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Pteropine orthoreovirus (PRV), potentially of bat origin, is reported to be a causative agent of emerging respiratory tract infections among humans in Southeast Asia. We evaluated the efficacy of serologic assays using the major outer capsid and cell attachment proteins (CAP) of PRV strains in the screening, confirmation and identification of three groups of human PRV infections; Indonesian/Japanese, Indonesian/Hong Kong and Malaysian strains. The different serologic assays were tested using rabbit polyclonal antisera raised against these proteins of selected PRV strains, and validation was carried out using sera from a Miyazaki-Bali/2007 PRV-infected patient and the patient's contacts. The results of this study showed that rabbit polyclonal antisera raised against the CAP of the Miyazaki-Bali/2007 PRV strain showed the highest reactivity to the Miyazaki-Bali/2007 PRV and to a lesser extent, cross-reactivity with the HK23629/07 and Melaka PRVs, respectively. Neutralization activity against the Miyazaki-Bali/2007 PRV was observed using rabbit anti-Miyazaki-Bali/2007 PRV CAP (320) but not with rabbit anti-HK23629/07 (

Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

After outbreak of severe acute respiratory syndrome (SARS) in 2003, many international airport quarantines adopted fever-based screening to identify infected individuals using infrared thermography to control global pandemic. Unfortunately, the sensitivity of fever-based screening system did not exceed 70.4% at Narita International Airport. In order to achieve accurate onboard entry screening for highly contagious infectious diseases, the authors developed a portable system designed for onboard entry screening with linear discriminant analysis. Within several tens of seconds, the system automatically discriminates infected individuals from normal subjects using measured heart rate, respiratory rate, as well as facial surface temperature determined by thermography. The size of system is small enough to be placed on airplane tray tables. The authors tested on 68 subjects including 12 influenza patients to evaluate the system. The result showed sensitivity of 91.7% and specificity of 92.9%. The system seems to be promising for onboard infection screening to safeguard public health.

Background Enzyme-linked immunosorbent assays (ELISA) are considered the gold standard in the demonstration of various immunological reactions with an application in the detection of infectious diseases such as during outbreaks or in patient care. Objective This study aimed to produce an ELISA-based diagnostic with an increased sensitivity of detection compared to the standard 96-well method in the immunologic diagnosis of infectious diseases. Methods A '3DStack' was developed using readily available, low cost fabrication technologies namely nanoimprinting and press stamping with an increased surface area of 4 to 6 times more compared to 96-well plates. This was achieved by stacking multiple nanoimprinted polymer sheets. The flow of analytes between the sheets was enhanced by rotating the 3DStack and confirmed by Finite-Element (FE) simulation. An Immunoglobulin G (IgG) ELISA for the detection of antibodies in human serum raised against Rubella virus was performed for validation. Results An improved sensitivity of up to 1.9 folds higher was observed using the 3DStack compared to the standard method. Conclusions The increased surface area of the 3DStack developed using nanoimprinting and press stamping technologies, and the flow pattern between sheets generated by rotating the 3DStack were potential contributors to a more sensitive ELISA-based diagnostic device.

In places of mass gathering, rapid infection screening prior to definite diagnosis is vital during the epidemic season of a novel influenza. In order to assess the possibility of clinical application of a newly developed non-contact infection screening system, we conducted screening for influenza patients.The system is operated by a screening program via a linear discriminant analysis using non-contact derived variables, i.e., palmar pulse derived from a laser Doppler blood-flow meter, respiration rate determined by a 10-GHz microwave radar, and average facial temperature measured by thermography. The system was tested on 57 seasonal influenza (2008-2009) patients (35.7 degrees Cor = body temperatureor = 38.3 degrees C, 19-40 years) and 35 normal control subjects (35.5 degrees Cor = body temperatureor = 36.9 degrees C, 21-35 years) at the Japan Self-defense Forces Central Hospital.A significant linear discriminant function (p0.001) was determined to distinguish the influenza group from the control group (Mahalanobis D-square = 6.5, classification error rate10%). The system had a positive predictive value (PPV) of 93%, which is higher than the PPV value (PPVor = 65.4%) reported in the recent summary of studies using only thermography performed mainly in hospitals.The proposed system appears promising for application in accurate screening for influenza patients at places of mass gathering.

In order to conduct fast screening of passengers with infections such as severe acute respiratory syndrome (SARS) or pandemic influenza at a quarantine depot, we developed a non-contact screening system with a self-produced program to conduct a human screening within five seconds, via a linear discriminant function from non-contact derived variables, i.e. palmer pulse derived from a laser Doppler blood-flow meter, respiration rate determined by a 10-GHz microwave radar, and facial temperature measured by a thermography. The system evaluation was conducted on seven healthy male subjects (23+1 years). In order to achieve a pseudo-infection condition, the subjects maintained an ergo-meter exercise load (100 W, 10 minutes). Before (normal condition) and after (pseudo-infection condition) exercise, a significant linear discriminant function (p50.001) was determined to distinguish the pseudo-infection condition from the normal condition (Mahalanobis D-square 1/4 20.3, classification error rate55%). The proposed system appears promising for future application in fast screening of infection at a quarantine depot.

Enzyme-linked immunosorbent assays (ELISAs) are considered the gold standard for the detection of various immunological reactions and can be used for the detection of infectious diseases during outbreaks or in the care of individual patients. To be useful in the timely implementation of prevention and control measures against infectious diseases, a diagnostic modality should be rapid, accurate, and affordable. In the current study, we demonstrate the efficiency (90% less time and volume consumption compared with those of a standard 96-well ELISA), detection capability, and ease of operation of a field-portable, battery-operated ELISA system, approximately the size of a cellular phone (12 × 6 × 5.5 cm), in the serological diagnosis of measles and rubella viruses that has the potential for onsite testing such as during disease outbreaks.

The emergence of orthoreoviruses as the causative agent of human respiratory illness over the past few years has led to a demand to determine their viral genome sequences. The whole genome sequencing of such RNA viruses using traditional methods, such as Sanger dideoxy sequencing following rapid amplification of cDNA ends presents a laborious challenge due to the numerous preparatory steps required before sequencing can commence. We developed a practical, time-efficient novel combination method capable of reducing the total time required from months to less than a week in the determination of whole genome sequence of Pteropine orthoreoviruses (PRV); through a combination of viral RNA purification and enrichment, adaptor ligation, reverse transcription, cDNA circularization and amplification and next generation sequencing. We propose to call the method “modified rolling circular amplification with adaptor ligation – next generation sequencing (mRCA-NGS)”. Here, we describe the technological focus and advantage of mRCA-NGS and its expansive application, exemplified through the phylogenetic understanding of the Miyazaki-Bali/2007 PRV.

Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

Pteropine orthoreovirus, potentially of bat origin, has been reported to cause respiratory tract infections among human beings in Southeast Asia. Twelve IgG ELISA-positive cases with antibodies against Pteropine orthoreovirus were detected among 272 human serum samples collected between March and June 2014 from in and around Hue City, Central Vietnam. These 12 cases were IgM ELISA negative. Neutralizing antibodies were also detected among six of these cases with the highest titer of 1:1,280 in 2 cases (both female, 32 and 68 years old, respectively). This is the first report of human infection with Pteropine orthoreovirus in Central Vietnam. These findings indicate the need for surveillance on Pteropine orthoreovirus infections in Southeast Asia to enable prevention and control strategies to be developed should a change in virulence occur.

Street rabies virus (SRV)-infected T-lymphocyte-deficient (nude) mice, in contrast to euthymic mice, did not develop hindlimb paralysis prior to death. To document the role of T lymphocytes in rabies virus-associated paralysis, 10(8) spleen cells from normal immunocompetent euthymic mice were transferred to nude mice and the recipient mice were challenged with SRV. One hundred percent of the reconstituted mice developed paralysis and died. Depletion of T cells from the donor spleen suspension prior to transfer abrogated the development of paralysis but did not prevent the deaths of the recipient animals. Mice receiving 10(8) rabies virus-immune spleen cells did not become paralyzed and did not die. Nude mice inoculated with either rabies virus-immune or normal mouse serum prior to and following SRV inoculation did not develop paralysis. Immune serum protected the mice, whereas animals inoculated with normal serum died. Central nervous system inflammatory responses in nude mice immunologically reconstituted with normal spleen cells were characterized by diffuse cellular infiltrates in the parenchyma and extensive perivascular cuffing. Perivascular infiltrates included CD8+ and CD4+ T lymphocytes and Mac-1+ macrophage-microglial cells. Inflammatory cells in the parenchyma were limited to CD8+ lymphocytes and Mac-1+ cells. These observations indicate that paralysis of SRV-infected mice is dependent on T lymphocytes. Whether injury leading to paralysis is mediated by T lymphocytes or by an influence of T lymphocytes on macrophage-microglial cells or other cells remains to be determined.

Cells persistently infected with Evelyn-Rokitnicki-Abelseth (ERA) rabies virus were established. The cells were used as stimulator and target cells to compare H-2 restricted cytotoxic T lymphocyte (CTL) responses specific for rabies virus in A/WySnJ (H-2a), C57BL/6J (H-2b), BALB/cByJ (H-2d), A.SW/SnJ (H-2s) and SJL/J (H-2s) mice. Using a 51chromium release assay, it was determined that an effector/target (E/T) ratio of 5:1 was necessary to demonstrate specific lysis of ERA virus persistently infected mouse neuroblastoma (MNB) (H-2a), EL-4 (H-2b) and P815 (H-2d) cells. Effectors at an E:T ratio of only 0.05:1 specifically lysed an SV-40 transformed SJL/J mouse fibroblast (SSSV) (H-2s) target monolayer. The CTL destruction of the SSSV monolayer was observed visually following Giemsa staining. This is the first instance in which a detailed method for detection of murine anti-rabies virus CTLs has been reported. Furthermore, it is the first time target cells persistently infected with rabies virus were used as stimulator cells to amplify CTLs in vitro and as target cells in the CTL assay. It also is the initial report in which rabies specific CTLs were characterized in H-2d and H-2s rabies virus infected mice.

The prevalence of West Nile (WN) virus infection in Karachi, Pakistan, was unknown until 1982. It had been noticed that there were more than a few patients with encephalitides in Karachi, and it was supposed that Japanese encephalitis (JE) cases would be found among them. Therefore, a seroepidemiological study was conducted to define the prevalence of WN virus infection and the possible occurrence of JE virus infection in the Karachi area. Prevalences of haemagglutination inhibition (HI) and neutralization (NT) antibodies against WN virus were studied among 81 serum samples (in July, 33 samples; in September, 48) during 1983, and among 156 paired serum samples that were collected twice, in July and October of 1985. Nearly the same antibody-positive rates were obtained in July of both years (1983: HI 55%; 1985: HI 53%; NT 50%); the rates increased slightly during September/October (1983: HI 65%; 1985: HI 59%, NT 54%). Among 156 paired samples in 1985, 20 (13%) and 12 (8%) showed positive- or negative-antibody conversion between July and October. Two serum samples from each of 156 residents obtained in July had a significantly higher NT antibody titre against JE virus than against WN virus (in case 1, JE 1:80, WN less than 1:10; in case 2, JE 1:40, WN less than 1:10). This is the first report to show the prevalence of WN virus infection in Karachi, Pakistan.