Nucleotide sequence of SRV-1, a type D simian acquired immune deficiency syndrome retrovirus.

Simian acquired immune deficiency syndrome (SAIDS) in the macaque genus of monkeys at the California Primate Research Center is apparently caused by infection by a type D retrovirus. The complete nucleotide sequence (8173 base pairs) of a molecular clone of the prototype SAIDS virus isolate, SRV-1, reveals a typical retrovirus structure with long terminal repeats (346 base pairs) and open reading frames for the gag (663 codons), pol (867 codons), and env (605 codons) genes. SRV-1 also has a separate open reading frame of 314 codons between the gag and pol genes that defines the viral protease gene (prt) and a short open reading frame of unknown significance downstream from the env gene. The SRV-1 protease region shows a high degree of homology to its counterpart in the hamster intracisternal A-type particle genome; both these protease genes are about twice as long as the analogous region of other retroviruses. SRV-1 has no notable similarity in either genetic organization or sequence to the human AIDS retroviruses.

Inapparent carriers of simian acquired immune deficiency syndrome type D retrovirus and disease transmission with saliva.

Simian acquired immune deficiency syndrome (SAIDS) type D retrovirus (SRV) was isolated from saliva, urine, and peripheral blood mononuclear cells of a 6-year-old healthy rhesus monkey (Macaca mulatta) seronegative for antibodies to human T-lymphotropic virus (HTLV) type I, HTLV type III, and simian T-lymphotropic virus type III (STLV-III), identified as an inapparent SAIDS carrier in retrospective epidemiologic studies. This animal was linked to 34 cases of SAIDS over a 3-year period. Two juvenile rhesus monkeys inoculated iv with the SRV-containing saliva from this carrier became persistently infected with the retrovirus and developed SAIDS after 4-6 weeks. Both animals seroconverted to SRV, but neither had detectable preinoculation or postinoculation antibodies against HTLV type I, HTLV type III, or STLV-III. One of these animals died of SAIDS with disseminated cytomegalovirus infection after 24 weeks, and the other remains alive with persistent SRV viremia, generalized lymphadenopathy, and splenomegaly after a transient immunosuppression. Major clinical and pathological features associated with the newly described STLV-III were not observed. SRV was subsequently identified in saliva of 2 additional healthy carriers as well as monkeys with SAIDS. The findings of a carrier state in SAIDS and evidence for saliva transmission of the probable causative virus further support the usefulness of this animal model of nononcogenic immunosuppressive retroviral disease.

Immunodeficiency in rhesus monkeys associated with the original Mason-Pfizer monkey virus.

The Mason-Pfizer monkey virus (MPMV) was reisolated from a cryopreserved sample of the original MPMV-containing rhesus breast carcinoma, and complete integrated MPMV provirus was detected in chromosomal DNA of this tumor. Reanalysis of the in vivo pathogenicity and molecular character of MPMV reisolated from the rhesus breast tumor and analysis of the original MPMV after long-term in vitro propagation in human and rhesus cells show that the original MPMV produces an acquired immunodeficiency similar to that caused by the recently described simian acquired immune deficiency syndrome type D retroviruses, and the MPMV genome and its immunosuppressive effect in vivo have remained stable despite prolonged in vitro passage in human and rhesus cells.

Immune system-neuroendocrine dysregulation in spinal cord injury.

Multiple communicative pathways among the nervous, endocrine and immune systems facilitate physiological immunoregulation. Spinal cord injury (SCI) patients have decreased natural (NK cell) and adaptive (T cell) immune function and reduced blood levels of cellular adhesion molecules (CAMs) that participate in immune function and wound healing. We found decreased LFA-1 and VLA-4 on peripheral blood leukocytes in SCI patients and lower levels of CAMs in SCI patients with pressure ulcers than in those without them. SCI might affect immune cells and immune responsiveness by: (1) disrupting the outflow of signals from the sympathetic nervous system to lymphoid tissues and their blood vessels as well as the returning afferent signals from these tissues to the brain; (2) immunosuppression caused by the stressors affecting SCI patients; (3) interrupting returning signals to the CNS from the periphery thereby reducing facilitation of immunoregulatory CNS neurons and decreasing their activity; or a combination of all three. SCI patients may develop dysregulation of the sympathetic nervous system that is intimately involved in immune function. Chronic stress mediates immunosuppression by corticosteroids, catecholamines, endorphins and met-enkephalin. The hypothalamus coordinates the response to stress through the release of soluble products from the sympathetic nervous system and hypothalamic-pituitary-adrenal axis. Whereas the nervous and endocrine systems are not concerned with immunological specificity, they do influence the intensity, kinetics and localization of immune responses. Products of an activated immune system may generate feedback circuits capable of inhibiting, enhancing or regulating neuronal input. Immune system cells can produce neurologically active peptides including ACTH, CRF, growth hormone, thyrotropin, prolactin, human chorionic gonadotropin, endorphin, enkephalins, substance P, somatostatin and VIP. Cytokines are likely important mediators of the HPA response to immune stimuli.

Evaluation of the mutagenic and genotoxic activities of anti-hepatitis B analogs of beta-L-adenosine by the Ames test and the Comet assay.

beta-L-2'-deoxyadenosine (beta-L-dA), beta-L-2',3'-dideoxyadenosine (beta-L-ddA) and its two bis (S-acyl-2-thioethyl; SATE) phosphotriester derivatives, beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(MeSATE) and beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(tButylSATE) have been previously shown to exhibit potent and selective anti-hepatitis B activity in vitro. None of the four compounds was mutagenic up to 100 microg in the Ames test (microtechnique) using Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation. In addition, the genotoxicity of beta-LdA and the three other compounds was evaluated in human lymphocytes using the Comet assay, at doses up to 5 microg with or without the addition of a microsomal S9 fraction. None of the four compounds induced DNA strand breakage with and without metabolic activation. In summary, the data clearly demonstrate that the purine nucleoside beta-L-dA, beta-L-ddA and the two prodrugs, beta-L-ddAMP-bis(MeSATE) and beta-L-ddAMP-bis(tButylSATE) are not mutagenic in the Ames test and do not induce DNA damage in human lymphocytes, as assessed by the Comet assay.

Antiviral activity and intracellular metabolism of bis(tButylSATE) phosphotriester of beta-L-2',3'dideoxyadenosine, a potent inhibitor of HIV and HBV replication.

The beta-L-nucleoside analogue beta-L-2',3'-dideoxy adenosine (beta-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide beta-L-2',3'-dideoxyadenosine-5'-mono phosphate (beta-L-ddAMP) (Placidi et al., 2000). However, the nucleotide beta-L-2',3'-dideoxyadenosine-5'-triphosphate (beta-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 microM without inhibiting human DNA polymerases alpha, beta, or gamma up to a concentration of 100 microM. These results suggested that prodrugs of beta-L-ddAMP may bypass the poor metabolic activation of beta-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of beta-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, beta-L-ddAMP-bis(tButylSATE) was synthesized. Beta-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of beta-L-ddAMP-bis(tButylSATE) demonstrated that beta-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of beta-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCs, respectively. The intracellular concentrations of beta-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.

Antiviral beta-L-nucleosides specific for hepatitis B virus infection.

Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.

Comparison of the clearance of radiolabelled nose drops and nasal spray as mucosally delivered vaccine.

The distribution and nasal clearance of 99Tcm-labelled albumin (18.5 MBq), used as a mucosal vaccine surrogate for FluMist, was determined in three volunteers. The subjects were randomized in a cross-over clinical study design to receive either large-particle aerosal (nasal spray) followed by nose drops, or nose drops followed by the nasal spray, 1 week apart. Gamma scintigraphy was used to measure the distribution and clearance. The 'vaccine' delivered as drops was cleared from the nose into the oesophagus and upper stomach at very variable rates. In contrast, the nasal spray was uniformly distributed and cleared from the nasopharynx with a 50% mean clearance time of 50 min (range 40-60 min) and was not detected in the lungs.

Pathogenesis of simian AIDS in rhesus macaques inoculated with the SRV-1 strain of type D retrovirus.

Type D retrovirus was isolated from rhesus macaques with simian acquired immunodeficiency syndrome (SAIDS) and transmitted to healthy rhesus macaques with tissue culture medium containing the virus. The clinical, immunologic, and lymph node morphologic changes were observed in 9 rhesus macaques for 52 weeks after inoculation. A spectrum of clinical signs developed including early death, persistent SAIDS, and apparent remission. Animals that died or developed persistent SAIDS had characteristic lymphoid depletion, persistently depressed peripheral blood mononuclear cell (PBMC) mitogenic response, and decreased serum immunoglobulins. The SAIDS retrovirus (SRV) was recovered from PBMC of 8 of the animals after inoculation. Virus could not be recovered from PBMC of one animal in remission, but this animal developed serum-neutralizing antibodies to SRV after inoculation. Seven of the animals seroconverted to SRV after inoculation, all 9 were seronegative for human T-lymphotropic virus-III, and 5 animals tested were seronegative to human T-lymphotropic virus-I. These findings support the etiologic role of the type D retrovirus in SAIDS and further define the pathogenesis of this disease.

Anti-HBV specific beta-L-2'-deoxynucleosides.

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Fertility awareness / natural family planning for adolescents and their families: report of multisite pilot project.

Fertility awareness is experiential learning about cyclic fertility. This awareness, used as a family planning method, differs from contraception because it does not isolate the procreative capacity of either partner. The acceptability and effect of teaching fertility awareness on teen sexual activity and decision making was tested in a multisite pilot program which taught fertility awareness via the prospective marker of the cervical mucus (ovulation method of natural family planning). 200 US and 35 Guatemalan volunteer women ages 15-17 in a structured 1 year curriculum, monitored cycle charting and explored the implications of experiencing one's signs of fertility. Control subjects were recruited from the general population and from family planning clinics. 9% of the US study group were sexually active prior to entry. By cycle 12, 1/2 had discontinued activity. Conception rate was 0.0044. The continuation rate dropped from 90% at cycle 7 to 71% at cycle 8 due to scheduling constraints for 2 classes and to 57% at cycle 12. Postprogram follow-up of early leavers showed only 1/3 the expected rate of onset of sexual activity and pregnancy. Parent involvement correlated positively with postponement and/or discontinuation of sexual activity. Reported movement away from peer group pressure appeared 3 months after entry.

The development of live attenuated cold-adapted influenza virus vaccine for humans.

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attenuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups.

RNA tumor virus phosphoproteins: primary structural analysis and identification of phosphopeptides.

Two-dimensional tryptic peptide mapping was used to compare the peptide sequences of the phosphoprotein (pp12) of cloned ecotropic and amphotropic wild mouse leukemia viruses, strains 1504 and 292. The maps of two ecotropic isolates were very similar to one another, as were the maps of two amphotropic isolates. There was also extensive similarity between the maps of this protein from ecotropic and amphotropic viruses, although characteristic peptide differences were readily recognized. These differences were consistent with the general type specificity of oncovirus phosphoproteins. The pp12 of the field isoalte of 292 virus contained five phosphopeptides, and the non-phosphorylated and variously phosphorylated species of this pp12 showed identical peptide maps, indicating differential phosphorylation of a single polypeptide.

Cloning and sequence analysis of murine cytomegalovirus protease and capsid assembly protein genes.

The murine cytomegalovirus UL80 open reading frame was cloned and the predicted amino acid sequence compared with those from other herpesviruses. The open reading frame encodes a fused protease-capsid assembly protein precursor and maintains conserved features including the active site serine, conserved regions CD1 through CD5, the release and maturation sites, and a potential internal cleavage site within the protease. However, the murine cytomegalovirus protease differs in comparison with the other proteases because it contains a unique 15-16 amino acid insertion between CD3 and CD1. The assembly protein sequences are relatively divergent, but they can be arranged into groups defined by herpesvirus subfamily, with each group possessing a conserved motif at its carboxyl terminus.

Molecular diversity among five different endogenous primate retroviruses.

Genetically transmitted retroviruses of Old and New World monkeys include type C viruses isolated from baboons (M7), macaque (MAC-1), and owl monkeys (OMC-1) and type D viruses from langurs (PO-1-Lu) and squirrel monkeys (SMRV, M534). Each of these isolates is unrelated to the others by nucleic acid hybridization criteria and contains a unique array of virion-associated proteins which can be resolved by agarose gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. The major structural protein of each virus has a distinct primary structure, as determined by two-dimensional tryptic peptide analysis, and is antigenically different from the others. The major virion phosphoproteins of endogenous primate type C viruses (pp15) are also different from those of type D viruses (pp13-pp14). Immunological and structural analyses show that the endogenous langur virus and the horizontally transmitted Mason-Pfizer virus of rhesus monkeys are closely related to one another, consistent with the sequence homology detected in their RNA genomes. Although certain radioimmunoassays detect interspecies antigenic determinants common to either the p30 or gp70 proteins of some of these viruses, no one assay has yet been designed which can detect all groups of endogenous primate retroviridae. The data lead to the conclusion that primates contain a minimum of three different sets of genetically transmitted type C and type D retroviral genes.