Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by similar to100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an similar to5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type lie residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 mug of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN,viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

Field evaluation of a sentinel mosquito (Diptera : culicidae) trap system to detect Japanese encephalitis in remote Australia

Incursions of Japanese encephalitis (JE) virus into northern Queensland are currently monitored using sentinel pigs. However, the maintenance of these pigs is expensive, and because pigs are the major amplifying hosts of the virus, they may contribute to JE transmission. Therefore, we evaluated a mosquito-based detection system to potentially replace the sentinel pigs. Single, inactivated JE-infected Culex annulirostris Skuse and C. sitiens Wiedemann were placed into pools of uninfected mosquitoes that were housed in a Mosquito Magnet Pro (MM) trap set under wet season field conditions in Cairns, Queensland for 0, 7, or 14 d. JE viral RNA was detected (cycling threshold [CT] = 40) in 11/ 12, 10/14, and 2/5 pools containing 200, 1,000, and 5,000 mosquitoes, respectively, using a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability to detect virus was not affected by the length of time pools were maintained under field conditions, although the CT score tended to increase with field exposure time. Furthermore, JE viral RNA was detected in three pools of 1,000 mosquitoes collected from Badu Island using a MM trap. These results indicated that a mosquito trap system employing self-powered traps, such as the MosquitoMagnet, and a real-time PCR system, could be used to monitor for JE in remote areas.

HIV/tuberculosis co-infection: a request for a better surveillance

The increasing endemicity of tuberculosis resulting from causes such as immigration, poverty, a declining public health infrastructure and co-infection by HIV/Mycobacterium tuberculosis, is leading to a change in tuberculosis control programmes. One of the main reasons for the resurgence of tuberculosis is HIV infection - the risk of tuberculosis is greater in HIV patients than in the majority of the population as can be seen from numerous research projects. The need for systematic testing for HIV infection in all tuberculosis patients by undertaking confidential HIV tests on admission to a tuberculosis programme is brought out. This measure would increase the number of cases diagnosed and provide data for better surveillance of the co-infection.

Epidemiology of malaria, schistosomiasis, geohelminths, anemia and malnutrition in the context of a demographic surveillance system in Northern Angola

Background: Malaria, schistosomiasis and geohelminth infection are linked to maternal and child morbidity and mortality in sub-Saharan Africa. Knowing the prevalence levels of these infections is vital to guide governments towards the implementation of successful and cost-effective disease control initiatives. Methodology/Principal Findings: A cross-sectional study of 1,237 preschool children (0–5 year olds), 1,142 school-aged children (6–15 year olds) and 960 women (.15 year olds) was conducted to understand the distribution of malnutrition, anemia, malaria, schistosomiasis (intestinal and urinary) and geohelminths in a north-western province of Angola. We used a recent demographic surveillance system (DSS) database to select and recruit suitable households. Malnutrition was common among children (23.3% under-weight, 9.9% wasting and 32.2% stunting), and anemia was found to be a severe public health problem (i.e., .40%). Malaria prevalence was highest among preschool children reaching 20.2%. Microhematuria prevalence levels reached 10.0% of preschool children, 16.6% of school-aged children and 21.7% of mothers. Geohelminth infections were common, affecting 22.3% of preschool children, 31.6% of school-aged children and 28.0% of mothers. Conclusions: Here we report prevalence levels of malaria, schistosomiasis and geohelminths; all endemic in this poorly described area where a DSS has been recently established. Furthermore we found evidence that the studied infections are associated with the observed levels of anemia and malnutrition, which can justify the implementation of integrated interventions for the control of these diseases and morbidities.

The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturation

Abstract - Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules. Methodology/Principal findings: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line. Conclusions/Significance: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

Performance of rubella suspect case definition: implications for surveillance

OBJECTIVE: To assess the performance of the rubella suspect case definition among patients with rash diseases seen at primary care units. METHODS: From January 1994 to December 2002, patients with acute rash, with or without fever, were seen at two large primary health care units and at a public general hospital in the municipality of Niterói, metropolitan area of Rio de Janeiro, Brazil. Data from clinical and serologic assessment were used to estimate the positive predictive values of the definition of rubella suspect case from the Brazilian Ministry of Health and other combination of signs/symptoms taking serologic status as the reference. Serum samples were tested for anti-rubella virus IgM using commercially available enzyme immunoassays. Positive predictive values and respective 95% confidence intervals were calculated. RESULTS: A total of 1,186 patients with an illness characterized by variable combinations of rash with fever, arthropathy and lymphadenopathy were studied. Patients with rash, regardless of other signs and symptoms, had 8.8% likelihood of being IgM-positive for rubella. The Brazilian suspect case definition (fever and lymphadenopathy in addition to rash) had low predictive value (13.5%). This case definition would correctly identify 42.3% of the IgM-positive cases, and misclassify 26.1% of the IgM-negative cases. CONCLUSIONS: These results support the recommendation to investigate and collect clinical specimens for laboratory diagnosis of all cases of rash, for surveillance purposes. Although this strategy may increase costs, the benefits of interrupting the circulation of rubella virus and preventing the occurrence of congenital rubella syndrome should pay off.

Surveillance of mother-to-child HIV transmission: socioeconomic and health care coverage indicators

OBJECTIVE: To identify clustering areas of infants exposed to HIV during pregnancy and their association with indicators of primary care coverage and socioeconomic condition. METHODS: Ecological study where the unit of analysis was primary care coverage areas in the city of Porto Alegre, Southern Brazil, in 2003. Geographical Information System and spatial analysis tools were used to describe indicators of primary care coverage areas and socioeconomic condition, and estimate the prevalence of liveborn infants exposed to HIV during pregnancy and delivery. Data was obtained from Brazilian national databases. The association between different indicators was assessed using Spearman's nonparametric test. RESULTS: There was found an association between HIV infection and high birth rates (r=0.22, p<0.01) and lack of prenatal care (r=0.15, p<0.05). The highest HIV infection rates were seen in areas with poor socioeconomic conditions and difficult access to health services (r=0.28, p<0.01). The association found between higher rate of prenatal care among HIV-infected women and adequate immunization coverage (r=0.35, p<0.01) indicates that early detection of HIV infection is effective in those areas with better primary care services. CONCLUSIONS: Urban poverty is a strong determinant of mother-to-child HIV transmission but this trend can be fought with health surveillance at the primary care level.

Digital disease detection and participatory surveillance: overview and perspectives for Brazil

ABSTRACT This study aimed to describe the digital disease detection and participatory surveillance in different countries. The systems or platforms consolidated in the scientific field were analyzed by describing the strategy, type of data source, main objectives, and manner of interaction with users. Eleven systems or platforms, developed from 1996 to 2016, were analyzed. There was a higher frequency of data mining on the web and active crowdsourcing as well as a trend in the use of mobile applications. It is important to provoke debate in the academia and health services for the evolution of methods and insights into participatory surveillance in the digital age.

People counting system using existing surveillance video camera

The Casa da Música Foundation, responsible for the management of Casa da Música do Porto building, has the need to obtain statistical data related to the number of building’s visitors. This information is a valuable tool for the elaboration of periodical reports concerning the success of this cultural institution. For this reason it was necessary to develop a system capable of returning the number of visitors for a requested period of time. This represents a complex task due to the building’s unique architectural design, characterized by very large doors and halls, and the sudden large number of people that pass through them in moments preceding and proceeding the different activities occurring in the building. To achieve the technical solution for this challenge, several image processing methods, for people detection with still cameras, were first studied. The next step was the development of a real time algorithm, using OpenCV libraries and computer vision concepts,to count individuals with the desired accuracy. This algorithm includes the scientific and technical knowledge acquired in the study of the previous methods. The themes developed in this thesis comprise the fields of background maintenance, shadow and highlight detection, and blob detection and tracking. A graphical interface was also built, to help on the development, test and tunning of the proposed system, as a complement to the work. Furthermore, tests to the system were also performed, to certify the proposed techniques against a set of limited circumstances. The results obtained revealed that the algorithm was successfully applied to count the number of people in complex environments with reliable accuracy.

Surveillance technology and strategy for management of Portugal's EEZ

EEZ International, Summer 2003, p. 43-47

Surveillance of arbovirus infections in the atlantic forest region, State of São Paulo, Brazil: I. detection of hemagglutination-inhibition antibodies in wild birds between 1978 and 1990

We report data related to arbovirus antibodies detected in wild birds periodically captured from January 1978 to December 1990 in the counties of Salesópolis (Casa Grande Station), Itapetininga and Ribeira Valley, considering the different capture environments. Plasmas were examined using hemagglutination-inhibition (HI) tests. Only monotypic reactions were considered, except for two heterotypic reactions in which a significant difference in titer was observed for a determined virus of the same antigenic group. Among a total of 39,911 birds, 269 birds (0.7%) belonging to 66 species and 22 families were found to have a monotypic reaction for Eastern equine encephalitis (EEE), Venezuelan equine encephalitis (VEE), Western equine encephalitis (WEE), Ilheus (ILH), Rocio (ROC), St. Louis encephalitis (SLE), SP An 71686, or Caraparu (CAR) viruses. Analysis of the data provided information of epidemiologic interest with respect to these agents. Birds with positive serology were distributed among different habitats, with a predominance of unforested habitats. The greatest diversity of positive reactions was observed among species which concentrate in culture fields.

Plague surveillance in Brazil: 1983 - 1992

Plague caused by Yersinia pestis, has persisted in Brazil in several natural foci spread throughout rural areas in the States of Ceara, Paraiba, Pernambuco, Piaui, Rio Grande do Norte, Alagoas, Bahia, Minas Gerais and Rio de Janeiro. Nationwide surveillance of plague in Brazil based on serological testing started in 1983. We now present an update report of the examinations carried out in our laboratory from 1983 to 1992. The passive hemagglutination test for antibodies against fraction 1A antigen of Y. pestis and the passive hemagglutination inhibition control were employed for testing a total of 220,769 sera. Samples analyzed included 2,856 sera from clinically diagnosed plague cases or suspects, 49,848 sera from rodents of 24 species and 2 species of small wild carnivores (marsupials), 122,890 sera from dogs, and 45,175 sera from cats. Specific antibodies were found in 92 (3.22%) human sera; 143 (0.29%) sera from rodents of 8 species and from the two species of marsupials, 1,105 (0.90%) sera from dogs and 290 (0.64%) sera from cats. The presence of significant levels of specific anti-F1A antibodies among rodents and wild or domestic carnivores (dogs and cats) indicates that all the Brazilian plague foci remain active in spite of the absence of human cases in some of them.

Sigla WEBGIS portal: an open source Web-based GIS application for the portuguese bluetongue Entomologiacal surveillance program.

Dissertação apresentada como requisito parcial para obtenção do grau de Mestre em Ciência e Sistemas de Informação Geográfica

A new model for EEZ surveillance and management in Portugal

Management Information Systems 2000, p. 103-111