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In the Republic of Ireland (ROI), BRCA1/BRCA2 genetic testing has been traditionally undertaken in eligible individuals, after pre-test counselling by a Clinical Geneticist/Genetic Counsellor. Clinical Genetics services in ROI are poorly resourced, with routine waiting times for appointments at the time of this pilot often extending beyond a year. The consequent prolonged waiting times are unacceptable where therapeutic decision-making depends on the patient's BRCA status. "Mainstreaming" BRCA1/BRCA2 testing through routine oncology/surgical clinics has been implemented successfully in other centres in the UK and internationally. We aimed to pilot this pathway in three Irish tertiary centres. A service evaluation project was undertaken over a 6-month period between January and July 2017. Eligible patients, fulfilling pathology and age-based inclusion criteria defined by TGL clinical, were identified, and offered constitutional BRCA1/BRCA2 testing after pre-test counselling by treating clinicians. Tests were undertaken by TGL Clinical. Results were returned to clinicians by secure email. Onward referrals of patients with uncertain/pathogenic results, or suspicious family histories, to Clinical Genetics were made by the treating team. Surveys assessing patient and clinician satisfaction were sent to participating clinicians and a sample of participating patients. Data was collected with respect to diagnostic yield, turnaround time, onward referral rates, and patient and clinician feedback. A total of 101 patients underwent diagnostic germline BRCA1/BRCA2 tests through this pathway. Pathogenic variants were identified in 12 patients (12%). All patients in whom variants were identified were appropriately referred to Clinical Genetics. At least 12 additional patients with uninformative BRCA1/BRCA2 tests were also referred for formal assessment by Clinical Geneticist or Genetic Counsellor. Issues were noted in terms of time pressures and communication of results to patients. Results from a representative sample of participants completing the satisfaction survey indicated that the pathway was acceptable to patients and clinicians. Mainstreaming of constitutional BRCA1/BRCA2 testing guided by age- and pathology-based criteria is potentially feasible for patients with breast cancer as well as patients with ovarian cancer in Ireland.

Objectives:Large numbers of new medical devices and diagnostics are developed and health services need to identify which ones offer real advantages. The National Institute for Health and Care Excellence (NICE) has introduced a system for assessing technologies that are often notified by companies, based on claims made for their benefits to patients, the National Health Service, and the environment.Methods:Detailed scrutiny of claims made for the benefits of products and the corresponding evidence, seeking associations between these and the selection of products for full evaluation to produce NICE guidance.Results:Between 2009 and 2015 a NICE committee considered 169 technologies, of which it selected 74 (44 percent) for full evaluation, based on the claims of benefit and the evidence available. An average of 7.5 claims were made per technology; the total number did not influence selection but presence of studies supporting all the claims (p < .001) or any of the claims (p < .05) had a positive influence, as did claims for quicker patient recovery (p < .001). A greater number of studies to support the claims made selection more likely (p < .001), as did cohort studies (p < .05) and surveys (p < .05) but, unexpectedly, not randomized trials. The Medical Device Directive class had no influence.Conclusions:This study presents categories of claims that may be useful to those developing new products and to others engaged in health technology assessment. It illustrates the importance of relevant evidence and of having a clear vision of the place of new products in care pathways from an early stage.

Objectives: The aim of this study was to review 5 years of activity from a new system devised by the National Institute for Health and Care Excellence (NICE), for assessing medical devices and diagnostics aimed at identifying and speeding adoption of technologies with clinical and cost advantages, compared with current practice in the United Kingdom healthcare system.Methods: All eligible notified technologies were classified using the Food and Drug Administration and Global Medical Device Nomenclature nomenclatures. Decisions about selecting technologies for full assessment to produce NICE recommendations were reviewed, along with the reasons given to companies for not selecting products.Results: Between 2009 and 2014, 186 technologies were notified (46 percent therapeutic and 54 percent diagnostic). Thirty-nine were judged ineligible (no regulatory approval), and 147 were considered by an independent committee. Of these, eighty (54 percent) were not selected for full assessment, most commonly because of insufficient evidence (86 percent): there were uncertainties specifically about benefits to the health service (54 percent), to patients (39 percent), and about cost (24 percent). The remaining 67 were selected and assessed for Medical Technology guidance (52 percent) (noninferior and/or lower cost consequences than current practice), for Diagnostics guidance (43 percent) or other NICE recommendations about adoption and use. Classifying technologies by two different systems showed no selection bias for any technology type or disease area.Conclusions: Identifying new or under-used devices and diagnostics with potential benefits and promoting their adoption is important to health services in the United Kingdom and worldwide. This new system offers a means of fostering both uptake and further research. Lack of research data on new products is a major obstacle to evaluation.

BackgroundThere is a paucity of UK data to aid healthcare professionals in predicting which patients hospitalised with community-acquired pneumonia (CAP) are at greatest risk of 30-day readmission and to determine which readmissions may occur soonest.MethodsAn analysis of CAP cases admitted to nine UK hospitals participating in the Advancing Quality Pneumonia Programme.ResultsAn analysis was performed of 12 157 subjects hospitalised with CAP in the Advancing Quality Programme Database. 26% of those discharged were readmitted within 30 days with readmission predicted by comorbidity including non-metastatic cancer, diabetes with complications and chronic kidney disease. 41% and 66% of readmissions occurred within 7 and 14 days of discharge, respectively. Patients readmitted within 14 days were more likely to have metastatic cancer (6.6% vs 4.5%; p=0.03) compared with those readmitted at 15–30 days.ConclusionsA quarter of patients hospitalised for CAP are readmitted within 30 days; of those, two-thirds are readmitted within 2 weeks. Further research is required to determine whether such readmissions might be preventable through imple menting measures including in-hospital cross-specialty comorbidity management, convalescence in intermediate care, targeted rehabilitation and advanced care planning.

Scoliosis-structural lateral curvature of the spine-affects around four children per 1,000. The MAGEC system comprises a magnetically distractible spinal rod implant and an external remote controller, which lengthens the rod; this system avoids repeated surgical lengthening. Rod implants brace the spine internally and are lengthened as the child grows, preventing worsening of scoliosis and delaying the need for spinal fusion. The Medical Technologies Advisory Committee at the National Institute for Health and Care Excellence (NICE) selected the MAGEC system for evaluation in a NICE medical technologies guidance. Six studies were identified by the sponsor (Ellipse Technologies Inc.) as being relevant to the decision problem. Meta-analysis was used to compare the clinical evidence results with those of one conventional growth rod study, and equal efficacy of the two devices was concluded. The key weakness was selection of a single comparator study. The External Assessment Centre (EAC) identified 16 conventional growth rod studies and undertook meta-analyses of relevant outcomes. Its critique highlighted limitations around study heterogeneity and variations in baseline characteristics and follow-up duration, precluding the ability to draw firm conclusions. The sponsor constructed a de novo costing model showing that MAGEC rods generated cost savings of £9,946 per patient after 6 years, compared with conventional rods. The EAC critiqued and updated the model structure and inputs, calculating robust cost savings of £12,077 per patient with MAGEC rods compared with conventional rods over 6 years. The year of valuation was 2012. NICE issued a positive recommendation as supported by the evidence (Medical Technologies Guidance 18).

Virtual Touch™ Quantification (VTq) is a software application used with Siemens Acuson ultrasound scanners to assess the stiffness of liver tissue. The National Institute for Health and Care Excellence (NICE) Medical Technologies Advisory Committee (MTAC) selected VTq for evaluation and invited the company to submit clinical and economic evidence. King's Technology Evaluation Centre, an External Assessment Centre (EAC) commissioned by NICE, independently assessed the evidence submitted. The EAC conducted its own systematic review, meta-analysis and economic analysis to supplement the company's submitted evidence. The meta-analyses comparing VTq and transient elastography (TE) with liver biopsy (LB) provided pooled estimates of liver stiffness and stage of fibrosis for the study populations (hepatitis B, hepatitis C or combined populations). When comparing significant fibrosis (Metavir score F ≥ 2) for both hepatitis B and C, VTq had slightly higher values for both sensitivity and specificity (77 and 81 %) than TE (76 and 71 %). The overall prevalence of cirrhosis (F4, combined populations) was similar with VTq and TE (23 vs. 23 %), and significant fibrosis (F ≥ 2) was lower for VTq than for TE (55 vs. 62 %). The EAC revised the company's de novo cost model, which resulted in a cost saving of £53 (against TE) and £434 (against LB). Following public consultation, taking into account submitted comments, NICE Medical Technology Guidance MTG27 was published in September 2015. This recommended the adoption of the VTq software to diagnose and monitor liver fibrosis in patients with hepatitis B or hepatitis C.

We have modeled highly active antiretroviral therapy (HAART) for AIDS in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type-1 (HIV-1). Seven RT-SHIV-infected macaques were treated with a combination of efavirenz (200 mg orally once daily), lamivudine (8 mg/kg subcutaneously once daily), and tenofovir (30 mg/kg subcutaneously once daily). Plasma viral RNA levels in all animals were reduced by more than 1,000-fold after 4 weeks and, in six of the seven animals, were reduced to undetectable levels after 10 weeks. Virus loads increased slightly between 12 and 16 weeks of treatment, associated with problems with the administration of efavirenz. After a change in the method of efavirenz administration, virus loads declined again and remained undetectable in the majority of animals for the duration of therapy. Treatment was stopped for three animals after 36 weeks of therapy, and virus loads increased rapidly. Posttreatment RT-SHIV isolates had no mutations associated with resistance to any of the three drugs. Efavirenz treatment was stopped, but lamivudine and tenofovir treatment for two other macaques was continued. The virus load in one of these two animals rebounded; virus from this animal was initially free of drug-resistance mutations but acquired the K65R mutation in reverse transcriptase at 11 weeks after efavirenz treatment was withdrawn. These results mimic HAART of HIV-1-infected humans. The RT-SHIV/rhesus macaque model should be useful for studies of tissue reservoirs and sites of residual replication that are not possible or practical with humans.

Human herpesvirus type 8 vMIP-II has one of the broadest ranges of chemokine receptor binding and therefore a multiplicity of biologic effects, both immunologic and antiviral. These properties make vMIP-II an attractive effector gene to be expressed from gene therapy vectors. The present studies were concerned with both therapeutic approaches: (1) an anti-simian immunodeficiency virus (SIV) biologic, and (2) an effector gene in SIV-based vectors. Regarding its antiviral properties, vMIP-II expressed from bacteria and SIV-based vectors bound the surface of CEMx174 cells and specifically suppressed SIVmac251 infection. A CCR3 monoclonal antibody partially inhibited vMIP-II binding, suggesting that both SIVmac251 and vMIP-II utilize a similar CCR3-like receptor for CEMx174 cell binding. Replication competent SIV-based vectors containing forward and reverse vMIP-II produced neither identifiable vMIP-II nor virions for the first 21 days. Virus replication occurred after this period. Significant sequence alterations in the forward vMIP-II containing replication competent vector transcripts were responsible for the failure of vMIP-II expression. The genetic basis for the initial failure to replicate virus and its later restoration was not determined but appeared in the II-PIMv containing vectors to coincide with deletions and compensatory rearrangements in nef 3′ of the polypurine tract. Cells transfected with SIVmac239Δ3ΔLTR- vMIP-II vectors expressed biologically active vMIP-II that bound CEMx174 cells and suppressed SIVmac251 infection. These data suggest that replication defective SIV vectors expressing immunobiolgic genes such as vMIP-II may prove useful in gene therapies, particularly in augmenting immune responses in chronically infected individuals.

16alpha-Bromo-epiandrosterone (epiBr), a synthetic derivative of the natural hormone dehyroepiandrosterone (DHEA), was evaluated for its effects on feline immunodeficiency virus (FIV) infection in experimental cats. The rationale for this study was based on the ability of DHEA to significantly reduce the mortality to viral infections in mice. DHEA and epiBr also have demonstrable in vitro anti-viral activity for both HIV-1 and FIV. Preliminary pharmacokinetic studies in cats demonstrated that subcutaneously injected epiBr was rapidly absorbed, completely metabolized, and nontoxic. Metabolites were excreted in both urine and feces, with the latter having the most complex pattern of breakdown products. Cats were then divided into four groups; two groups were infected with FIV and two uninfected. Two groups, one infected and one uninfected were treated on 5 consecutive days of weeks 0, 4, 8, 12 and 16 with epiBr. The remaining two groups were mock treated with the drug vehicle alone. Treatment started 1 week prior to infection and extended for 4 weeks after infection. Cats were observed for 20 weeks post-FIV infection. Infected cats had identical decreases in blood neutrophil and lymphocyte counts following, regardless of whether they were treated with epiBr or vehicle alone. The CD4/CD8 T-cell ratio was decreased following FIV exposure, but was significantly more decreased for the epiBr treated animals from week 2 post-infection onward. CD4+ T cells were decreased in FIV-infected cats treated with epiBr compared to their untreated cohort, while CD8+ T cells tended to be higher in treated animals. FIV infected cats that were treated with epiBr had over one-log higher virus loads at week 2 post-infection than non-epiBr treated cohorts. In spite of this enhanced initial viremia, the subsequent levels of virus in the blood were significantly lower in epiBr treated versus untreated animals. EpiBr treated cats had significantly higher FIV-p24 antibody responses than control cats receiving vehicle alone, although primary and secondary antibody responses to a T-cell dependent non-FIV antigen, keyhole limpet hemocyanin (KLH), were unaffected. EpiBr treatment significantly decreased the expected FIV-induced suppression of IL-12 p40 mRNA levels in peripheral blood mononuclear cells (PBMCs) observed at weeks 4, 5, 8, 9 and 16 post-infection, but had no influence on FIV-induced changes in IL-4, IL-6, IL-10, IFN-gamma, MIP-1alpha and RANTES.

We used a focal infectivity assay with HeLa H1-JC.37 cells to directly compare susceptibilities of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) to protease inhibitors. SIVmac239 was inhibited by indinavir, saquinavir, and ritonavir, with 50% effective concentrations (means ± standard deviations) of 39 ± 8, 55 ± 3, and 13 ± 5 nM, respectively. The corresponding values for inhibition of HIV-1 were 66 ± 4, 47 ± 10, and 25 ± 14 nM, respectively.

Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIV mac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(−)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (−)-FTC, (−)-β- d -(2 R ,4 R )-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (−)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC–MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >10 4 copies of RNA/ml. RT-SHIV loads at the start of treatment ( V 0 ) were similar in all treated cohorts ( P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency ( P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred rapidly following cessation of any treatment. The enhanced HAART (four- or five-drug combination) showed significant improvement in viral suppression compared to the three-drug combination, but no combination was sufficient to eliminate viral reservoirs.

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⁺ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by realtime TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

We report the cloning, expression and characterization of biologically active feline tumour necrosis factor-alpha (fTNF-alpha). Messenger RNA was extracted from feline peritoneal macrophage cultures and used to synthesize cDNA for polymerase chain reaction (PCR) amplification. The PCR products were cloned into the plasmid vector pCRII and sequenced, showing 99.3% homology with a published fTNF-alpha gene sequence. Subcloning into the vector pGEX-2T and subsequent expression resulted in a 43 kDa fusion protein of fTNF-alpha and glutathione S-transferase (GST). Thrombin cleavage of the fusion protein yielded a 17 kDa protein. This protein cross-reacted with a monoclonal anti-human TNF-alpha antibody in Western blotting, but not with a polyclonal anti-murine TNF-alpha serum. Recombinant fTNF-alpha (rfTNF-alpha) and rfTNF-alpha-GST had a CD50 of 15 ng ml-1 and 230 ng ml-1, respectively, in the L929 cytotoxicity assay. Cats given rfTNF-alpha-GST intravenously manifested the typical biological effects of TNF-alpha, including fever, depression, and piloerection. The rfTNF-alpha-GST upregulated IL-2 receptor and MHC-II antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on TNF-alpha receptor and MHC-I antigen expression.

Five species of bacteria known to be naturally-occurring pathogens of cats were screened for their ability to grow in feline macrophages in vitro, and to induce antibodies and delayed type hypersensitivity (DTH) responses in vivo. Two of these organisms, L. monocytogenes and S. marcescens, were selected for further study based on clear-cut differences in their in vitro and in vivo behavior. Listeria was macrophage tropic, induced DTH, and evoked poor antibody responses post-recovery, whereas Serratia remained extracellular, did not induce a DTH reaction, and produced high titer of antibodies. Young specific pathogen free cats were then inoculated subcutaneously into the drainage areas of the right and left popliteal and auricular lymph nodes with either L. monocytogenes or S. marcescens. Each of the four lymph nodes were then removed in sequence over a two week period, weighed, cultured for viable bacteria, and RNA extracted for Th1/Th2 cytokine mRNA quantitation. Antibody responses and delayed type hypersensitivity responses were also measured. Identical to pilot studies, cats infected with Serratia developed very high levels of antibody compared to Listeria infected cats but no DTH, while Listeria infected cats produced negligible or low titers of antibodies and strong DTH. Immunity to Listeria occurred around 168 h post infection as evidenced by the disappearance of living bacteria from the nodes, while immunity to Serratia took over 264 h. Pronounced lymph node hyperplasia occurred in both infections, but persisted longer for Serratia. Enlargement of Serratia infected nodes was associated with marked follicular, primary and secondary germinal center and medullary hyperplasia. Germinal center formation in Listeria stimulated nodes was much less intense and dense accumulations of macrophages dissected between follicles downward from the subcapsular sinuses. Although functional and histologic studies showed a clear-cut cell-mediated vs. humoral response in the respective Listeria and Serratia infections, preferential cytokine mRNA upregulation was observed for only two of the five major Th1/Th2 cytokines measured. Interferon-gamma, a Th1 cytokine, was much more elevated in the Listeria stimulated nodes, but TNF-alpha (also a Th1 cytokine) was more elevated in Serratia infected nodes. Interleukin-12, an important Th1 cytokine, was elevated to equal levels in both infections as were the Th2 cytokines IL-4 and IL-10.

We have developed a method to quantitate feline cytokine gene expression using competitive RT-PCR. Feline cytokine specific primers were developed that encompass an intron, thus allowing differentiation of cDNA vs. genomic DNA amplification products. The PCR products of the primers were verified by sequencing and Southern blot analysis. For quantitation, a non-homologous RNA competitor was created for each cytokine of interest. The competitor was designed to yield an RT-PCR product 10-20% larger than the native sequence, thereby allowing differentiation of the two products by electrophoresis on an agarose gel. Both competitor and native sequences used the same primer sequences for RT (oligo dT) and PCR (cytokine specific). The amplification efficiency of the competitor and native sequence was shown to be identical which allowed comparison at any point during the amplification, including the plateau phase. The quantity of starting cytokine mRNA was determined by interpolation from a standard curve. As little as 1 microgram of total cellular RNA was required per cytokine determination. The assay can routinely quantify as few as 1000 copies of template and spans a range of up to 4 log.

Molecularly cloned viruses are considered essential reagents for characterizing viral domains responsible for infectivity and disease pathogenesis in the host. The infectivity and hematological alterations associated with two molecularly cloned isolates of feline immunodeficiency virus (FIV-pPPR and FIV-pF34) and an uncloned isolate (FIV-PPR) were assessed by inoculation of cats. Inoculated cats were tested for viral antibody, viremia, and clinical pathological disease. Peripheral blood mononuclear cells isolated from inoculated cats were assayed for virus infection by virus isolation, amplification of proviral DNA (by polymerase chain reaction), and in situ hybridization for viral RNA. Over 50% of the cats inoculated with cloned virus FIV-pF34 failed to seroconvert even when coinfected with feline leukemia virus; these cats were consistently virus positive only by amplification of proviral DNA. All cats inoculated with cloned virus FIV-pPPR seroconverted and were found virus positive by at least two of three virus detection assays. Both cloned viruses were less capable of suppressing CD4:CD8 ratios when compared to the biological isolates from which they were cloned. Isolates which replicate efficiently in feline peripheral blood mononuclear cells (PBMC), i.e., FIV-pPPR or biological FIV-PPR, caused greater virus load and lower CD4:CD8 ratios when compared to cloned FIV-pF34, which replicates efficiently in feline adherent cell lines and macrophages but poorly in primary feline PBMC. Molecular clones FIV-pF34 and FIV-pPPR will be useful reagents for characterization of viral determinants of virus load and possibly, cell tropism in vivo.

Sixteen adolescent specific pathogen free cats were inoculated with the Petaluma strain of feline immunodeficiency virus (FIV) and two cats were then necropsied at each of 5, 10, 21, 28, 42, 56, 70, and 84 day time points following infection. Lymphadenopathy gradually increased starting at Day 10 and persisted for the duration. Gross clinical signs of fever, mild to severe malaise, anorexia, diarrhea, dehydration, and generalized soreness appeared around Day 42, peaked at Day 56, and disappeared by Days 70-84 post-infection. Leukopenia, associated initially with a mild lymphopenia and later by both a mild lymphopenia and a severe neutropenia, appeared 14-28 days following infection, troughed at Day 56, and persisted thereafter. The CD4+:CD8+ T cell ratio started to decrease around Day 28, reaching a nadir at Days 56-70. This decrease was due to a decline in the absolute numbers and percentage of CD4+ T cells and an increase in the percentage of CD8+ T cells. Significant histopathologic lesions included myeloid hyperplasia between Days 56-70 post-infection; thymitis with cortical involution and follicular hyperplasia starting at Day 42; lymphoid hyperplasia of peripheral and mesenteric nodes, spleen and tonsils beginning around Day 42; typhlitis most evident from Day 56 onward, and an interstitial nephritis and pneumonitis that was most intense after Day 42. Virus was isolated from peripheral blood mononuclear cells (PBMC) beginning 2 weeks post-infection, and plasma viremia appeared 1 week later. Plasma and PBMC-associated viremia peaked at 42-56 days following infection and decreased abruptly thereafter. Proviral DNA was detectable as early as 5 days after infection in blood leukocytes and after 10 days in other organs. The central nervous system, lungs, thymus, tonsils and mesenteric lymph nodes were the earliest sites of virus localization. Antibodies to the FIV capsid protein appeared 14 days following infection and reached peak levels by Days 42-56. Abnormalities occurring during the primary stage of FIV infection were consistent with those described for acute simian and human immunodeficiency virus-induced disease.

Several novel fluorinated acyclic nucleoside phosphonates [i.e. 9-(3-fluoro-2-phosphonylmethoxy propyl)adenine (FPMPA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)-2,6-diaminopurine (FPMPDAP)] were evaluated for their inhibitory effect against feline immunodeficiency virus (FIV) replication in Crandell feline kidney (CrFK) cells and feline peripheral blood lymphocytes (PBL) in vitro. Whereas 3-azido-3-deoxythymidine (AZT) was not able to achieve complete suppression of viral antigen expression and reverse transcriptase activity in the FIV-infected cell culture supernatants at 25 μM, FPMPA, FPMPDAP, and the 9-(2-phosphonylmethoxyethyl)purine derivatives PMEA and PMEDAP fully protected FIV-infected cells at μM. Both FPMPA and FPMPDAP were endowed with a higher antiviral potency and/or therapeutic selectivity than PMEA and PMEDAP in inhibiting FIV infection, mainly due to a markedly lower toxicity for the cell cultures.

Measuring depression among persons with stroke faces many challenges; diagnostic tools are lengthy and do not measure the extent of depression; screening tools are not stroke-specific; and metrics from the available indices do not provide a value that is mathematically or clinically meaningful. Purpose To provide evidence for the development of a stroke specific Geriatric Depression Scale screening measure (SS-GDS) through Rasch methodology. Methods Secondary analyses of a randomized controlled trial post-stroke. Interviews from 91 subjects aged 71 (SD 10) over three time points or 240 interviews were analyzed. Rasch Analysis helped transform the 30-item GDS onto a logit scale. Unidimensionality, item fit, redundancy, and differential item functioning (DIF) were assessed. Results Seventeen items fit the model to form a hierarchical measure ranging in difficulty from +1.2 to −1.8 logits. Preliminary psychometric properties of reliability, validity, and responsiveness were adequate. Two items that demonstrated DIF, one for language and one for gender, were split. Conclusion The 17-item SS-GDS Rasch measure was developed to screen for post-stroke depression (PSD) and provide an important step toward quantifying PSD. If revalidated in a larger sample, the SS-GDS could provide a mathematically valid index to screen for depression in stroke survivors. Copyright © 2010 John Wiley & Sons, Ltd.

To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2-58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans.

Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4+ and CD8+ T-lymphocytes. A brief decrease in the CD 4+ CD 8+ T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8+ T-lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD 4+ CD 8+ T-lymphocyte ratios returned to baseline within 4–8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV-1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenic responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV-1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection.

Antigenic epitopes on the major core (gag) protein of isolates of simian and human immunodeficiency virus (SIV and HIV) were compared using a panel of eleven mouse monoclonal antibodies (Mabs) that recognized nine distinct gag epitopes. Viral isolates used for comparison were HIV-1IIIb, HIV-2ROD, and SIV isolates from macaque (SIVmac), sooty mangabey (SIVsm-UCD), African green monkey (SIVagm), and stump-tailed macaque (SIVstm-UCD). The relatedness of the various HIV and SIV isolates, as determined by Mabs to core protein epitopes, paralleled that ascertained by genetic sequencing.

The ability of two vaccine preparations (UV-psoralen inactivated SIV administered intramuscularly and live-attenuated SIV inoculated intravaginally) to prevent genital transmission of virulent SIV in rhesus macaques was tested. Two of six whole-inactivated SIV vaccinated macaques, three of five live-attenuated SIV vaccinated macaques, and four of six controls became persistently infected after two separate intravaginal inoculations with a 50% animal infectious dose of virulent SIV. No association was observed between levels of SIV-specific antibodies in serum or vaginal secretions prior to challenge and subsequent infection with virulent SIV.

Highly active antiretroviral therapy (HAART) enables long-term suppression of plasma HIV-1 loads in infected persons, but low-level virus persists and rebounds following cessation of therapy. During HAART, this virus resides in latently infected cells, such as resting CD4+T cells, and in other cell types that may support residual virus replication. Therapeutic eradication will require elimination of virus from all reservoirs. We report here a comprehensive analysis of these reservoirs in fluids, cells, and tissues in a rhesus macaque model that mimics HAART in HIV-infected humans. This nonhuman primate model uses RT-SHIV, a chimera of simian immunodeficiency virus containing the HIV-1 reverse transcriptase (RT). Methods were developed for extraction, preamplification, and real-time PCR analyses of viral DNA (vDNA) and viral RNA (vRNA) in tissues from RT-SHIV-infected macaques. These methods were used to identify viral reservoirs in RT-SHIV-infected macaques treated with a potent HAART regimen consisting of efavirenz, emtricitabine, and tenofovir. Plasma virus loads at necropsy ranged from 11 to 28 copies of vRNA per ml. Viral RNA and DNA were detected during HAART, in tissues from numerous anatomical locations. Additional analysis provided evidence for full-length viral RNA in tissues of animals with virus suppressed by HAART. The highest levels of vDNA and vRNA in HAART-treated macaques were in lymphoid tissues, particularly the spleen, lymph nodes, and gastrointestinal tract tissues. This study is the first comprehensive analysis of the tissue and organ distribution of a primate AIDS virus during HAART. These data demonstrate widespread persistence of residual virus in tissues during HAART.

An infectious, virulence-attenuated molecular clone of simian immunodeficiency virus (SIV), SIVMAC-1A11, was derived from an SIV isolate that causes fatal immunodeficiency in rhesus macaques. When inoculated intravenously in rhesus macaques, SIVMAC-1A11 induced transient viremia (1 to 6 weeks) without clinical disease and a persistent humoral antibody response. The antibodies were directed mainly against the viral envelope glycoproteins, as determined by immunoblots and virus neutralization. The potential of this virulence-attenuated virus to protect against intravenous challenge with a pathogenic SIVMAC strain was assessed. Five rhesus macaques were each given two intravenous inoculations with SIVMAC-1A11 7 months apart. Three of the five immunized monkeys and four naive control animals were then challenged with 100 to 1,000 100% animal infectious doses of pathogenic SIVMAC. All seven animals became persistently viremic following the challenge. Four of four unimmunized animals developed severe clinical signs of simian acquired immunodeficiency syndrome by 38 to 227 days after challenge and were euthanatized 91 to 260 days postchallenge. However, no signs of illness were seen in immunized monkeys until 267 to 304 days postchallenge, when two of three immunized animals developed mild thrombocytopenia and lymphopenia; one of these animals died with clinical signs of simian immunodeficiency disease at 445 days after challenge. The two SIVMAC-1A11-immunized monkeys that were not challenged were healthy and antibody positive 22 months after the initial immunization. Thus, although live SIVMAC-1A11 was immunogenic and did not induce any disease, it failed to protect rhesus macaques against infection with a moderately high dose of pathogenic virus. However, immunization prevented severe, early disease and prolonged the lives of monkeys subsequently infected with pathogenic SIV.

Objective: To assess the safety of nurses and paramedics offering telephone assessment, triage, and\ud advice as an alternative to immediate ambulance despatch for emergency ambulance service callers\ud classified by lay call takers as presenting with "non-serious" problems (category C calls).\ud Design: Data for this study were collected as part of a pragmatic randomised controlled trial reported\ud elsewhere. The intervention arm of the trial comprised nurse or paramedic telephone consultation using a\ud computerised decision support system to assess, triage, and advise patients whose calls to the emergency\ud ambulance service had been classified as "non-serious" by call takers applying standard priority despatch\ud criteria. A multidisciplinary expert clinical panel reviewed data from ambulance service, accident and\ud emergency department, hospital inpatient and general practice records, and call transcripts for patients\ud triaged by nurses and paramedics into categories that indicated that despatch of an emergency\ud ambulance was unnecessary. All cases for which one or more members of the panel rated that an\ud emergency ambulance should have been despatched were re-reviewed by the entire panel for an\ud assessment of the "life risk" that might have resulted.\ud Setting: Ambulance services in London and the West Midlands, UK.\ud Study population: Of 635 category C patients assessed by nurses and paramedics, 330 (52%) cases that\ud had been triaged as not requiring an emergency ambulance were identified.\ud Main outcome measures: Assessment of safety of triage decisions.\ud Results: Sufficient data were available from the routine clinical records of 239 (72%) subjects to allow\ud review by the specialist panel. For 231 (96.7%) sets of case notes reviewed, the majority of the panel\ud concurred with the nurses’ or paramedics’ triage decision. Following secondary review of the records of\ud the remaining eight patients, only two were rated by the majority as having required an emergency\ud ambulance within 14 minutes. For neither of these did a majority of the panel consider that the patient\ud would have been at "life risk" without an emergency ambulance being immediately despatched.\ud However, the transcripts of these two calls indicated that the correct triage decision had been\ud communicated to the patient, which suggests that the triage decision had been incorrectly entered into the\ud decision support system.\ud Conclusions: Telephone advice may be a safe method of managing many category C callers to 999\ud ambulance services. A clinical trial of the full implementation of this intervention is needed, large enough to\ud exclude the possibility of rare adverse events.

The methionine-to-valine mutation in codon 184 (M184V) in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) confers resistance to (−)-2′-deoxy-3′-thiacytidine (3TC; lamivudine) and increased sensitivity to 9-[2-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir). We have used the SIV model to evaluate the effect of the M184V mutation on the emergence of resistance to the combination of 3TC plus PMPA. A site-directed mutant of SIVmac239 containing M184V (SIVmac239-184V) was used to select for resistance to both 3TC and PMPA by serial passage in the presence of increasing concentrations of both drugs. Under these selection conditions, the M184V mutation reverted in the majority of the selections. Variants resistant to both drugs were found to have the lysine-to-arginine mutation at codon 65 (K65R), which has previously been associated with resistance to PMPA in both SIV and HIV. Similarly, in rhesus macaques infected with SIVmac239-184V for 46 weeks and treated daily with (−)-2′-deoxy-5-fluoro-3′-thiacytidine [(−)-FTC], there was no reversion of M184V, but this mutation reverted to 184 M in all three animals within 24 weeks of treatment with (−)-FTC and PMPA. Although the addition of PMPA to the (−)-FTC therapy induced a decrease in virus loads in plasma, these loads eventually returned to pre-PMPA levels in each case. All animals receiving this combination developed the K65R mutation. These results demonstrate that the combination of PMPA with 3TC or (−)-FTC selects for the K65R mutation and against the M184V mutation in SIV RT.

Drug-resistant mutants with a methionine-to-valine substitution at position 184 of reverse transcriptase (M184V) emerged within 5 weeks of initiation of therapy in four newborn macaques infected with simian immunodeficiency virus (SIVmac251) and treated with lamivudine (3TC) or emtricitabine [(−)-FTC] (two animals per drug). Thus, this animal model mimics the rapid emergence of M184V mutants of HIV-1 during 3TC therapy of human patients. One animal of each treatment group developed fatal immunodeficiency at 12 weeks of age, which is similar to the rapid disease course seen in most untreated SIVmac251-infected infant macaques. To further evaluate the effect of the M184V mutation on viral fitness and virulence, groups of juvenile macaques were inoculated with the molecular clone SIVmac239 with either the wild-type sequence (group A [n= 5]) or the M184V sequence (SIVmac239-184V; group B [n= 5] and group C [n= 2]). The two SIVmac239-184V-infected animals of group C did not receive any drug treatment, and in both animals the virus population reverted to predominantly wild type (184M) by 8 weeks after inoculation. The other five SIVmac239-184V-infected animals (group B) were treated with (−)-FTC to prevent reversion. Although virus levels 1 week after inoculation were lower in the SIVmac239-184V-infected macaques than in the SIVmac239-infected animals, no significant differences were observed from week 2 onwards. Two animals in each group developed AIDS and were euthanized, while all other animals were clinically stable at 46 weeks of infection. These data demonstrate that the M184V mutation in SIV conferred a slightly reduced fitness but did not affect disease outcome.

RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host.

The goal of this study was to identify a strain of feline immunodeficiency virus (FIV) that would be more virulent for adult cats than the prototype FIV-A p etaluma and, thereby, enhance the FIV infection model for HIV-1 related research. Diehl et al. reported that one clade C strain of FIV, FIV-C pg ammar, was more virulent than other known FIV isolates. Mortalities from 58 to 100% were reported for kittens 12 weeks of age and less following intravenous inoculation. A more variable and somewhat less virulent disease course was observed in neonatal to 8–10-week-old kittens infected orally, intravaginally or intrarectally with this same isolate ( Obert and Hoover, 2000 ). However, no studies have been done with FIV-C pg ammar in adult cats. Therefore, the virulence of FIV-C pg ammar for young adult cats was compared to that of FIV-A p etalulma, the original FIV isolate. One group of five cats were inoculated intraperitoneally with 470 TCID 50 of FIV-C pg ammar in the form of pooled plasma from acutely infected cats, while a second group was infected with plasma containing the 750 TCID50 of FIV-A p etaluma. The cats were observed for 20 weeks for gross signs of disease, hematologic abnormalities, time of antibody appearance, and plasma and peripheral blood mononuclear cell (PBMC) associated virus levels. Viral RNA and proviral DNA were measured by a real-time PCR, sensitive to 50 copies per milliliter. The only outward sign of disease was lymphadenopathy, which occurred at a similar time and intensity in both groups of cats. Cats infected with FIV-C pg ammar were more likely to be neutropenic and lymphopenic during the first 10–12 weeks of infection than cats infected with FIV-A p etaluma. Both groups of cats showed similar overall declines in absolute mean CD4 cell counts and identical concomitant increases in CD8 cells. CD4/CD8 cell ratios were also similar. Antibody, as measured by an ELISA against recombinant FIV-TM antigen, appeared in all cats by 4 weeks post-infection. The most significant differences were in plasma viral RNA and PBMC proviral DNA levels. Cats infected with FIV-C pg ammar had up to 100 times higher mean levels of viral RNA during the first few weeks of infection than cats infected with FIV-A p etaluma. This difference was also mirrored in levels of proviral DNA in PBMC, which were significantly higher in the FIV-C pg ammar infected cats. Plasma viral RNA and PBMC proviral DNA levels were virtually identical in both groups of cats at 20 weeks post-infection. However, proviral DNA in tissues such as thymus and popliteal lymph nodes was 10-fold or so higher in FIV-C pg ammar infected cats at 20 weeks and histopathologic lesions were more severe. Based on these various parameters, we concluded that FIV-C pg ammar was more virulent than FIV-A p etaluma in young adult cats during the 20-week study period. However, we were not able to recreate the severe and rapidly progressive disease previously reported for kittens, suggesting an age-related resistance similar to that observed previously for FIV-A p etaluma ( George et al., 1993 ).

Specific pathogen-free cats were inoculated subcutaneously into the drainage areas of the left auricular and popliteal lymph nodes with living Yersinia pseudotuberculosis. Inflammation was evident at the inoculation sites and the regional lymph nodes were palpably enlarged at 48 h post-infection. Lymph node enlargement was due to marked paracortical lymphoid hyperplasia and variable neutrophil infiltrates. Yersinia was cultured from the regional lymph nodes and/or spleens of three of the six cats, indicating systemic spread of bacteria. Specific T-helper 1 and 2 (Th1, Th2) cell-associated cytokine mRNA levels were compared in regional lymph nodes, peripheral blood mononuclear cells (PBMC) and spleen at 48 h post-inoculation. Relative to unstimulated control tissues, there was a significant increase in TNF-alpha, IFN-gamma, IL-12, and IL-10 mRNAs in spleen with down-regulation of IL-4. Significant up-regulation of TNF-alpha and down-regulation of IL-4 were also observed in PBMC. Paradoxically, 48 h stimulated lymph nodes showed only minimal differences in cytokine mRNA expression when compared to lymph nodes from mock-inoculated control animals or unchallenged contralateral lymph nodes from the same animal. This study demonstrated that cats, like mice, respond to an intracellular pathogen such as Y pseudotuberculosis with a predominantly Th1-type immune response. The cytokine responses in regional lymph nodes and spleen were asynchronous, while cytokine stimulation in cells of the spleen was mirrored by PBMC.

Acute feline calicivirus (FCV) infection caused a more severe disease in chronically feline immunodeficiency virus (FIV) infected than in non-FIV infected cats. FIV infected cats shed significantly higher amounts of FCV through their saliva after FCV challenge than the non-FIV infected cats. However, there was no difference in the duration of FCV shedding. None of the cats exposed to FCV developed chronic FCV carriage. Both groups of cats mounted similar titers of neutralizing antibodies to FCV. Although FIV infected cats started out with significantly lower total lymphocyte and neutrophil numbers than the non-FIV infected cats, the transient lymphopenia and neutrophilia attributable to the FCV infection was of similar intensity in both groups of animals. There was no evidence that the underlying FIV-related disease or viremia was influenced by acute FCV infection. Acute FCV infection did not significantly alter the CD 4+ CD 8+ T lymphocyte ratio in FIV infected compared to non-FIV infected cats. The ongoing humoral IgG response to FIV was not affected by the FCV infection. There was no significant change in the proportion of FIV infected peripheral blood mononuclear cells during 8 subsequent weeks after FCV challenge as determined by polymerase chain reaction.

The coding sequences of the core proteins p17 and p28 of caprine arthritis-encephalitis virus (CAEV) were amplified using the polymerase chain reaction and cloned into the plasmid expression vector p-GEX-2T. Both p17 and p28 were expressed as fusion proteins with glutathione S-transferase. The recombinant proteins were affinity purified from induced bacterial lysates using glutathione-agarose beads. The purified proteins were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against CAEV in goat sera and milk samples. Three different ELISA tests were developed based on p17, p28 or the combination of these two recombinant proteins (p17 + p28). A comparison was made to an ELISA based on purified whole virus particles and to agar immunodiffusion test (AGID). Sera with conflicting results in the different ELISA tests were examined by Western blotting. There was a high correlation between the ELISA tests based on p17 + p28 recombinant proteins and whole virus ELISA, with an estimated kappa value of 0.92. Only 72-75% of the sera that tested positive in these two ELISA tests were positive in AGID. Antibodies to CAEV were detected in significantly more animals when serum samples were tested compared to milk samples. Based on the time and materials required to prepare the reagents, the recombinant based ELISA test was less expensive than the whole virus ELISA.

Neonatal, young adult, and aged specific pathogen-free cats were experimentally infected with cat-passaged Petaluma strain of feline immunodeficiency virus. The primary stage of illness occurred 6-8 weeks following infection in cats of all ages, but it differed in severity and clinical signs. Generalized lymphadenopathy persisted for the entire 42-week study period in neonatally infected cats, was transient in young adults, but inapparent in aged cats. Only two aged cats became chronically and severely ill during the study. One aged cat died with severe necrotizing transmural enteritis, while a second developed chronic generalized staphylococcal pyoderma that was partially controlled with antibiotics. Neutropenia appeared 6-8 weeks following infection in cats of all ages, but was more severe in newborn and aged cats than in young adults. A persistent decrease in CD4+/CD8+ T lymphocyte ratios, due to both increased CD8+ and decreased CD4+ T lymphocytes, occurred in the neonatal and aged cats. Decreased CD4+/CD8+ T lymphocyte ratios in the young adult cats was due solely to decreased CD4+ T lymphocytes. Antibody response to FIV virus, as measured by ELISA to recombinant FIV p24 antigen, was lower in aged cats than the other age groups during the first 6 weeks after infection. Antibody levels were not significantly different among the three age groups thereafter. Although there are some differences between FIV infection of cats and HIV infection of human beings, age at infection influences the severity of disease in both species.

Assays that use rhesus macaque whole blood and an antigen capture enzyme-linked immunosorbent assay for the simian immunodeficiency virus (SIV) p27 core protein were developed for the isolation of SIV from the blood of infected animals, the titration of infectivity of SIV inocula, and the quantitation of virus neutralizing antibodies in serum. These assays required small amounts of whole blood, were adaptable to a microtiter format, and used substrates mainly of rhesus macaque origin.

Eight rhesus macaques were immunized four times over a period of 8 months with a psoralen-UV-light-inactivated whole simian immunodeficiency virus vaccine adjuvanted with threonyl muramyl dipeptide. Eight unvaccinated control animals received adjuvant alone. Only the vaccinated animals made antibodies before challenge exposure to the viral core and envelope as determined by Western blotting (immunoblotting) and virus-neutralizing antibodies. Ten days after the final immunization, one-half of the vaccinated and nonvaccinated monkeys were challenged exposed intravenously (i.v.) and one-half were challenge exposed via the genital mucosa with virulent simian immunodeficiency virus. All of the nonvaccinated control monkeys became persistently infected. In spite of preexisting neutralizing antibodies and an anamnestic antibody response, all of the immunized monkeys also became persistently infected. However, there was evidence that the clinical course in immunized i.v. infected animals was delayed. All four mock-vaccinated i.v. challenge-exposed animals died with disease from 3 to 9 months postchallenge. In contrast, only one of four vaccinated i.v. challenge-exposed monkeys had died by 11 months postchallenge.

Sera from 526 old-world primates representing 50 different species were screened by ELISA for antibodies to human T-lymphotropic viruses I and III, and simian retrovirus type 1 (SRV-1). About one-fourth of the sera were positive by ELISA. There was a tendency, however, for the same sera to be positive for all three human and simian retroviruses. Only about one in five of the ELISA antibody-positive sera were confirmed to be positive by Western blotting. False-positive ELISA antibody tests were particularly common among sera from mandrills, crab-eating macaques, lion-tailed macaques, African green monkeys, and DeBrazza's and moustached guenons. Sera that were falsely positive in ELISA antibody tests to the three human and simian retroviruses were found to contain antibodies that reacted at comparable intensity with feline leukemia, infectious peritonitis and panleukopenia viruses. The false anti-viral activity of these sera was found to be due to antibodies that reacted with non-viral proteins that were copurified with all five virus preparations. These proteins were present in normal cat and human cells used to grow the various viruses and in gelatin. The implications of nonspecific cell-protein antibodies in primate sera were discussed in the light of this and previous seroepidemiologic studies of man and old-world monkeys.

Antisera from cats naturally exposed and resistant to feline leukemia virus lysed feline lymphoma cells in vitro with cat complement. Lymphoma cell lysis was detected by 51Cr release and by viable cell counts with phase-contrast microscopy. The rate of target cell lysis varied according to the nature of the antiserum and the origin of the complement used. Cat alloantisera, raised by immunization with target cells, lysed lymphoma cells rapidly with cat complement, but immune sera from cats naturally infected with feline leukemia virus caused slow lysis requiring up to 20 hr to reach maximum effect. Slow lysis of lymphoma cells in homologous serum mixtures was complement dependent, specific for leukemia virus-infected cells, and was found to occur in cat-immune whole serum. When 143 cat sera were examined for presence or absence of lytic antibody by 51Cr release and for antibody to feline oncornavirus-associated cell membrane antigen by immunofluorescence, the qualitative correlation obtained varied according to the complement used in the 51Cr release assay. With heterologous absorbed complement sources the correlation was 79% with guinea pig and 65% with rabbit; with homologous complement the correlation was 92%. The close association seen between high antibody levels and tumor resistance in this outbred species under natural conditions serves to emphasize the potential significance of these observations.

Monoclonal antibodies can be used in sandwich enzyme-linked immunosorbent assays to measure viral antigens. Such an assay was developed to detect the core protein, p24, of human T-cell lymphotropic virus type III and lymphadenopathy-associated virus, etiologic agents of the acquired immunodeficiency syndrome (AIDS). Another AIDS-associated virus, AIDS-associated retrovirus type 2 (ARV-2) could not be detected in this assay because of the low affinity of one of the monoclonal antibodies to ARV-2 p24. Detection of ARV-2 was accomplished with a monoclonal antibody-rabbit polyclonal antibody sandwich enzyme-linked immunosorbent assay. These two assays were used to efficiently detect AIDS-related viruses in lymphocyte cell cultures and to distinguish strains of the viruses.

The human immunodeficiency virus (HIV) was isolated from the blood of 192 of 410 seropositive individuals. Original isolations were made in peripheral blood mononuclear cell (PBMC) cultures, and only one-fifth of the HIV isolates could be adapted to replicate in continuous T-cell lines. Of the 192 HIV isolates, 42 had the characteristic p24 antigen marker of the acquired immunodeficiency syndrome-associated retrovirus type 2 strain of HIV (HIV ARV-2) and 150 resembled the human T-cell lymphotropic virus type III strain of HIV (HIV HTLV-III). Significantly, primary PBMC cultures from two patients yielded multiple variants. When these variants were exposed to continuous T-cell lines, only one of them continued to replicate. The remaining variants were lost and could not be reisolated following passage back into PBMC cultures. We conclude the following from these studies: PBMC cultures are more efficient at isolating HIV than continuous T-cell lines are; some patients harbor more than one genetic variant of HIV in the blood at the same time; and continuous T-cell lines are likely to yield only a portion of the HIV variants originally present in the blood.

By combining the high resolution of sodium dodecylsulfate polyacrylamide gel electrophoresis with the sensitivity of enzyme-linked immunosorbent assay (ELISA) antibodies specific for different feline leukemia virus components are characterized. Based on the same principle, Concanavalin A binding sites of FeLV components are also detected.