Adeno-associated virus type 2 in US children with acute severe hepatitis.

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA1,2. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA3-7, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.

Adeno-associated virus 2 infection in children with non-A-E hepatitis.

An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.

The early history of tumor virology: Rous, RIF, and RAV.

One hundred years ago Peyton Rous recovered a virus, now known as the Rous sarcoma virus (RSV), from a chicken sarcoma, which reproduced all aspects of the tumor on injection into closely related chickens. There followed recovery of causal viruses of tumors of different morphology from 4 more of 60 chicken tumors. Subsequent studies in chickens of the biology of the first RSV isolated moved slowly for 45 y until an assay of ectodermal pocks of the chorioallantoic membrane of chicken embryos was introduced. The inadequacies of that assay were resolved with the production of transformed foci in cultures of chicken fibroblasts. There followed a productive period on the dynamics of RSV infection. An avian leukosis virus (ALV) was found in some chicken embryos and named resistance-inducing factor (RIF) because it interferes with RSV. Its epidemiology in chickens is described. Another ALV was found in stocks of RSV and called Rous-associated virus (RAV). Cells preinfected with RAV interfere with RSV infection, but RSV does not produce infectious virus unless RAV is added during or after RSV infection. Intracellular RAV provides the infectious coat for the otherwise defective RSV. The coat determines the antigenicity, host range, and maturation rate of RSV. RSV particles carry reverse transcriptase, an enzyme that converts their RNA into DNA and allows integration into the cell's DNA, where it functions as a cellular gene. This was the bridge that joined the biological era to the molecular era. Its relation to oncogenes and human cancer is discussed.

Integrative effect of defective interfering RNA accumulation and helper virus attenuation is responsible for the persistent infection of Japanese encephalitis virus in BHK-21 cells.

Persistence of RNA viruses is often, but not always, associated with the production of defective interfering (DI) particles. To investigate possible roles of DI particles and helper viruses in RNA virus persistence, persistent infection with Japanese encephalitis virus (JEV) was established in baby hamster kidney (BHK-21) cells. At the 6th and 7th serial undiluted passages of JEV on BHK-21 cells, viral persistence was established spontaneously with DI RNA generation. Seven cell clones exhibiting persistent infection were obtained from the initial BHK-21 cell batches exhibiting JEV persistence, and maintained for over 400 days. Most cell clones produced infectious particles (10(1) -10(5) PFU/ml) continuously, expressed viral proteins, and resisted homologous superinfection. Two helper viruses, chvBS6-3 and chvBS7-1, were isolated from two of the seven cell clones, and characterized to investigate their roles in JEV persistence. While chvBS6-3 was restored to its full cytopathicity in the absence of DI RNA, chvBS7-1 exhibited almost no cytopathicity, regardless of DI RNA co-replication. Attenuation of chvBS7-1 did not appear to be due to inadequate adsorption or genome replication, but due to inefficient egress of the assembled progeny virions, suggesting altered helper virus emergence during JEV persistence in BHK-21 cells. These observations suggest that at least two mechanisms are involved in JEV persistence; a DI RNA-dependent mechanism, where DI RNA co-replication nullifies the helper virus's cytopathicity, or a DI RNA-independent mechanism, where the helper virus is self-attenuated. This study provides a useful in vitro tool for understanding the mechanisms underlying RNA virus persistent infections.

A nonimmunosuppressive helper virus allows high efficiency induction of B cell lymphomas by reticuloendotheliosis virus strain T.

We have documented the effect of two nondefective helper viruses, reticuloendotheliosis virus A (REV-A) and chick syncytial virus (CSV) infection on bursal tissue. REV-A infection results in bursal atrophy, destroying both its structural and functional integrity. In contrast, the bursae in CSV-infected chicks, while reduced slightly in size, appear both structurally and functionally normal. REV-A-induced bursal atrophy is not a result of viral replication in the B-lymphocyte as (a) both viruses are capable of inducing, with equal efficiency, the formation of preneoplastic lesions containing proliferating B lymphocytes and (b) it appears that equivalent amounts of viral antigen are expressed in the bursae of chicks infected with either virus. We have examined the phenotype of tumors induced by the replication-defective virus REV-T when replicated by the two different helper viruses, REV-A and CSV. In REV-T(REV-A)-infected chicks, the majority of tumors that develop are negative for IgM expression. In contrast, the majority of tumors induced by REV-T(CSV) infection are IgM+. This finding is confirmed by recovery of IgM- cell lines from REV-T(REV-A)-infected chicks and IgM+ cell lines from REV-T(CSV)-infected chicks. In addition, repopulation studies show that bursal-derived cells that are IgM+ serve as target cells for REV-T(CSV)-induced lymphomas. This study demonstrates, therefore, that REV-T can induce IgM+, B cell lymphomas with high efficiency. We conclude that infections by the helper viruses, REV-A and CSV, differ dramatically in their effects on the composition of the population of cells that serve as targets for REV-T-induced neoplasia.

Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.

Moloney Murine Leukemia Virus (MoMuLV) causes T cell neoplasms in rodents but is not known to be a pathogen in primates. The core protein and enzyme genes of the MoMuLV genome together with an amphotropic envelope gene are utilized to engineer the cell lines that generate retroviral vectors for use in current human gene therapy applications. We developed a producer clone that generates a very high concentration of retroviral vector particles to optimize conditions for gene insertion into pluripotent hematopoietic stem cells. This producer cell line also generates a much lower concentration of replication-competent virus that arose through recombination. Stem cells from rhesus monkeys were purified by immunoselection with an anti-CD34 antibody, incubated in vitro for 80-86 h in the presence of retroviral vector particles with accompanying replication-competent virus and used to reconstitute recipients whose bone marrow had been ablated by total body irradiation. The retroviral vector genome was detected in circulating cells of five of eight transplant recipients of CD34+ cells and in the circulating cells of two recipients of infected, unfractionated bone marrow mononuclear cells. Three recipients of CD34+ cells had a productive infection with replication-competent virus. Six or seven mo after transplantation, each of these animals developed a rapidly progressive T cell neoplasm involving the thymus, lymph nodes, liver, spleen, and bone marrow. Lymphoma cells contained 10-50 copies of the replication-competent virus, but lacked the retroviral vector genome. We conclude that replication-competent viruses arising from producer cells making retroviral vectors can be pathogenic in primates, which underscores the importance of carefully screening retroviral producer clones used in human trials to exclude contamination with replication-competent virus.

Immunodeficiency and clonal growth of target cells induced by helper-free defective retrovirus.

The murine acquired immunodeficiency syndrome is induced by a defective retrovirus. To study the role of virus replication in this disease, helper-free stocks of defective Duplan virus were produced. These stocks were highly pathogenic in absence of detectable replicating murine leukemia viruses (MuLVs) other than xenotropic MuLV. They induced expansion of the infected cell population (over 1000-fold), and this cell expansion was oligoclonal in origin and, most likely, arose through cell division. These results suggest that this defective virus is oncogenic, inducing a primary neoplasia associated with an acquired immunodeficiency syndrome as a paraneoplastic syndrome. These data emphasize the need to determine whether virus replication is necessary for the progression of other immunodeficiency diseases, including acquired immunodeficiency syndrome, and whether these diseases also represent paraneoplastic syndromes.

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses.

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the ß-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

Isolation, growth, and purification of defective adenovirus deletion mutants.

Defective adenovirus deletion mutants can be grown by complementation in the presence of helper viruses that supply essential functions missing in the deletion mutant. In general, the deletion mutant then must be separated physically from the helper for use in subsequent experiments. This chapter includes suggestions for selection of helper viruses, protocols for the production of stocks by complementation, and procedures for physical separation of deletion mutants from their helpers.

Quantification of high-capacity helper-dependent adenoviral vector genomes in vitro and in vivo, using quantitative TaqMan real-time polymerase chain reaction.

First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehicles for transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviral vectors in preclinical trials have been limited by immune-related adverse side effects. This has been, in part, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untoward immune adverse side effects elicited when high doses of these vectors were used. HC-Ads have several advantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makes them invisible to the host's immune system. Another advantage is their large cloning capacity (up to approximately 35 kb). However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus (RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently, the development of accurate, simple, and reproducible methods to standardize and validate adenoviral preparations for the presence of contaminant genomes is required. By using a molecular method that allows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, and RCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of these three genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro, and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistribution of HC-Ad and improve the safety and efficacy of clinical trials.

Establishment of two canine sarcoma cell lines: productive infection with feline leukemia virus.

Two canine sarcoma cell lines (11028, 11031) were established in vitro and have been transferred 213 and 306 times, respectively, since 1970. These cell lines had a chromosome pattern consistent with their canine origin. Both cultures were infected with feline leukemia virus (FeLV), which caused a morphologic and karyotypic changes. The cells became rounded after infection with FeLV and both cultures showed the presence of a single, large, acrocentric chromosome considered to be a marker chromosome. All tumors were transplanted into newborn beagle pups, but only the 11028-FeLV formed metastatic tumors. No helper or focus-forming activity or virus particles were found in the uninfected cultures. Helper activity and virus were demonstrated in both sarcoma lines after infection with FeLV, though no focus-forming activity was noted. Helper activity of progeny virus could be assayed on either cat or dog embryo cells.

Growth of murine sarcoma virus-transformed rat kidney cells in nude mice: absence of induction of host endogenous viruses.

Clonal isolates of the normal rat kidney cell line (NRK) transformed by a defective murine sarcoma virus (Kirsten strain) were injected into nude mice of BALB/c background to determine whether the growth of these cells as tumors was accompanied by the induction of host endogenous type C viruses. All the virus-transformed clones produced rapidly growing tumors in nude mice, but neither the induction of mouse endogenous viruses nor the rescue and spread of the transforming sarcoma virus were observed during the growth of tumors. The degree of expression of the tumor virus structural proteins in the transformed cells did not determine the cellular phenotype with regard to tumorigenicity in nude mice, nor did it modify the cellular growth properties in vitro. Consistent with earlier observations with simian virus 40-transformed mouse and rat cells, the ability of sarcoma virus-transformed NRK cells to initiate tumor growth in nude mice appeared to be correlated with anchorage-independent growth in vitro.

Sarcoma-negative leukemia-positive transformed cell culture established from a murine sarcoma virus-induced rat bone tumor.

Inoculation of the Soehner-Dmochowski isolate of the Moloney strain of murine sarcoma virus (MSV), designated MSV-SD, consistently leads to the development of bone tumors in the susceptible New Zealand black (NB) rats. Two separate cell cultures have been established from 2 individual MSV-SD-induced NB rat bone tumors. Cells of 1 bone tumor culture, designated RBT-E, are in early in vitro passages. These cells form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at a greatly enhanced rate, 5 times that of normal nontransformed rat embryo cells. Cells of the RBT-E culture release both MSV and murine leukemia virus (MuLV) and therefore contain sarcoma-positive leukemia-positive transformed cells. The other rat bone tumor culture, designated RBT-L, produced MSV at early passages. RBT-L culture has been passaged over 130 times in vitro. Cells of the RBT-L culture form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at an enhanced rate (3 times that of rat embryo cells), indicating the presence of transformed cells within the RBT-L culture. However, cells of the RBT-L culture at late passages (Passage 130 or more) produce only MuLV and no detectable MSV activity (as shown by the lack of tumor-inducing activity and the lack of focus-forming activities by direct assay or by infectious center assay). Attempts to rescue MSV activity from RBT-L cells by cocultivation with MuLV-producing mouse cells were not successful. The MuLV found in the RBT-L cells, however, is a competent helper virus capable of rescuing the MSV genome from MSV-SD-induced hamster bone tumor cells. All the available evidence supports the notion that late passages of the RBT-L culture contain transformed cells that do not produce conventionally detectable MSV. These cells are referred to as sarcoma-negative leukemia-positive cells. The sarcoma-negative leukemia-positive cells represent a different kind of MSV-induced transformed cells and provide a unique system for studies in search of MSV markers such as MSV-specific antigens and MSV-specific nucleotide sequences.

A DNA-based method to assay total and infectious particle contents and helper virus contamination in high-capacity adenoviral vector preparations.

High-capacity adenoviral (HC-Ad) vectors are devoid of all viral genes. Therefore, these vectors feature reduced toxicity, immunogenicity, and increased capacity for foreign DNA. HC-Ad vectors are produced in E1-transformed cell lines in the presence of an E1-deleted helper virus that provides in trans all viral functions necessary for vector production. By cre/loxP- or FLPe/Frt-mediated recombination the packaging signal of the helper virus is excised during vector production resulting in nonpackagable helper virus genomes. Although recombinase-mediated excision of the packaging signal from the helper virus genome is highly efficient, a small number of helper virus genomes with retained packaging signals are still packaged into capsids. For clinical trials, HC-Ad vector preparations have to be characterized accurately with respect to the number of (1) total HC-Ad vector particles, (2) infectious HC-Ad vector particles, and (3) the number of contaminating helper virus particles. We describe a fast and versatile DNA-based biologic assay for determination of these three parameters by standard laboratory methods. This assay is a useful tool for determining bioactivity data of adenoviral vector preparations and, importantly, allows their comparison among different studies.

Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.

Gene transfer vectors based on the replication-defective human parvovirus, adeno-associated virus type 2 (AAV-2), are viable candidates for in vivo and ex vivo human use. However, widespread testing of AAV vectors has been limited by difficulties in generating pure, high-titer vector stocks that are fully characterized. To address these issues, we have developed a single-step purification scheme using heparin affinity chromatography. Recovery from the crude lysate starting material exceeds 70%, and the end product rAAV vector is highly purified and appears to be free of adenovirus and cellular contaminates. Importantly, purified vectors retain predicted in vivo biologic activity. Concurrently, we have developed simple and rapid approaches for vector quantification using real-time PCR. These new methods, combined with the use of stable producer cell lines for rAAV production, make the commercial production of rAAV vectors for human use truly viable and pragmatic.

Assessment of recombinant adenoviral vectors for hepatic gene therapy.

Recombinant adenoviral vectors have recently been used to transfer genes into a number of different cell types in vitro and in vivo. A recombinant adenoviral vector bearing the Escherichia coli beta-galactosidase (beta-gal) gene was used to quantitate the frequency of hepatocyte transduction in the mouse after direct viral infusion into the portal vein. When 10(10) adenoviral particles were infused, over 95% of the hepatocytes were transduced in vivo as determined by x-gal staining. The transduction protocol is relatively safe in that there is no detectable helper virus production in transduced animals and that very few extrahepatic cells are transduced by this method. There is also no evidence of significant liver pathology unless substantially greater quantities of virus are used. However, the transduced hepatocytes do not appear to persist in vivo because the percentage of hepatocytes expressing beta-gal declined over time. Four months after the procedure, 0.5-10% of the hepatocytes contain detectable beta-gal activity in vivo. The change in beta-gal-positive cells correlates with decreasing amounts of adenoviral DNA. Thus, current recombinant adenoviral vectors may have clinical applications in gene therapy for acute hepatic disorders.

Long-term expression of human adenosine deaminase in mice after transplantation of bone marrow infected with amphotropic retroviral vectors.

Three retroviral vectors, containing a human adenosine deaminase (ADA) cDNA linked to either the simian virus 40 (SV40) early promoter, the human cytomegalovirus (CMV) immediate early promoter, or the Moloney murine leukemia virus (MoMLV) promoter, were tested for their ability to express ADA following infection and transplantation of murine bone marrow. Virus was produced by using PA317 amphotropic retrovirus packaging cells. The titer of each of the vectors was similar and no helper virus was detected. Human ADA was expressed in the blood of some animals for 6 months after transplantation of infected marrow, and vector DNA was found in the spleen and in bone marrow from these animals. The percentage of animals expressing human ADA (33%) and the amount of human ADA in blood (1-5% of total ADA) was similar for each of the vectors. These results show that amphotropic vectors are capable of infecting pluripotent hematopoietic stem cells having long-term repopulating ability, and that a variety of promoters allow gene expression following differentiation of these early cells.

Evaluation of the respiratory epithelium of normals and individuals with cystic fibrosis for the presence of adenovirus E1a sequences relevant to the use of E1a- adenovirus vectors for gene therapy for the respiratory manifestations of cystic fibrosis.

Lung disease associated with disorders such as cystic fibrosis (CF) may be amenable to somatic gene therapy in which there is delivery of the normal gene directly to the respiratory epithelium using E1a- adenovirus (Ad) type 2- or 5-based vectors. For safety reasons, the Ad vectors are rendered replication deficient by deletion of the E1a region. Because there is the theoretical possibility of an E1a- replication-deficient vector replicating as a result of recombination or complementation with Ad 2/5 E1a sequences present in the target cell, this study is directed toward evaluating respiratory epithelium of normals and individuals with CF for the presence of E1a sequences. Using Ad 2/5 E1a-specific primers and the polymerase chain reaction to evaluate DNA recovered from freshly isolated nasal and bronchial epithelium recovered by brushing, E1a sequences were detected in respiratory epithelium of 19 of 91 normals (21%). In the E1a-positive samples, the average of E1a copy number was 55 +/- 18/10(3) recovered cells. In CF individuals, 7 of 52 (13%) had detectable E1a sequences in the respiratory epithelium, with E1a copy number in the positive samples of 80 +/- 21/10(3) recovered cells. These results demonstrate that there are detectable Ad 2/5 E1a sequences in the respiratory epithelium of a small percentage of normals and individuals with CF. Because of the theoretical potential of such sequences supporting replication of E1a- Ad vectors, human gene therapy protocols for CF utilizing such vectors should consider evaluating study individuals for the presence of Ad 2/5 E1a sequences in the respiratory epithelium.

Involvement of peritoneal macrophages and spleen stromal cells in X-irradiation-induced reticulum cell neoplasms in C57BL/6 mice.

Some correlation was observed between the occurrence of FA-positive PE-MO and spleen stromal cells (removed from X-irradiated RCN-bearing old-adult B6 mice) and the generation of RCN. No significant correlation was found between the viral content of lymphoid organs from the same mice and the occurrence of RCN. The main viral particle detected in lymphoid organs from radiation-induced RCN-bearing mice was the xenotropic virus. Ecotropic viruses were detected in a few spleens and Payer patches from such mice. These ecotropic viruses showed very poor lymphomagenic activity and required 400R X-ray as a cofactor. No dualtropic viruses were detected. However, inoculation of ecotropic (SFA2) helper virus to X-irradiated old-adult B6 mice, resulted in an efficient rescue of lymphomagenic viruses, enriched with phenotypically mixed, dualtropic viruses. Some of these DT viral preparations were cloned and seemed to consist mainly of xenotropic sequences. Thus, inoculation of helper viruses influenced the generation and selection of DT viruses. Such viral preparations, enriched with DT viruses, had a better lymphomagenic activity compared to endogenous ecotropic viruses, isolated from radiation-induced RCN-bearing mice. Indirect evidence suggested the involvement of a defective (xenotropic and possibly adjacent cellular genes) particle in lymphoma induction. To conclude, a possible mechanism for the development of radiation-induced RCN is suggested, emphasizing the role of MO in such a process.

A new one-step RT-PCR method for virus quantitation in murine AIDS.

The causative agent of murine AIDS (MAIDS) in C57BL/6 mice, is a defective murine leukemia virus (BM5d) that requires the replication-competent helper virus (BM5e). Since this animal model of immunodeficiency, which shows many similarities to human AIDS, is also used to test the efficacy and toxicity of antiretroviral drugs, a method that allows the quantitative detection of both viruses would be very useful also if hampered potentially by endogenous viral sequences usually present in mice. While BM5d alone could induce the disease, the effect of BM5e on the immune system of diseased mice is unclear. A specific and reliable one-step RT-PCR method was developed for the co-amplification, with the same efficiency, of BM5d or BM5e with ss-actin used as an internal standard. The standard curves produced with cloned cDNA sequences (ss-actin and BM5d or BM5e) assure that all samples are analyzed during the exponential phase of the reaction. Using this new assay which provided a dynamic range of at least four-log-unit, the ratio of initial absolute amounts of the virus and ss-actin RNA was determined, obtaining quantitative information on virus-specific cellular-transcript in the lymph nodes and spleen during the natural history of the disease and during therapeutic regimens.