Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

Microplate-reverse hybridization method to determine dengue virus serotype.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3'-noncoding region universal primers.

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3'-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%).

Stability of p53 protein in rat cells transformed by various viral transforming genes.

To clarify the correlation between metabolic stabilization of p53 protein and cellular transformation, we transformed the normal rat cell line F2408 with various viral transforming genes and examined the expression and stability of p53 protein in these transformed cells by pulse-chase immunoprecipitation experiments. As expected, the level of p53 in the SV40-transformed rat cell line was higher than that in the normal cell line and its stability was also increased. In contrast, in cells transformed with the E7 and E6 genes of human papillomavirus type 16, the level and stability of p53 were similar to those in the normal cells. In cells transformed by the middle T or large T of polyomavirus, v-K-ras, and v-src, the levels and stabilities of p53 were also not elevated, although the level of p53 was increased in activated c-H-ras-transformed cells without an increase in its stability. These results show that increased stability and expression of the p53 protein are not common events in viral transformation of the rat cell line. In addition, we demonstrated with a monoclonal antibody specific for the mutant form of p53 that cellular transformation by viral transforming genes does not involve mutational activation of p53 to an oncogenic form.

Determination of the functional difference between human papillomavirus type 6 and 16 E7 proteins by their 30 N-terminal amino acid residues.

Human papillomavirus type 16 (HPV 16) is often found in cervical carcinomas, while HPV 6 is frequently associated with benign genital lesions. We have compared the abilities of the E7 transforming proteins of HPV 6 and 16 to transform various established and primary rodent cells by using the same heterologous promoter system. HPV 16 E7 efficiently induced anchorage-independent growth of all the rodent cell lines tested and immortalized or cooperated with ras in transforming primary rat cells. On the other hand, the transforming activity of HPV 6 E7 was lower and was restricted. By construction of chimeras of HPV 6 and 16 E7, we found that the difference in transforming activity between the two E7 proteins was mainly determined by the difference in their 30 N-terminal amino acid residues, although some activities seem to be slightly affected by differences in their residual C-terminal portions.

Cloning and characterization of human papillomavirus type 52 from cervical carcinoma in Indonesia.

Human papillomavirus (HPV) type 52 from a cervical carcinoma in Indonesia was molecularly cloned and characterized. By hybridization with cervical carcinoma DNAs from Indonesian patients, HPV 52 was detected in 3 of 52 cases (6%), whereas HPV 16 and 18 were detected in 8 and 7 cases, respectively (15% and 13%). Sequence analysis revealed that the E6-E7 ORFs contained several DNA binding motifs (Cys-X-X-Cys) like previously sequenced HPVs. The E6 ORF also contained splice donor and acceptor signals, which may allow the expression of E6* protein. The E7 ORF encoded an amino acid sequence that is conserved in some DNA tumor viruses and is involved in binding to Rb protein and in cellular transformation. Transfection of a subgenomic fragment of HPV 52 under the control of a heterologous promoter showed that the E7 ORF alone induced anchorage-independent growth of established rodent cells and immortalized primary rat embryo fibroblasts (REF), and that in cooperation with activated ras, it induced malignant transformation of REF. The E6 ORF also induced, less efficiently, anchorage-independent growth. These results strongly suggest that HPV 52, like HPV 16 and 18, has oncogenic potential.

Isolation of cellular revertants from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16.

Three revertants defective in the ability to form colonies in semisolid medium were isolated from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16 (HPV16). These revertants appeared to be defective in a cellular factor(s) necessary for transformation by HPV16-E6E7 genes since they still expressed a comparable amount of HPV16-E6E7 mRNA and E7 protein to the parental cells, harbored rescuable transforming virus, and were resistant to retransformation by HPV16-E6E7 genes. All these reverted phenotypes of the three mutants were recessive on somatic cell hybridization with normal cells, because all the hybrids showed transformed phenotypes.