Low Levels Of Viral DNA Research Articles

A mutation in the latency-related gene of bovine herpesvirus 1 disrupts the latency reactivation cycle in calves.

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle, and infection is usually initiated via the ocular or nasal cavity. Following acute infection, the primary site for BHV-1 latency is the sensory neuron. Reactivation from latency occurs sporadically, resulting in virus shedding and transmission to uninfected cattle. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA, suggesting that it mediates some aspect of latency. An LR mutant was constructed by inserting three stop codons near the beginning of the LR-RNA, suggesting that expression of LR proteins would be altered. The LR mutant grew with wild-type (wt) efficiency in bovine kidney cells (MDBK). When calves were infected with the LR mutant, a dramatic decrease (3 to 4 logs) in ocular, but not nasal, viral shedding occurred during acute infection relative to the wt or the LR-rescued virus (M. Inman, L. Lovato, A. Doster, and C. Jones, J. Virol. 75:8507-8515, 2001). In this study, we examined the latency reactivation cycle in calves infected with the LR mutant and compared these results to those from calves infected with wt BHV-1 or the LR-rescued virus. During acute infection, lower levels of infectious virus were detected in trigeminal ganglion homogenates from calves infected with the LR mutant. As judged by in situ hybridization, BHV-1-positive neurons were detected in trigeminal ganglia of calves infected with the wt but not the LR mutant. Although LR-RNA was detected by reverse transcription-PCR in calves latently infected with the LR mutant, a semiquantitative PCR analysis revealed that lower levels of viral DNA were present in trigeminal ganglia of calves infected with the LR mutant. Dexamethasone treatment of calves latently infected with wt BHV-1 or the LR-rescued virus, but not the LR mutant, consistently induced reactivation from latency, as judged by shedding of infectious virus from the nose or eyes and increases in BHV-1-specific antibodies. In summary, this study demonstrates that wt expression of LR gene products plays an important role in the latency reactivation cycle of BHV-1 in cattle.

Two closely related but distinct retroviruses are associated with walleye discrete epidermal hyperplasia.

Walleye discrete epidermal hyperplasia (WEH) is a hyperproliferative skin disease that is prevalent on adult walleye fish throughout North America. We have identified two retroviruses associated with WEH, designated here as walleye epidermal hyperplasia virus type 1 and type 2 (WEHV1 and WEHV2), that are closely related to one another (77% identity) and to walleye dermal sarcoma virus (64% identity) within the polymerase region. WEHV1 and/or WEHV2 viral DNA was readily detected by PCR in hyperplastic tissue samples, but only low levels of viral DNA were detected in uninvolved skin. Southern blot analysis showed one to three copies of integrated WEHV2 viral DNA in lesions but did not detect WEHV2 viral DNA in uninvolved skin from the same fish. Northern blots detected abundant levels of WEHV1 and/or WEHV2 virion RNA transcripts of approximately 13 kb in hyperplastic tissue, but virion RNA was not observed in uninvolved skin and muscle. These results suggest that WEHV1 and WEHV2 are the causative agents of discrete epidermal hyperplasia.

Identification of sequences downstream of the primer binding site that are important for efficient replication of human immunodeficiency virus type 1

Reverse transcription of retroviruses is initiated from an 18-nucleotide (nt) primer binding site (PBS), located within the 5' region of viral genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. We constructed proviral DNA clones of human immunodeficiency virus (HIV) that had selective deletions of either a 7-nt segment found immediately downstream of the PBS or an extended nontranslated 54-nt stretch located immediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had been transfected with these various constructs. We found that similar levels of minus-strand strong-stop DNA as well as DNA produced after template switching were expressed in MT-4 cells infected with COS-7-derived wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54-nt stretch. Furthermore, the molecular clone containing the 7-nt deletion was able to replicate with wild-type kinetics, while that containing the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3' end of this 54-nt segment was largely responsible for these effects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA derived from either a wild-type HIV construct or our various deletion mutants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of viral mRNA transcripts were detected, which was not the case for the 54 nt-deletion mutant. However, these various mRNAs possessed similar stabilities, as shown through studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5' nontranslated RNA, located downstream of the PBS, is involved in efficient expression of each of viral DNA, mRNA, and infectious virus.

The Role of AV2 (“Precoat”) and Coat Protein in Viral Replication and Movement in Tomato Leaf Curl Geminivirus

We analyzed various mutants of tomato leaf curl virus-India to investigate the role of ORFs AV3, AV2, and coat protein (CP) in viral replication, movement, and symptom development. The results of these studies indicate that ORF AV3 does not encode a protein. Plants inoculated with infectious DNA which contained deletions in AV2 developed very mild symptoms and accumulated only low levels of both single-stranded (ss) and double-stranded (ds) viral DNA, whereas inoculated protoplasts accumulated both ss and dsDNA to wild-type levels, showing that AV2 is required for efficient viral movement. However, both plants and protoplasts inoculated with substitution, frameshift, and other similar mutations in AV2 accumulated low levels of viral DNA. The low levels of accumulation of DNA of these mutants were apparently not due to a defect in AV2 synthesis. Mutations in the CP caused a marked decrease in ssDNA accumulation in plants and protoplasts while increasing dsDNA accumulation in protoplasts. Mutations in both AV2 and CP behaved like AV2 mutants in plants and like CP mutants in protoplasts. The results demonstrated that multiple functions provided by AV2, BV1, and BC1 are essential for viral movement, and that changes in A-component virion-sense mRNA structure or translation affect viral replication.

A polymerase chain reaction for the detection of nucleopolyhedroviruses in infected insects: the fate of the Spodoptera littoralis virus in Locusta migratoria.

The Spodoptera littoralis nucleopolyhedrovirus (SlNPV) is a potential pest control agent of Spodoptera spp. As part of our studies to establish the use of this virus, a polymerase chain reaction (PCR)-based method was developed for the detection of viral DNA in infected insects. PCR amplification of the polyhedrin sequences enabled the detection of low levels of viral DNA directly from viral occlusion bodies or from total larval DNA. The use of different sets of synthetic DNA primers allowed us to differentiate between SlNPV and the Autographa californica NPV (AcNPV) and to identify a new AcNPV variant isolated from a cotton pest, Pectinophora gossypiella NPV. The PCR method was also used to test for the possible infection of Locusta migratoria larvae by SlNPV, reported by Bensimon et al., 1987. The progress of SlNPV infection in L. migratoria larvae was monitored by PCR for 2 weeks. The reaction revealed decreasing amounts of viral DNA in infected larvae. During this time, no signs of disease were observed in the infected locusts.

4 Viral serology and detection

Viral detection is an important part of clinical hepatology. For many years practical clinical tests have been serological but recently newer molecular techniques have become available for virus detection, although these have yet to become routine and some, such as PCR of viral nucleic acid in blood or tissue are not yet consistently reliable. Serology remains the mainstay at present for routine diagnosis. the IgM response representing acute infection and the IgG response immunity, although more sophisticated molecular techniques have been applied experimentally. A second agent of epidemic enteral hepatitis, the hepatitis E virus, has recently been cloned and sequenced and serological tests for this virus are available, although experience in their use is necessarily limited and a commercial IgM assay has yet to be produced. Serological tests for the hepatitis B virus are well developed. The IgM anticore response differentiates acute infection from chronic, the latter being characterized by the persistence of hepatitis B surface antigen for over six months. Chronic carriers are at risk of liver damage and this risk is best assessed by the amount of viral DNA circulating, which can be determined using a hybridization assay. More sensitive techniques such as the branched chain DNA assay or PCR can detect lower levels of viral DNA but their clinical relevance remains to be established. The hepatitis D virus is defective and relies on hepatitis B to replicate. Serology for antibody and antigen is well established although PCR for circulating viral genome may come to supplant hepatic viral antigen as a test for hepatitis D replication. For hepatitis C serology is feasible only for antibodies, not antigens; although early tests were prone both to false positives and false negatives, current versions are more reliable. PCR has been much used for detection of hepatitis C RNA in blood and tissues and a bDNA assay is now commercially available. Cytomegalovirus detection is confounded by the problem of distinguishing asymptomatic viral replication from disease. Serology is helpful, especially in primary infections, but viral culture is a widely used method. PCR (especially quantitative modifications) or the pp65 antigenaemia assay are experimental approaches which may prove specific enough for general use.

Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain.

Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of reverse transcriptase activity. These data indicate that in addition to integration, IN may have an effect on the proper course of events in the viral life cycle that precede integration.

Persistence of hepatitis B virus DNA in the liver after loss of HBsAg in chronic hepatitis B

To determine whether patients with chronic hepatitis B who lose hepatitis B virus DNA and HBsAg from the serum completely resolve the hepatitis and virus infection, we evaluated serum and liver tissue from 11 patients who had lost HBsAg. These patients were evaluated for clinical, histological and serological features and for hepatitis B virus DNA by use of hybridization and polymerase chain reaction techniques. Liver biochemical test results were normal in all except two patients who had mild aminotransferase elevations. All sera were negative for hepatitis B virus DNA by direct hybridization, and only one was positive transiently by polymerase chain reaction. Liver histology was abnormal in all patients, but the changes were mild and markedly improved compared with biopsy specimens taken before loss of HBsAg. Liver tissue from 10 patients was positive for hepatitis B virus DNA by polymerase chain reaction but not by direct hybridization. These findings indicate that loss of HBsAg is associated with marked improvements in clinical and serum biochemical features of chronic hepatitis B, yet mild degrees of hepatitis and low levels of viral DNA may persist in the liver.

Persistence of hepatitis B virus DNA in the liver after loss of HBsAg in chronic hepatitis B

To determine whether patients with chronic hepatitis B who lose hepatitis B virus DNA and HBsAg from the serum completely resolve the hepatitis and virus infection, we evaluated serum and liver tissue from 11 patients who had lost HBsAg. These patients were evaluated for clinical, histological and serological features and for hepatitis B virus DNA by use of hybridization and polymerase chain reaction techniques. Liver biochemical test results were normal in all except two patients who had mild aminotransferase elevations. All sera were negative for hepatitis B virus DNA by direct hybridization, and only one was positive transiently by polymerase chain reaction. Liver histology was abnormal in all patients, but the changes were mild and markedly improved compared with biopsy specimens taken before loss of HBsAg. Liver tissue from 10 patients was positive for hepatitis B virus DNA by polymerase chain reaction but not by direct hybridization. These findings indicate that loss of HBsAg is associated with marked improvements in clinical and serum biochemical features of chronic hepatitis B, yet mild degrees of hepatitis and low levels of viral DNA may persist in the liver (HEPATOLOGY 1993;18:1313–1318.

Infected cell specific protein and viral DNA synthesis in productive and abortive infections ofSpodoptera frugiperda nuclear polyhedrosis virus

This study examines viral protein and DNA synthesis in Spodoptera frugiperda multicapsid nuclear polyhedrosis virus (SfMNPV) infections of S. frugiperda and Trichoplusia ni cells. A total of 28 infected cell specific polypeptides (ICSPs) were detected in the productive S. frugiperda cells. Of these, 14 were identified as structural polypeptides. Based on the change in their rate of synthesis during the replication cycle, these ICSPs were grouped into four classes. Only a 97k and a 29k ICSP were detected in SfMNPV infections of T. ni cells. Inhibition of host protein synthesis occurred in productive infections only, beginning at 10 h postinfection (p.i.) and reaching maximal levels by 20 h p.i. The rate of viral DNA synthesis in the productive cells was maximal between 8 to 16 h postinfection, and only low levels of viral DNA were synthesized in T.ni cells. The data suggest that the productive SfMNPV/S.frugiperda cell infection has a gene expression program similar but not identical to that of Autographa californica MNPV infections. The SfMNPV/T.ni cell infection is nonpermissive and is restricted at the earliest phase of the viral gene expression program.

Hepatitis B virus replication and clinical outcome in carriers of HBsAg. Perspectives of treatment with DNA inhibitors.

HBV-DNA was measured by the spot hybridization technique in serial serum samples obtained from 47 HBsAg carriers followed up for a mean of 4 years. The levels of HBV-DNA were compared to the conventional HBV serology and immunopathology to determine the relation of active HBV replication to the outcome of hepatitis and the suitability of Italian HBsAg carriers for treatment with DNA inhibitors. HBV-DNA was found in 26 carriers (53%) and persisted with comparable serum levels in 24 of them throughout the follow up. The occurrence rate of an unfavorable outcome as determined by histological evidence of cirrhosis was 6% versus 44% (p less than 0.01) in carriers with active viral infection (greater than 1 ng/ml of HBV-DNA) and in patients with absent or low levels of viral DNA (less than 1 pg/ml), respectively. Progression of the liver disease could not be predicted on the basis of active HBV replication and was presumably related to factors other than synthesis of HBV. In many patients with inactive viral infection a primary pathogenic factor was the HBV-associated delta, an agent with a putative RNA genome against which DNA inhibitors have no rationale and possibly no effects. The majority of Italian carriers do not appear suitable for treatment with DNA inhibitors and they should be considered for a different therapy.