Conventional Reverse Transcriptase-polymerase Chain Research Articles

Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

Genetic characterization and phylogenetic analysis of foot and mouth disease virus vaccine strains and recent field isolate

Foot and Mouth Disease (FMD) control is a national and regional responsibility. FMD outbreaks were detected in three farms in Port-Said Governorate in 2020, although animals in these farms were vaccinated with the local polyvalent inactivated vaccine. Samples from tongue epithelium and vesicular fluid were collected from these farms, where FMDV isolation was performed on BHK-21 cell culture. Viral RNA was extracted from the virus isolates, and vaccine strains were then screened for FMDV using conventional Reverse transcriptase polymerase chain reaction. The substitution rates and phylogenetic relationship between the field isolate and the vaccine strains were determined using DNA sequence and bioinformatics analyses. The results illustrate that the sequence analysis for the 1D region of FMDV field isolate was serotype A‑Africa topotype, Genotype IV, and closely related to isolated A‑Africa topotype Genotype IV by Animal Health Research Institute, 2020 with nucleotide similarity ranging from 97.1% to 99.74%, and revealing genetic variation of 5.63-7.88% from previously Egyptian isolated A‑Africa topotype, Genotype IV in 2016 and 2018. High genetic variations were determined from the locally used vaccine strain serotype A of the Asia topotype, lineage Iran‑05, in the major antigenic sites of the VP1 region with a nucleotide difference of 26.34%. Depending on these findings, the Veterinary Serum and Vaccine Research Institute produced an emergency inactivated monovalent vaccine using the newly isolated strain, and we recommended adding it to the subsequent prepared vaccine batches.

Molecular Detection of Respiratory Syncytial Virus in Infants and Young Children by the Conventional Reverse Transcriptase Polymerase Chain Reaction

Molecular Detection of Respiratory Syncytial Virus in Infants and Young Children by the Conventional Reverse Transcriptase Polymerase Chain Reaction

A novel genotype of avian hepatitis E virus identified in chickens and common pheasants (Phasianus colchicus), extending its host range

In 2019, outbreaks of hepatitis-splenomegaly syndrome (HSS) were observed in six commercial layer chicken flocks, belonging to three different Polish farms, and characterized by increased mortality, hemorrhagic hepatitis with attached blood clots on the liver surface, and splenomegaly. Diseased flocks were initially investigated for the presence of avian hepatitis E virus (aHEV) – the etiological agent of HSS – by conventional reverse transcriptase polymerase chain reaction, which revealed aHEV sequences clustering separately from all known aHEV genotypes. Additionally, an aHEV genome was identified for the first time in common pheasants, from a flock in France, using Next Generation Sequencing. This genome clustered together with the Polish aHEVs here investigated. Complete genome aHEV sequences from the HSS outbreaks confirmed the divergent cluster, with a shared nucleotide sequence identity of 79.6–83.2% with other aHEVs, which we propose to comprise a novel aHEV genotype – genotype 7. Histology and immunohistochemistry investigations in the liver and spleen established an association between aHEV and the observed lesions in the affected birds, consolidating the knowledge on the pathogenesis of aHEV, which is still largely unknown. Thus, the present investigation extends the natural host range and genotypes of aHEV and strengthens knowledge on the pathogenesis of HSS.

Influenza virus and its subtypes circulating during 2018–2019: A hospital-based study from Assam

PurposeInfluenza virus can cause serious respiratory illness sometimes resulting in epidemics and pandemics associated with significant morbidity and mortality across the globe. Hence, continuous surveillance of the activity of the influenza virus and its subtypes is necessary to help the policy makers to take effective and appropriate decisions regarding its control. The study aimed to determine distribution of influenza viruses in Assam of north-east India having subtropical climate that may lead to viral subtype divergence. MethodsClinically suspected ninety cases with Influenza like illness (ILI) were included, irrespective of age and sex during the period 1st July 2018 to 30th June 2019. Aseptically collected Nasopharyngeal swabs in viral transport media (VTM) were tested by conventional Reverse Transcriptase Polymerase Chain Reaction (RT PCR) for detection of Influenza A and Influenza B viruses which were further processed for detection of subtypes such as H1N1 pdm09, H3N2 and Influenza B (Yamagata and Victoria lineage). Normally distributed continuous variables were summarised by mean and standard deviation. All categorical variables were summarised as percentages. ResultsInfluenza activity was seen in 42.2% of ILI cases with male predominance (57.9%). Influenza A was the predominant type (84.2%). Among the subtypes, A(H1N1) pdm09 was predominant (76.3%) followed by Influenza B (Victoria lineages) (15.8%) and AH3N2 (7.9%). Significant difference was observed between different subtypes with regard to age distribution only. Influenza activity in Assam showed two seasonal peaks; the primary one from May to July and the secondary from November to February. ConclusionThe study described the distribution of different Influenza viruses and its subtypes in Assam along with their seasonal activities. These findings will help to formulate the policy for its prevention and control in Assam as well as to monitor the efficacy of the current influenza vaccine.

PCR to CRISPR: Role of Nucleic Acid Tests (NAT) in detection of COVID-19.

COVID-19 infection has emerged as an unparalleled pandemic with morbidity and mortality tolls challenging diagnostic approaches and therapeutic interventions, and raising serious questions for healthcare policy-makers. From the diagnostic perspective, Reverse transcriptase polymerase chain reaction remains the gold standard. However, issues associated with gene primer variation in different countries, low analytical sensitivity, cross-reactivity with certain human coronaviruses have raised serious concerns within the scientific community. Alongside longer turnaround times, requirements of sophisticated equipment and trained technicians are the other challenges for conventional reverse transcriptase polymerase chain reaction testing. The recent biotechnological boom has now allowed newer nucleic acid testing options for diagnosing severe acute respiratory syndrome Coronovairus 2 (SARS-CoV2) with much better diagnostic efficiency, reduced turnaround times and possible benefit for use as a point-of-care test. Isothermal techniques with simple equipment requirements along with uniform temperature for analysis have emerged to be more sensitive and specific with turnaround times as low as 10-15 minutes. Similarly, Cluster Regularly Interspaced Short Palindromic Repeats have also been seen to play a very decisive role in COVID-19 diagnostics with much superior diagnostic efficiency and feasibility as a point-of-care test and its possible use for sequencing. The current narrative review was planned to consolidate data for all possible nucleic acid testing options under research/clinical use, and to provide a comparative assessment from the perspective of both the clinician and the laboratory.

Molecular Epidemiology of Rotavirus Strains in Symptomatic and Asymptomatic Children in Manhiça District, Southern Mozambique 2008-2019.

Group A rotaviruses remain the leading cause of diarrhoea in children aged <5 years. Mozambique introduced rotavirus vaccine (Rotarix®) in September 2015. We report rotavirus genotypes circulating among symptomatic and asymptomatic children in Manhiça District, Mozambique, pre- and post-vaccine introduction. Stool was collected from enrolled children and screened for rotavirus by enzyme-immuno-sorbent assay. Positive specimens were genotyped for VP7 (G genotypes) and VP4 (P genotypes) by the conventional reverse transcriptase polymerase chain reaction. The combination G12P[8] was more frequently observed in pre-vaccine than in post-vaccine introduction, in moderate to severe diarrhoea (34%, 61/177 vs. 0, p < 0.0001) and controls (23%, 26/113 vs. 0, p = 0.0013) and mixed genotypes (36%, 24/67 vs. 7% 4/58, p = 0.0003) in less severe diarrhoea. We observed changes in post-vaccine compared to pre-vaccine introduction, where G3P[4] and G3P[8] were prevalent in moderate to severe diarrhoea (10%, 5/49 vs. 0, p = 0.0002; and 14%, 7/49 vs. 1%, 1/177, p < 0.0001; respectively), and in less severe diarrhoea (21%, 12/58 vs. 0, p = 0.003; and 24%, 14/58 vs. 0, p < 0.0001; respectively). Our surveillance demonstrated the circulation of similar genotypes contemporaneously among cases and controls, as well as switching from pre- to post-vaccine introduction. Continuous surveillance is needed to evaluate the dynamics of the changes in genotypes following vaccine introduction.

Co-inections of domestic and wild birds with avian influenza and Newcastle disease viruses: implications for control and genetic mutations.

Co-infections of birds' upper respiratory tract by avian pathogens are common and cause increasing economic losses. This study determines co-infection status of avian influenza (AI) and Newcastle disease (ND) in birds in two Nigerian states with different highly pathogenic avian influenza (HPAI) records and where modified stamping out policy contained the virus for seven years after which the virus resurged with higher infectivity in 2015. A cross-sectional study sampling 910 apparently healthy domestic and 90 wild birds from wild habitats, commercial poultry farms, households and live bird markets (LBMs) was conducted. Cloacal and tracheal swabs were tested for AI H5 and ND viruses using conventional reverse transcriptase polymerase chain reaction (RT-PCR). Overall detection rates of 3% and 18% for AI and ND were obtained. There was an equivalence of 8.5% ND detection rate in poultry and wild birds in the two states. Co-infection (0.2%) of a local chicken from a live bird market (LBM) and crown crane (Balearica regulorum) from a household to AI H5 and ND viruses occurred, respectively. Exposure of birds to AI and ND was significantly detected in apparently healthy domestic and wild birds. The probability of these viruses exchanging genetic materials to resurge with increasing virulence is foreseen. Therefore, routine AI and ND control measures should incorporate virus surveillance and instituting appropriate preventive measures in domestic and wild birds held in households, commercial farms and LBMs.

Detection of Newcastle Disease Virus (NDV) in Laughing Doves and the Risk of Spread to Backyard Poultry

Abstract Newcastle disease (ND) is a highly infectious viral disease of birds caused by the Newcastle disease virus (NDV) and doves have been incriminated in previous outbreaks of the disease that have discouraged backyard poultry productions. This survey was done to detect and characterize the NDV from 184 swabs from the cloacae and pharynxes of 67 trapped laughing doves and 25 backyard poultry birds. The study utilized haemagglutination assay (HA) followed by haemagglutination inhibition (HI) tests on HA positive samples to screen field samples. Conventional reverse transcriptase polymerase chain reaction (RT-PCR) was conducted on the HI positives to characterize the NDV. This study revealed that of 134 dove samples screened, 88 (65.7 %) were HA positive. Of these HA positives subjected to HI testing, 37 (42.1 %) were HI positive. Interestingly, 21 (56.8 %) of the HI positives were also RT-PCR positive: 8 lentogenic, 12 velogenic, while one had both lentogenic and velogenic NDV. Comparatively, of the 50 chicken samples screened, 23 (46 %) were HA positive; and of these, HA positives subjected to HI testing, 16 (69.6 %) were HI positive. Only 4 (25 %) of the HI positives were RTPCR positive: 3 lentogenic and a velogenic NDV. From this study it was concluded that laughing doves were demonstrated to be infected with either lentogenic or velogenic NDV or both. The use of red blood adsorption-de-adsorption concentration of NDV enhanced the RT-PCR detection using the fusion gene primers NDV-F 4829 and NDV-R 5031. The detection of not only lentogenic but velogenic NDV in laughing doves poses a great risk to backyard poultry production.

A Pilot Study to Assess Transfusion Transmission of Hepatitis C Virus.

Alexandria University blood bank adopted double screening tests: a fully automated chemiluminescence immunoassay followed by nucleic acid testing. The aim of the study was to assess the efficiency of dual check of HCV in preventing transfusion related infection among patients admitted to PICU. A prospective cohort study was carried on patients admitted to PICU during 6months. The included patients performed HCV RNA detection on admission by conventional reverse transcriptase polymerase chain reaction (RT-PCR) technique. Only negative cases were recruited, then patients receiving blood or its product were checked after 4weeks from discharge by RT-PCR for HCV RNA. A total of 33 patients received 108 transfusions: 9 patients of them deceased during PICU stay and the remaining 24 patients were all found to be negative for HCV. The dual screening of HCV should be implemented in all blood banks of Egypt especially for critically ill pediatric patients.

A Survey on Human T-cell Lymphotropic Virus Type 1 (HTLV-1) and Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Coinfection in Tehran, Iran.

Background:Xenotropic murine leukemia virus-related virus (XMRV) is a gamma retrovirus, which has been detected in patients with prostate cancer, chronic fatigue syndrome, and general population with a number of acquired infections such as infection with human T-cell lymphotropic virus (HTLV) and human immunodeficiency virus (HIV). The aim of this study was to determine the HTLV-1 and XMRV coinfection for the first time in Iranian patients who were admitted to the Tehran hospitals.Materials and Methods:Two hundred and ninety one patients suspected with HTLV-1 were referred to the hospitals affiliated to the Iran University of Medical Sciences, Tehran, Iran from April 2012 to October 2016. Genomic deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) from peripheral blood mononuclear cells/cerebrospinal fluids was extracted by High Pure Viral Nucleic Acid Kit (Roche, Germany). After complementary DNA synthesis, conventional reverse transcriptase polymerase chain reaction was used for the detection of HTLV-1 or XMRV-infected patients. Statistical Package for Social Sciences (SPSS) software, version 16 (SPSS, Chicago, IL, USA) was used for statistical analyses.Results:Of the 291 patients suspected of HTLV infection, 123 (42.3%) were male with a mean age of 38±15 years. HTLV-1 RNA was found in 93 (31.9%) specimens comprising 40 men (41.3%) and 53 women (56.9%). Of the 93 patients who were HTLV-1 positive, one sample (1%) was positive for XMRV env gene.Conclusion:These findings suggest that the lack of significant detection of XMRV in patients who were HTLV-1 positive could not be associated with complications of HTLV-1. Although this is a preliminary report from Iranian patients with HTLV-1, further studies are needed to show the actual prevalence of XMRV infection by geographical distribution and various populations.

Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus.

BackgroundAvian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. To date, conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. However, these assays are time consuming, inconvenient, and cannot detect the virus quantitatively. In this study, a rapid and sensitive SYBR Green real-time RT-PCR assay was developed to detect avian HEV RNA quantitatively in serum, liver, spleen, and fecal samples from chickens.ResultsBased on the sequence of the most conserved HEV gene, ORF3, the primers for the assay were designed, and the standard plasmid was constructed. The detection limit of the assay was shown to be 10 copies/μl of standard plasmid/reaction, with a corresponding cycle-threshold value of 29.3. The standard curve exhibited a dynamic linear range across at least 7 log units of DNA copy number. The specificity and reproducibility of this assay was high, showing that the assay detected avian HEV RNA specifically and with little variability. Compared to conventional RT-PCR, the current assay is more sensitive for detecting avian HEV in serum, liver, spleen, and fecal samples from chickens.ConclusionsA rapid, specific, and reproducible SYBR Green real-time RT-PCR assay was developed for the diagnosis of avian HEV infection in chickens. This assay can accurately detect avian HEV RNA in serum, liver, spleen, and fecal samples with more sensitivity than conventional RT-PCR.

Co-positivity of anti-dengue virus and anti-Japanese encephalitis virus IgM in endemic area: co-infection or cross reactivity?

ObjectiveTo report high co-positivity of anti-dengue virus (DV) and anti-Japanese encephalitis virus (JEV) IgM in an area endemic for both the viruses and to discuss the possibilities of co-infection. MethodsSerum samples from the patients who presented with fever, suspected central nervous system infection and thrombocytopenia, were tested for anti-DV IgM and anti-JEV IgM antibodies. Conventional reverse transcriptase polymerase chain reaction was done for detection of DV RNA and JEV RNA. ResultsOf 1 410 patient sera tested for anti-DV and anti-JEV antibodies, 129 (9.14%) were co-positive for both. This co-positivity was observed only in those months when anti-JEV IgM positivity was high. Titers of both anti-DV IgM and anti-JEV IgM were high in most of the co-positive cases. Among these 129 co-positive cases, 76 were tested by conventional reverse transcriptase polymerase chain reaction for both flaviviruses, of which eight cases were co-positive for DV and JEV. ConclusionsCo-infection with more than one flavivirus species can occur in hyperendemic areas.

Prognostic significance of the expression levels of the p16, p15, and p14 genes in dogs with high-grade lymphoma

The prognostic significance of the inactivation of the p16, p15, and p14 genes has been reported in lymphoid malignancies in humans. To evaluate the relationship between inactivation of the p16, p15, and p14 genes and prognosis in canine high-grade lymphoma, primary tumor cell samples obtained from 71 dogs with high-grade lymphoma were examined for the expression levels of these genes. Quantitative and conventional reverse transcriptase–polymerase chain reaction (RT-PCR) analyses were used to measure the amounts of p16, p15, and p14 mRNAs. The methylation status of the CpG island of the p16 gene was evaluated using methylation-specific PCR. Overall survival (OS) was compared using the Kaplan–Meier method. The log-rank test and Cox proportional hazard model were used to evaluate factors that influenced OS.Of 62 dogs examined, p16, p15, and p14 mRNA levels were found to be undetectable in 21, 18, and 10 dogs, respectively. In 20/68 dogs analyzed, the CpG island of the p16 gene was shown to be methylated. The prognostic significance of inactivation of the p16, p15, and p14 genes as well as various conventional factors obtained from medical records was examined. p16 expression status and anatomic form/immunophenotype were found to correlate with OS in the dogs with high-grade lymphoma. p16 mRNA level over its cut-off value correlated with a poor prognosis; however, the expression levels of p15 and p14 mRNAs and p16 methylation status did not influence the prognosis in dogs with high-grade lymphoma.

Optimisation of reverse transcription loop-mediated isothermal amplification assay for the rapid detection of pandemic (H1N1) 2009 virus

Conventional reverse transcriptase polymerase chain reaction (RT-PCR) and optimized of a closed tube reverse-transcription loop-mediated isothermal amplification (RT-LAMP) were used for detection of pandemic (H1N1) 2009 virus and the optimized of a closed tube RT-LAMP methods were compared with the conventional RT-PCR with respect to specificity and sensitivity. In this study, optimized RT-LAMP detected 2 copies of target RNA by visual detection with modified dye. Reaction time, temperature and quantity of each reagent were optimised for the detection of the virus. The sensitivity of detection limit by optimised RT-LAMP was 100 times as that of conventional RT-PCR. Amplification of DNA can be identified by visualization with modified dye, which reduces the cross-contamination caused by opening tube. The sensitivity of visual detection was equivalent to that of electrophoresis analysis. Additionally, the method was specific as no cross-reaction was observed among samples from human blood, Escherichia coli and other related viruses including human seasonal influenza A, subtypes H1N1, H1N2 and H3N2 viruses. These results demonstrate that the optimized RT-LAMP assay for pandemic (H1N1) 2009 virus RNA was a valuable tool with simplicity, rapidity and specificity, as well as its superiority for the screening and surveillance of influenza in developing countries. Key words: Pandemic (H1N1) 2009 virus, loop-mediated isothermal amplification (LAMP), reverse transcriptase polymerase chain reaction (RT-PCR), HA gene.

Proteotyping of the Parainfluenza Virus with High-Resolution Mass Spectrometry

Parainfluenza viruses (PIVs) are one of the most common causes of respiratory tract infections in children and can be life-threatening when the airway becomes obstructed. Infection results in a spectrum of respiratory disease symptoms that makes diagnosis difficult. A new proteotyping approach employing high-resolution mass spectrometry is shown to be able to distinguish common human serotypes of the PIV from the perspective of all surface and internal viral proteins. The detection of signature peptides, conserved in sequence and unique in mass, within the spectra of these protein or whole virus digests enables the parainfluenza virus to be identified and typed and for it to be distinguished from the influenza virus. Given that the approach is more rapid and direct than conventional reverse transcriptase polymerase chain reaction (RT-PCR), and that it can be implemented with high sample throughout at a comparable sensitivity, it affords an effective new means with which to characterize the virus at the molecular level.

Expression of survivin in human oocytes and preimplantation embryos

To determine whether [1] survivin is expressed in human oocytes and embryos; [2] embryos grown in vitro secrete survivin protein; and [3] survivin levels are correlated with embryo cleavage rates. Experimental. University-affiliated IVF clinic. Couples undergoing IVF-ET cycles. Conventional reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, immunohistochemistry, Western blot on oocytes, embryos and control choriocarcinoma JEG-3 cells, and ELISA analysis of conditioned culture media. Detection of survivin mRNA and protein in oocytes and preimplantation embryos and in JEG-3 cancer cells. Detection of survivin concentrations in embryo culture media. Survivin mRNA and protein were expressed during human oocyte maturation, from germinal vesicle to metaphase II stage, and throughout embryo development, from pronuclear stage to blastocyst stage. Survivin was localized predominantly in the cytoplasm of all cells examined and in the oocytes on the chromatin of metaphase chromosomes and midbodies. Western blot analysis of human oocyte and cancer cell extracts detected a full-length (primary) survivin band of 16.5 kDa. Survivin was also detected in conditioned media samples from embryo cultures and showed a positive correlation with embryo cleavage rates. Our data have demonstrated for the first time that human oocytes/embryos not only express but also secret survivin, suggesting that survivin may play an important role in human oogenesis and embryogenesis.

Investigation on the 19S ATPase proteasome subunits (Rpt1–6) conservation and their differential gene expression in Schistosoma mansoni

The ubiquitin-proteasome system is responsible for degradation of the majority of intracellular proteins in eukaryotic cells. The 26S proteasome proteolytic complex is composed of a 20S core particle responsible for protein degradation and the 19S lid which plays a role in the recognition of polyubiquitinated substrates. The 19S regulatory particle (Rps) is composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. In this study, we analyzed the expression profile of 19S Rpt subunits in the larvae and adult stage of the Schistosoma mansoni life cycle. Conventional reverse transcriptase polymerase chain reaction (RT-PCR) revealed that the majority of the 19S Rpt subunits amplified at the expected molecular masses for various investigated stages. In addition, SmRpt1, SmRpt2, and SmRpt6 transcript levels were increased in 3h-cultured schistosomula and reasonably maintained until 5h in culture, as revealed by qRT-PCR. Phylogenetic analysis of 19S Rpt subunits showed high structural conservation in comparison to other Rpt orthologues. The mRNA expression profile of 19S Rpt subunits did not correlate with 26S proteasome proteolytic activity as judged by a (14)C-casein-degrading assay, in the early cultured schistosomula. Taken together, these results revealed a differential expression profile for 19S Rpt subunits whose transcript levels could not be directly associated to 26S proteasome activity.

Epidemiological and clinical features of human rabies cases in Bali 2008-2010

BackgroundPreviously thought to be rabies free, Bali experienced an outbreak of animal and human rabies cases in November 2008. We describe the epidemiological and clinical data of human rabies cases occurring in the first two years of the outbreak.MethodsWe analysed the patient records of all rabies cases from the Sanglah General Hospital in Denpasar, and district hospitals in Buleleng and Tabanan. A conventional reverse transcriptase polymerase chain reaction was developed to detect the rabies virus genome in saliva, corneal swabs, and ante- and post-mortem cerebrospinal fluid (CSF).ResultsThere were 104 human rabies cases in Bali during November 2008-November 2010. Patients' mean age was 36.6 years (range 3-84 years; SD 20.7), most were male (56.7%), and originated from rural districts. Almost all (92%) cases had a history of dog bite. Only 5.8% had their wounds treated and received an anti-rabies vaccine (ARV) after the bite incident. No patients received rabies immunoglobulin (RIG). The estimated time from dog bite to the onset of signs and symptoms was 110.4 days (range 12-720 days; SD 118.2). The mean length of medical care until death was 21.8 hours (range 1-220 hours; SD 32.6). Less than 50% of patients had prodromal symptoms. The most frequent prodromal symptom was pain or paraesthesia at the bite site (37.6%). The two most common central nervous system infection signs were agitation (89.2%) and confusion (83.3%). Signs of autonomic nervous system dysfunction included hydrophobia (93.1%), hypersalivation (88.2%), and dyspnea (74.4%). On admission, 22 of 102 patients (21.6%) showed paralytic manifestations, while the rest (78.4%) showed furious rabies manifestations. The case-fatality rate was 100%. The rabies virus genome was detected in 50 of 101 patients (49.5%) with the highest detection rate from post-mortem CSF samples.ConclusionsRabies is a major public health problem in Bali. Human fatalities occur because of a lack of knowledge regarding rabies risk, the poor management of dog bites, and the limited availability of RIG. Increasing public awareness of dog bite management, increasing the availability of ARV and RIG, and implementing an island-wide dog vaccination campaign will help prevent human rabies cases.

Functional role of voltage‐gated Na+ channels in rat mesenteric arteries

We have previously reported that voltage-gated Na+ channels (NaV) are expressed in freshly dissociated smooth muscle cells from the mouse portal vein, which displays spontaneous, rhythmic contractions. However, the physiological function of NaV in smooth muscle is controversial. Here, we explored the role of NaV in arterial vascular contractility. Isolated mesenteric arteries from male Wistar rats (200–300g) were mounted in a wire-myograph for tension recording. The NaV activator veratridine elicited concentration-dependent (1–30μM), but endothelium-independent, contractions. These contractions (at 10μM, 8.8±0.7mN; n=8) were greatly inhibited by 1μM tetrodotoxin (TTX), a NaV blocker (1.3±0.9mN, n=4) and abolished in Na+-free physiological salt solution (−0.2±0.1mN, n=4). To probe the expression of NaV isoforms (NaV1.1–1.9), we used conventional reverse transcriptase polymerase chain reaction (RT-PCR) with mRNA extracted from the isolated vessels. The presence of TTX-sensitive NaV isoforms (1.2, 1.3, 1.6 and 1.7) was evident. Based on these data, we propose that functional NaV are expressed in arteries without spontaneous rhythmic contractions and may play a role in the regulation of vascular tone. We acknowledge the financial support from St George's University of London and British Heart Foundation