A series of 23 Amaryllidaceae isoquinoline alkaloids and related synthetic analogues were isolated or synthesized and subsequently evaluated in cell culture against the RNA-containing flaviviruses (Japanese encephalitis, yellow fever, and dengue viruses), bunyaviruses (Punta Toro, sandfly fever, and Rift Valley fever viruses), alphavirus (Venezuelan equine encephalomyelitis virus), lentivirus (human immunodeficiency virus-type 1) and the DNA-containing vaccinia virus. Narciclasine [1], lycoricidine [2], pancratistatin [4], 7-deoxypancratistatin [5], and acetates 6-8, isonarciclasine [13a], cis-dihydronarciclasine [14a], trans-dihydronarciclasine [15a], their 7-deoxy analogues 13b-15b, lycorines 16 and 17, and pretazettine [18] exhibited consistent in vitro activity against all three flaviviruses and against the bunyaviruses, Punta Toro and Rift Valley fever virus. Activity against sandfly fever virus was only observed with 7-deoxy analogues. In most cases, however, selectivity of the active compounds was low, with toxicity in uninfected cells (TC50) occurring at concentrations within 10-fold that of the viral inhibitory concentrations (IC50). No activity was observed against human immunodeficiency virus-type 1, Venezuelan equine encephalomyelitis virus, or vaccinia viruses. Pancratistatin [4] and its 7-deoxy analogue 5 were evaluated in two murine Japanese encephalitis mouse models (differing in viral dose challenge, among other factors). In two experiments (low LD50 viral challenge, variant I), prophylactic administration of 4 at 4 and 6 mg/kg/day (2% EtOH/saline, sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 100% and 90%, respectively. In the same model, prophylactic administration of 5 at 40 mg/kg/day in hydroxypropylcellulose (sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 80%. In a second variant (high LD50 viral challenge), administration of 4 at 6 mg/kg/day (ip, twice daily for 9 days, day -1 to +7) resulted in a 50% survival rate. In all cases, there was no survival in the diluent-treated control mice. Thus, 4 and 5 demonstrated activity in mice infected with Japanese encephalitis virus but only at near toxic concentrations. To our knowledge, however, this represents a rare demonstration of chemotherapeutic efficacy (by a substance other than an interferon inducer) in a Japanese-encephalitis-virus-infected mouse model.

We measured levels of antibodies to Japanese encephalitis virus (JEV) in serum and in cerebrospinal fluid (CSF) specimens obtained from 32 patients with acute encephalitis by using "antibody-capture" solid-phase enzyme-linked immunoassays specific for IgM or IgG to JEV. The proportions of confirmed cases with IgM to JEV detectable in CSF were 68% (obtained on day 1), 100% (day 7), 96% (day 30), and 72% (day 180). For IgG in CSF the proportions were 47% (day 1), 89% (day 7), 100% (day 30), and 100% (day 180). Twenty-five CSF samples were obtained from control patients with other diseases with possible nervous system involvement (but none with a clinical diagnosis of viral encephalitis); none had detectable IgM to JEV. Five JEV-infected but asymptomatic siblings of patients with encephalitis were also examined; all had high levels of IgM to JEV in serum, but none had detectable IgM to JEV in CSF.

Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at ؊20°C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at ؊20°C or at ؊70°C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that ؊20°C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.The introduction of highly active antiretroviral therapy and the demonstration of dramatic improvements in human immunodeficiency virus (HIV)-and AIDS-related mortality and morbidity in North America and Europe have fueled international efforts to expand access to care and treatment in lessdeveloped countries. Several major initiatives to provide treatment in resource-limited settings, including the U.S. President's Emergency Plan for AIDS Relief and the Global Fund against AIDS, TB and Malaria, are now in progress (16). The implementation of these programs requires the development of appropriate and effective patient-monitoring systems, including surveillance for antiretroviral drug resistance. Sentinel drug resistance surveillance systems are important public health tools that can provide information on trends in the prevalence of resistance at the population level and can be used to modify treatment guidelines.Plasma and serum are considered the preferred specimen types for HIV type 1 (HIV-1) drug resistance testing. However, these types of specimens are not optimal in resource-limited settings where the equipment necessary for PCR amplification and sequencing may not be available at collection sites and resistance testing requires transportation of the samples to a refer...

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