Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA.

INTRODUCTION: Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay. METHODS: The PVL quantification procedure was optimized by inclusion of an exogenous control hepatitis C virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660, and the LTR region of SIVagmSAB were also optimized. RESULTS: Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV RNA in the same samples using the 'industry standard' method of branched-DNA (bDNA) signal amplification. CONCLUSIONS: Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs.

Recombinant varicella vaccines induce neutralizing antibodies and cellular immune responses to SIV and reduce viral loads in immunized rhesus macaques.

The development of an effective AIDS vaccine remains one of the highest priorities in HIV research. The live, attenuated varicella-zoster virus (VZV) Oka vaccine, safe and effective for prevention of chickenpox and zoster, also has potential as a recombinant vaccine against other pathogens, including human immunodeficiency virus (HIV). The simian varicella model, utilizing simian varicella virus (SVV), offers an approach to evaluate recombinant varicella vaccine candidates. Recombinant SVV (rSVV) vaccine viruses expressing simian immunodeficiency virus (SIV) env and gag antigens were constructed. The hypothesis tested was that a live, attenuated rSVV-SIV vaccine will induce immune responses against SIV in the rhesus macaques and provide protection against SIV challenge. The results demonstrated that rSVV-SIV vaccination induced low levels of neutralizing antibodies and cellular immune responses to SIV in immunized rhesus macaques and significantly reduced viral loads following intravenous challenge with pathogenic SIVmac251-CX-1.

Detection of exogenous and endogenous avian leukosis virus in commercial chicken eggs using reverse transcription and polymerase chain reaction assay.

Avian leukosis retroviruses (ALV) cause lymphomas and other cancers in chickens. Previous studies have used enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) to detect ALV p27 group-specific antigens (GSA) in commercial chicken eggs. In the poultry industry eradication programme against exogenous ALV, ELISA assays are used to identify chickens infected with the virus. The inability of ELISA and IFA assays to discriminate between ALV GSA of endogenous or exogenous origin, and actual virus, have limited rigorous assessments of viral transmission dynamics. Here, we report the use of a newly developed reverse transcriptase-polymerase chain reaction (RT-PCR) assay, with direct sequencing of the RT-PCR product, to show endogenous and exogenous ALV in albumen from unfertilized chicken eggs. We found that 95% of 20 eggs from ALV-exposed commercial chickens and 14.2% of 240 egg samples from 20 randomly chosen New Orleans retail stores were ALV-positive by RT-PCR. In comparison, only 2.5% of the same egg samples from the retail stores were positive by ELISA. Corresponding direct sequencing of randomly chosen RT-PCR products showed that four of six egg samples contained endogenous ALV, while two of the six samples were positive for exogenous subgroup A ALV. The finding of endogenous subgroup E ALV in unfertilized chicken eggs emphasizes that the transmission of endogenous ALV is common and should be considered in the implementation of ALV eradication programmes by the poultry industry.

Intranasal monoclonal IgA antibody to respiratory syncytial virus protects rhesus monkeys against upper and lower respiratory tract infection.

Respiratory syncytial virus (RSV), the major cause of lower respiratory tract disease in infants, is thought to infect the upper airways before spreading to the lower respiratory tract. A rhesus monkey model of RSV infection after upper airway inoculation was used to test the protective effect of intranasal treatment with HNK20, a mouse monoclonal IgA antibody against RSV F glycoprotein. HNK20 was administered once daily for 2 days before RSV challenge and 4 days after challenge. Treatment with 0.5 mg/kg HNK20 reduced viral shedding in the nose, throat, and lungs by 3-4 log10/mL (P < or = .002). All monkeys developed RSV neutralizing antibody in serum, even in the absence of detectable viral replication. Neutralizing concentrations of monoclonal antibody remained in nasal secretions for > 1 day after treatment. These results suggest that nose-drop application of monoclonal antibody could provide convenient and effective protection against RSV infection in human infants at risk of severe lower respiratory tract disease.

Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin.

A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.

Polymyositis, arthritis, and uveitis in a macaque experimentally infected with human T lymphotropic virus type I.

OBJECTIVE: To establish a simian model of human T lymphotropic virus type I (HTLV-I) infection and disease. METHODS: Irradiated HTLV-I-producing cells were used to infect two 2-year-old rhesus macaque monkeys (Macaca mulatta). One monkey was also simultaneously inoculated with a cell-free suspension of simian immunodeficiency virus (SIV). Evidence of infection was monitored by serial clinical examinations and by serologic, molecular, and virologic assays. RESULTS: Both HTLV-I-inoculated monkeys became persistently infected following inoculation. Clinical disease was observed in the singly inoculated monkey, which developed arthritis (with synovial fluid positive for HTLV-I by culture and polymerase chain reaction), anterior chamber uveitis, and steroid-responsive polymyositis confirmed by electrophysiologic studies. The dually inoculated animal remained clinically healthy, despite high levels of SIV and HTLV-I virus expression and loss of HTLV-I-specific antibodies. CONCLUSION: These results indicate the utility of a nonhuman primate model for studying HTLV-I disease pathogenesis and the dynamics of SIV-1/HTLV-I retroviral coinfection.

The rat thymidine kinase gene 5' region: evolution of a promoter.

The nucleotide sequence of the rat thymidine kinase (tk) gene 5' region was determined and compared to the 5' sequences of the tk gene from mouse, human, hamster and chicken. This analysis revealed the highest degree of homology to be between the rat and the mouse sequences and correspondingly greater sequence variation relative to the other species. There has apparently been an especially high rate of change in regions that have been demonstrated to be protein binding regions in some of these promoters. This has led to changes in the potential transcription factors bound by some of these promoters.

Clustering and subfamily relationships of the Alu family in the human genome.

Thirteen and 10 sequences of the Alu family of repeated DNA elements found within the human thymidine kinase and beta-tubulin genes, respectively, were compared. These genes have approximately five times the expected density of Alu family members. The consensus sequence that could be drawn from these 23 Alu family members would differ slightly from others drawn from random Alu family sequences but only at very heterogeneous positions. The different Alu family members do show different pairwise percentage identities, with approximately 15% (7 of 48 Alu family members analyzed) of them clearly representing a separate subfamily of sequences. This analysis also confirms the species-specific differences between human and the prosimian Galago crassicaudatus Alu family members. These data are consistent with both the origin of these sequences in primates less than 65-70 Myr ago and amplification since that time to their present 500,000 copies. The data do not show any special relationships among densely clustered Alu family members.

Sequence, structure and promoter characterization of the human thymidine kinase gene.

The 12.9-kb human thymidine kinase gene (tk) has been sequenced in its entirety along with flanking regions. Consistent with the previously sequenced chicken tk sequence, the human tk is composed of seven exons. The intron sizes differ substantially, and are responsible for the four-fold greater size of the human relative to the chicken gene. Within the introns are found 13 Alu family repeated sequences and a polypyrimidine stretch. A functional promoter region has been located by fusing sequences from the 5' end of the tk gene to the chloramphenicl acetyl transferase (CAT) gene and assaying CAT activity following transfection into mouse L cells. Several putative transcription signals have been identified in the 5' end including 'TATAA' and 'CCAAT' sequences and 'G-C' elements, two of which are arranged in a 27-bp inverted repeat. There is also a 12-bp repeat, containing an inverted 'CCAAT' element. This repeat shows strong homology to a repeat in the chicken tk promoter as well as the 5' regions of other cell-cycle regulated genes, suggesting that it may be part of the promoter or a regulatory signal. The 5' flanking sequence is G + C-rich and has a high concentration of CpG dinucleotides.

Mouse mammary tumor virus gene expression regulated in trans by Lps locus.

Expression of mouse mammary tumor virus (MMTV) in the lactating mammary glands of uninfected mice varies between strains of mice in a manner largely independent of the proviral content. Previous linkage analysis in the mouse suggested that the Lps locus was associated with steady-state levels of MMTV RNA. The Lps locus mediates the mouse's response to the injection of lipopolysaccharide (LPS) in the responder mouse while mice with the deficient allele are incapable of responding. Injecting LPS-responder mice, C3HfB/HeN, and nonresponder mice, C3Hf/HeJ, with LPS resulted in a threefold increase in the level of MMTV RNA in responder mice but had no effect on nonresponders. The increased level was due to only one of the possible MMTV transcripts: the 1.7-kb transcript containing the open reading frame (orf) of the long terminal repeat (LTR). The level of MMTV-specific transcripts, then, is regulated by the Lps locus, a cellular gene which is not linked to any viral coding sequences and therefore must act in trans.

Cellular genes in the mouse regulate in trans the expression of endogenous mouse mammary tumor viruses.

The transcriptional activities of the eleven mouse mammary tumor virus (MMTV) proviruses endogenous to two sets of recombinant inbred (RI) mouse strains, BXD and BXH, were characterized. Comparison of the levels of virus-specific RNA quantitated in each strain showed no direct relationship between the presence of a particular endogenous provirus or with increasing numbers of proviruses. Association of specific genetic markers with the level of MMTV-specific RNA was examined by using multiple regression analysis. Several cellular loci as well as proviral loci were identified that were significantly associated with viral expression. Importantly, these cellular loci associated with MMTV expression segregated independently of viral sequences.

Mouse mammary tumor virus DNA methylation: tissue-specific variation.

Mouse mammary tumor virus-specific DNA sequences endogenous to the BALB/c mouse are shown to exhibit variable levels of methylation in a tissue-specific manner. In DNA from both lactating mammary gland and spleen, MMTV-specific sequences were hypomethylated at specific HpaII and HhaI sites. These variably methylated sites were found in the terminal repetitive sequences of the endogenous viral genomes. The specific hypomethylation of a HpaII site in Mtv-9 is associated with expression of a 1.6 kb transcript in the lactating mammary gland.