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Twenty years have passed since the emergence of hantavirus zoonosis in Panama at the beginning of this millennium. We provide an overview of epidemiological surveillance of hantavirus disease (hantavirus pulmonary syndrome and hantavirus fever) during the period 1999-2019 by including all reported and confirmed cases according to the case definition established by the health authority. Our findings reveal that hantavirus disease is a low-frequency disease, affecting primarily young people, with a relatively low case-fatality rate compared to other hantaviruses in the Americas (e.g., ANDV and SNV). It presents an annual variation with peaks every 4-5 years and an interannual variation influenced by agricultural activities. Hantavirus disease is endemic in about 27% of Panama, which corresponds to agroecological conditions that favor the population dynamics of the rodent host, Oligoryzomys costaricensis and the virus ( Choclo orthohantavirus ) responsible for hantavirus disease. However, this does not rule out the existence of other endemic areas to be characterized. Undoubtedly, decentralization of the laboratory test and dissemination of evidence-based surveillance guidelines and regulations have standardized and improved diagnosis, notification at the level of the primary care system, and management in intensive care units nationwide.

RNA structures can form functional elements that play crucial roles in the replication of positive-sense RNA viruses. While RNA structures in the untranslated regions (UTRs) of several picornaviruses have been functionally characterized, the roles of putative RNA structures predicted for protein coding sequences (or open reading frames [ORFs]) remain largely undefined. Here, we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome to predict 53 conserved RNA structures within the ORF. Forty-six of these structures were located in the regions encoding the nonstructural proteins (nsps). To investigate whether structures located in the regions encoding the nsps are required for FMDV replication, we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimize RNA secondary structures while preserving protein coding, native dinucleotide frequencies, and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV subgenomic replicon. We found that three of the RNA structures, all at the 3' termini of the FMDV ORF, were critical for replicon replication. In contrast, disruption of the other 43 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains. IMPORTANCE Some RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 46 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the nonstructural proteins, only three are essential for replication of an FMDV subgenomic replicon. Replicon replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify whether the remaining 43 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease, posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part by the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for virus replication as well as locations that can tolerate small genetic insertions. Such insertion sites can subsequently be adapted by the incorporation of commonly used epitope tags, facilitating their detection with commercially available reagents. In this study, we used random transposon-mediated mutagenesis to produce a library of 15 nt insertions in the FMDV nonstructural polyprotein. Using a replicon-based assay, we isolated multiple replication-competent as well as replication-defective insertions. We adapted the replication-competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins for use in a variety of downstream assays. Additionally, we showed that replication of some of the replication-defective insertion mutants could be rescued by co-transfection of a ‘helper’ replicon, demonstrating a novel use of random mutagenesis to identify intergenomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornavirus replication complexes.

Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.

The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Importance: At least 13 medication-associated diethylene glycol (DEG) mass poisonings have occurred since 1937. To our knowledge, this is the first longitudinal study characterizing long-term health outcomes among survivors beyond the acute poisoning period., Objective: To characterize renal and neurologic outcomes among survivors of a 2006 DEG mass-poisoning event in Panama for 2 years after exposure., Design, Setting, and Participants: This prospective longitudinal study used descriptive statistics and mixed-effects repeated-measures analysis to evaluate DEG-poisoned survivors at 4 consecutive 6-month intervals (0, 6, 12, and 18 months). Case patients included outbreak survivors with a history of (1) ingestion of DEG-contaminated medication, (2) hospitalization for DEG poisoning, and (3) an unexplained serum creatinine level of 1.5 mg/dL or higher (to convert to micromoles per liter, multiply by 88.4) during acute illness or unexplained exacerbation of preexisting end-stage renal disease., Main Outcomes and Measures: Demographics, mortality, dialysis dependence, renal function, neurologic signs and symptoms, and nerve conduction studies., Results: Of the 32 patients enrolled, 5 (15.6%) died and 1 was lost to follow-up, leaving 26 patients at 18 months. Three (9.4%) missed 1 or more evaluations. The median age was 62 years (range, 15-88 years), and 59.4% were female. Three (9.4%) patients had preexisting renal failure. Enrollment evaluations occurred at a median of 108 days (range, 65-154 days) after acute illness. The median serum creatinine level for the 22 patients who were not dialysis dependent at time 0 was 5.9 mg/dL (range, 1.8-17.1 mg/dL) during acute illness and 1.8 mg/dL (range, 0.9-5.9 mg/dL) at time 0. Among non-dialysis-dependent patients, there were no significant differences in the log of serum creatinine or estimated glomerular filtration rate over time. The number of patients with subjective generalized weakness declined significantly over time (P < .001). A similar finding was observed for any sensory loss (P = .05). The most common deficits at enrollment were bilateral lower extremity numbness in 13 patients (40.6%) and peripheral facial nerve motor deficits in 7 (21.9%). All patients with neurologic deficits at enrollment demonstrated improvement in motor function over time. Among 28 patients (90.3%) with abnormal nerve conduction study findings at enrollment, 10 (35.7%) had motor axonal involvement, the most common primary abnormality., Conclusions and Relevance: Neurologic findings of survivors tended to improve over time. Renal function generally improved among non-dialysis-dependent patients between acute illness and the first evaluation with little variability thereafter. No evidence of delayed-onset neurologic or renal disease was observed.

Background: Urinary biomarkers for bladder cancer detection are constrained by inadequate sensitivity or specificity. Here we evaluate the diagnostic accuracy of Mcm5, a novel cell cycle biomarker of aberrant growth, alone and in combination with NMP22., Methods: 1677 consecutive patients under investigation for urinary tract malignancy were recruited to a prospective blinded observational study. All patients underwent ultrasound, intravenous urography, cystoscopy, urine culture and cytologic analysis. An immunofluorometric assay was used to measure Mcm5 levels in urine cell sediments. NMP22 urinary levels were determined with the FDA-approved NMP22® Test Kit., Results: Genito-urinary tract cancers were identified in 210/1564 (13%) patients with an Mcm5 result and in 195/1396 (14%) patients with an NMP22 result. At the assay cut-point where sensitivity and specificity were equal, the Mcm5 test detected primary and recurrent bladder cancers with 69% sensitivity (95% confidence interval = 62-75%) and 93% negative predictive value (95% CI = 92-95%). The area under the receiver operating characteristic curve for Mcm5 was 0.75 (95% CI = 0.71-0.79) and 0.72 (95% CI = 0.67-0.77) for NMP22. Importantly, Mcm5 combined with NMP22 identified 95% (79/83; 95% CI = 88-99%) of potentially life threatening diagnoses (i.e. grade 3 or carcinoma in situ or stage ≥pT1) with high specificity (72%, 95% CI = 69-74%)., Conclusions: The Mcm5 immunoassay is a non-invasive test for identifying patients with urothelial cancers with similar accuracy to the FDA-approved NMP22 ELISA Test Kit. The combination of Mcm5 plus NMP22 improves the detection of UCC and identifies 95% of clinically significant disease. Trials of a commercially developed Mcm5 assay suitable for an end-user laboratory alongside NMP22 are required to assess their potential clinical utility in improving diagnostic and surveillance care pathways.

Introduction: In April 2009, a novel influenza A (H1N1) virus was identified in patients from Mexico and the United States. From 8 May through 25 June 2009, in the Republic of Panama, 467 cases infected with the same virus were identified, 13 of which were hospitalized at the Santo Tomas Hospital in Panama City. Up to the date of this report, no deaths have been reported in Panama. This study presents the first thirteen cases of Influenza A (H1N1) 2009 that were hospitalized in Panama City., Methodology: The Santo Tomas Hospital (HST), a third-level institution of the Ministry of Health (MINSA) for adult health care (patients above the age of 14), was designated as the reference center for treating these cases. For this purpose, the norms and criteria established by the system were followed and every patient (case) presenting flu-like symptoms was included (fever equal or greater than 38 masculineC (100.4 masculineF), cough, sore throat, rhinorrhea, lethargy in children under the age of one, and respiratory distress)., Results: Seventy-six patients were hospitalized as suspected cases for infection with the influenza A H1N1 2009 virus, of which 13 (17.1%) were confirmed as positive. The clinical picture was characterized by fever (100%), cough (92.3%), rhinorrhea (69.2%), malaise (53.8%), headache (53.8%), and only one case presented gastrointestinal symptoms (diarrhoea). The male:female ratio was 1:2.2., Conclusion: The knowledge and technology translation previously acquired through courses to the HST health care providers were the key in controlling the first influenza A (H1N1) 2009 cases.