and its reassortants. Hep G2 cells and other hepatic cell lines may prove to be useful tools to explore the hepatotropic potential of wild-type rotaviruses and candidate vaccine strains.

Solution hybridization and enzyme immunoassay for biotinylated DNA-RNA hybrids to detect enteroviral RNA in cell culture.

A non-isotopic hybridization assay is described for detection of enteroviral RNA in cell culture. Two biotin-labelled cDNA probes, corresponding to 1 kb from the 5' end and 3.5 kb from the 3' end of the coxsackievirus B3 genome, were hybridized in solution with protease and detergent-treated cell culture suspensions. Labelled DNA-RNA hybrids were captured on microtiter plates coated with anti-biotin antibody and bound hybrids were measured with a beta-galactosidase-labelled monoclonal antibody specific for DNA-RNA hybrids. Coxsackie B3 was detected at a concentration of 500 pfu ml-1. The limit of detection for other enteroviruses ranged from 10(3.3) to 10(5.8) pfu ml-1. The enteroviruses that could be detected included coxsackie B1 and 3, coxsackie A1-6 and 15, poliovirus types 1-3, and enteroviruses 7, 11, and 71. ECHO 22 was the only enterovirus, of those that were tested, that could not be detected. The solution hybridization reaction and enzyme immunoassay for DNA-RNA hybrids does not require the use of radiolabelled probes or extraction of RNA with phenol. The assay yields a quantitative endpoint, which avoids the subjectivity inherent in membrane-based methods. These features would make the assay more adaptable to clinical laboratories than other formats which have been devised for measurement of viral RNA.