Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

<p>Abstract</p> <p>Background</p> <p>Chandipura virus (CHPV), a member of family <it>Rhabdoviridae </it>was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.</p> <p>Methods</p> <p>Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [<it>in vivo </it>(mice) <it>in ovo </it>(eggs), <it>in vitro </it>(Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.</p> <p>Results</p> <p>Real-time one step RT-PCR was optimized using <it>in vitro </it>transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 10²-10¹⁰(r²= 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 10⁰PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 10²PFU/ml). Vero and PS cell-lines (1.2 × 10³PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.</p> <p>Conclusion</p> <p>On account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.</p

An Analysis on the Detection of Biological Contaminants Aboard Aircraft

The spread of infectious disease via commercial airliner travel is a significant and realistic threat. To shed some light on the feasibility of detecting airborne pathogens, a sensor integration study has been conducted and computational investigations of contaminant transport in an aircraft cabin have been performed. Our study took into consideration sensor sensitivity as well as the time-to-answer, size, weight and the power of best available commercial off-the-shelf (COTS) devices. We conducted computational fluid dynamics simulations to investigate three types of scenarios: (1) nominal breathing (up to 20 breaths per minute) and coughing (20 times per hour); (2) nominal breathing and sneezing (4 times per hour); and (3) nominal breathing only. Each scenario was implemented with one or seven infectious passengers expelling air and sneezes or coughs at the stated frequencies. Scenario 2 was implemented with two additional cases in which one infectious passenger expelled 20 and 50 sneezes per hour, respectively. All computations were based on 90 minutes of sampling using specifications from a COTS aerosol collector and biosensor. Only biosensors that could provide an answer in under 20 minutes without any manual preparation steps were included. The principal finding was that the steady-state bacteria concentrations in aircraft would be high enough to be detected in the case where seven infectious passengers are exhaling under scenarios 1 and 2 and where one infectious passenger is actively exhaling in scenario 2. Breathing alone failed to generate sufficient bacterial particles for detection, and none of the scenarios generated sufficient viral particles for detection to be feasible. These results suggest that more sensitive sensors than the COTS devices currently available and/or sampling of individual passengers would be needed for the detection of bacteria and viruses in aircraft

Whole Genomes of Chandipura Virus Isolates and Comparative Analysis with Other Rhabdoviruses

The Chandipura virus (CHPV) belonging to the Vesiculovirus genus and Rhabdoviridae family, has recently been associated with a number of encephalitis epidemics, with high mortality in children, in different parts of India. No full length genome sequences of CHPV isolates were available in GenBank and little is known about the molecular markers for pathogenesis. In the present study, we provide the complete genomic sequences of four isolates from epidemics during 2003–2007. These sequences along with the deduced sequence of the prototype isolate of 1965 were analysed using phylogeny, motif search, homology modeling and epitope prediction methods. Comparison with other rhaboviruses was also done for functional extrapolations. All CHPV isolates clustered with the Isfahan virus and maintained several functional motifs of other rhabdoviruses. A notable difference with the prototype vesiculovirus, Vesicular Stomatitis Virus was in the L-domain flanking sequences of the M protein that are known to be crucial for interaction with host proteins. With respect to the prototype isolate, significant additional mutations were acquired in the 2003–2007 isolates. Several mutations in G mapped onto probable antigenic sites. A mutation in N mapped onto regions crucial for N-N interaction and a putative T-cell epitope. A mutation in the Casein kinase II phosphorylation site in P may attribute to increased rates of phosphorylation. Gene junction comparison revealed changes in the M-G junction of all the epidemic isolates that may have implications on read-through and gene transcription levels. The study can form the basis for further experimental verification and provide additional insights into the virulence determinants of the CHPV

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections

Assessment of Knowledge of ASHAs about RCH Services in Rural Area

oai:ojs2.www.ijhsonline.com:article/2Background: The National Rural Health Mission was launched in year 2005 to improve rural health care delivery system. One of the key initiatives was to appoint accredited Social health Activist (ASHA) to bridge the gap in rural healthcare. ASHAs are being selected from the local community by the health department. Reproductive and Child Health (RCH) is the most important programme under NRHM. Thus the present study was undertaken to assess the knowledge of ASHAs about RCH services provided by them in rural area. Objectives: Assess the knowledge of ASHAs about RCH services. Material and methods: There are total ten blocks in Latur district. Out of each blocks one PHC was selected randomly for the study. All the trained ASHAs working under the selected PHC were enrolled in the study. Thus total 311 ASHAs were included in the study. A questionnaire was designed using induction training module used for ASHAs at their respective PHCs during the month of October 2013. Results: in this study showed that 49.2% ASHAs scored 25 -50% marks and 32.48% scored 50-75% marks. The mean score was 41.46 with SD 0.92. ASHAs from most of the blocks scored between 25-50% while ASHAs from two blocks (Renapur and Jalkot) scored 50-75%. It was observed that most of the ASHAs were in the age group of 25-30yrs (49.06%) followed by 31 -35 yrs. (33.02%)(X2 =8.42, df= 3, p&lt;0.05). Majority of ASHAs were having educational qualification between 8th to 12th standard. Conclusion: The study revealed the training given to ASHA workers there is still lacunae left in their knowledge regarding various aspects and Frequency of quality training for ASHAs must be strengthened

Utility of Pandemic H1N1 2009 Influenza Virus Recombinant Hemagglutinin Protein-Based Enzyme-Linked Immunosorbent Assay for Serosurveillance ▿

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against the pandemic H1N1 2009 influenza A virus, employing a recombinant hemagglutinin protein of the virus, was compared to the hemagglutination inhibition (HI) test using 783 serum samples. The results showed a concordance of 98.4%, suggesting the utility of the ELISA in serosurveillance. Two hundred sixty-nine (100%) serum samples with an HI titer of ≥20 were ELISA reactive

Acid base disturbances in uncontrolled type II Diabetes Mellitus.

Aim: To estimate pH, pCO2, pO2 and bicarbonate (HCO3─) levels in arterial blood of diabetes mellitus type II patients. Material and Method: 90 subjects above the age of 40 years were participated in the present study, subdivided in Group 1: 30 diabetic subjects suffering from diabetes mellitus type II as diagnosed by the physician and having random plasma glucose &lt; 400 mg/dl. Group 2: 30 diabetic subjects suffering from diabetes mellitus type II as diagnosed by the physician and having random plasma glucose ≥ 400 mg dl (uncontrolled). Control group: 30 non-diabetic subjects. Arterial blood samples were collected from radial artery in a heparinised syringe. Result: Arterial pH was not significantly decreased (&gt; 0.05) when Group 1 (7.38 ± 0 .09) was compared with control group (7.405 ± 0.044) but significantly decreased (&lt; 0. 05) when Group 2 (7.31 ± 0.11) was compared with control group (7.405 ± 0.044) as well as significantly decreased (&lt; 0.05) when Group 2 (7.31 ± 0.11) was compared with Group 1 (7.38 ± 0.09). Arterial pCO2 was not significantly decreased (&gt;0.05) when Group 1 (36.13 ± 14.20) was compared with control group (39.56 ± 4.50) but significantly decreased (&lt; 0. 05) when Group 2 (31.93 ± 9.50) was compared with control group (39.56 ± 4.50) as well as significantly decreased (&lt; 0.05) when Group 2 (31.93 ± 9.50) was compared with Group 1 (36.13 ± 14.20). &nbsp;Arterial pO2 was not significantly decreased (&gt;0.05) when Group 1 (88.06 ± 7.09) was compared with) control group (93.16 ± 5.18) but highly significantly decreased (&lt; 0.001) when Group 2 (81.23 ± 12.96) was compared with control group (93.16 ± 5.18) &nbsp;as well as significantly decreased (&lt; 0.05) when Group 2 (81.23 ± 12.96) was compared with Group 1 (88.06 ± 7.09). Plasma (HCO3─) was not significantly decreased (&gt;0.05) when Group 1 (20.99 ± 6.72) was compared with control group (22.65 ± 1.58) but significantly decreased (&lt; 0.05) when Group 2 (16.95 ± 5.69) was compared with control group (22.65 ± 1.58) as well as significantly decreased (&lt;0.05) when Group 2 (16.95 ± 5.69) was compared with Group 1 (20.99 ± 6.72). Conclusion: Uncontrolled hyperglycemia (group 2 cases) is significantly associated with acid base imbalance in type II diabetes mellitus patients