Specific Reverse Transcriptase-polymerase Chain Reaction Research Articles

Long persistence of severe acute respiratory syndrome coronavirus 2 swab positivity in a drowned corpse: a case report

BackgroundSince the beginning of the worldwide spread of severe acute respiratory syndrome coronavirus 2 to date, important knowledge has been obtained about the virus behavior in living subjects and on inanimate surfaces; however, there is still a lack of data on virus persistency on dead bodies and the risk of contagion from cadavers.Case presentationThe present case shows the persistency of the severe acute respiratory syndrome coronavirus 2 viral genome in nasopharyngeal swabs performed on a drowned Caucasian man, aged 41 years old, who was completely asymptomatic when he was alive, up to 41 days after death. Specific real-time reverse transcriptase-polymerase chain reaction (TaqMan 2019-nCoV Assay Kit v2; Thermo Fisher Scientific, Italy and Realquality RQ-SARS-CoV-2, AB Analytical) was used to evaluate the swabs.ConclusionsThis data reflect the importance of postmortem swabs in all autopsy cases, and not only in potential severe acute respiratory syndrome coronavirus 2-related death, and also highlight the necessity to evaluate virus positivity a long time after the moment of death, even if a low initial viral load was assessed.

Three-year Efficacy and Safety of Takeda’s Dengue Vaccine Candidate (TAK-003)

BackgroundTakeda’s live attenuated tetravalent dengue vaccine candidate (TAK-003) is under evaluation in a long-term clinical trial across 8 dengue-endemic countries. Previously, we have reported its efficacy and safety in both seronegative and seropositive participants and that its performance varies by serotype, with some decline in efficacy from first to second year postvaccination. This exploratory analysis provides an update with cumulative and third-year data.MethodsHealthy 4–16 year olds (n = 20099) were randomized 2:1 to receive TAK-003 or placebo (0, 3 month schedule). The protocol included baseline serostatus testing of all participants and detection of all symptomatic dengue throughout the trial with a serotype specific reverse transcriptase-polymerase chain reaction.ResultsCumulative efficacy after 3 years was 62.0% (95% confidence interval, 56.6–66.7) against virologically confirmed dengue (VCD) and 83.6% (76.8–88.4) against hospitalized VCD. Efficacy was 54.3% (41.9–64.1) against VCD and 77.1% (58.6–87.3) against hospitalized VCD in baseline seronegatives, and 65.0% (58.9–70.1) against VCD and 86.0% (78.4–91.0) against hospitalized VCD in baseline seropositives. Efficacy against VCD during the third year declined to 44.7% (32.5–54.7), whereas efficacy against hospitalized VCD was sustained at 70.8% (49.6–83.0). Rates of serious adverse events were 2.9% in TAK-003 group and 3.5% in placebo group during the ongoing long-term follow-up (ie, second half of the 3 years following vaccination), but none were related. No important safety risks were identified.ConclusionsTAK-003 was efficacious against symptomatic dengue over 3 years. Efficacy declined over time but remained robust against hospitalized dengue. A booster dose evaluation is planned.

SARS-CoV-2 in municipal wastewater treatment plant, collection network, and hospital wastewater.

The current outbreak of coronavirus disease (COVID-19) has led to creating a public health emergency conditions since 2019. COVID-19, which is caused by SARS-CoV-2, is spread via human-to-human transmission by direct contact or droplets. Through conducting this study, we were looking for detecting SARS-CoV-2 in wastewater produced in Iran country (Ardabil, Nir, Khalkhal, and Kowsar) (wastewater collection network, wastewater treatment plant, and hospital wastewater). In this research, samples (n=76) were collected from influent and effluent of municipal and hospital wastewater treatment plants, and some samples were also collected from Ardabil municipal wastewater manholes. The sampling duration included the white (lower risk of COVID-19) and red (high risk of COVID-19) conditions. Samples were stored at −20 °C for further diagnostic tests. The specific primer and probe real-time reverse transcriptase-polymerase chain reaction (real-time PCR) targeting ORF1ab and N genes (nucleoprotein gene) were applied to detect viral genomes of the SARS-CoV-2 virus in the wastewater samples. Out of 76 samples, a total of 15 samples (19.73%) collected from wastewater in Ardabil province (Ardabil, Nir, Khalkhal, and Kowsar), were positive in terms of SARS-CoV-2. Wastewater epidemiology can facilitate detection of the incidence of pathogens through metropolises, measurement of population prevalence without direct testing, and provision of information to the public health system about the efficiency of intervening efforts. Graphical abstract

SARS-CoV-2 During Abdominal Operations: Are Surgeons at Risk?

The safety of surgery in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive patients remains unclear. On the basis of data from other viral diseases, it has been assumed that coronavirus disease 2019 (COVID-19) has the potential risk of transmission during surgery. The presence of the virus within the peritoneal fluid and the peritoneal tissues is not known. This is a prospective cross-sectional study including adult patients with confirmed COVID-19 who underwent surgery at a single institution. Using specific real-time reverse transcriptase-polymerase chain reaction, the presence of SARS-CoV-2 was determined in abdominal fluid samples. Six patients with COVID-19 underwent abdominal surgery. Five patients were asymptomatic, and 1 had severe disease. SARS-CoV-2 was tested in 20 different samples, all of which resulted negative. SARS-CoV-2 was not found in the peritoneal cavity of 6 patients undergoing abdominal surgery. The risk of transmissibility of COVID-19 during surgery is still unclear.

Multi-centric evaluation of a stage-specific reverse transcriptase-polymerase chain reaction assay as a xenomonitoring tool for the detection of infective (L3) stage Wuchereria bancrofti in vectors.

Background & objectives:An infective stage specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay utilizing the abundant larval transcript-3 (Alt-3) gene of Wuchereria bancrofti was developed at ICMR-VCRC, Puducherry and found to be stage specific, and sensitive upon validation in the laboratory. This study was aimed at independently evaluating this assay for its utility as a monitoring/surveillance tool in the operational programme for elimination of lymphatic filariasis (LF) by four national research laboratories.Methods:Evaluation of the assay was carried out in a multi-centric mode in three phases. In phase I, a workshop was conducted to impart hands-on training to the scientists from the collaborating centres on the RT-PCR assay and in Phase II the assay was evaluated for specificity and sensitivity in detecting the infective (L3) stage larvae of W. bancrofti in its vector, Culex quinquefasciatus, using 50 coded pooled samples. Phase III evaluation was done on wild-caught mosquito vectors from selected endemic areas of Assam and Bhubaneswar States and Andaman Nicobar islands.Results:Phase I data indicated that the assay was able to detect all the pools of mosquito samples contaning L3 stage larvae of W. bancrofti as positive, even in the presence of other vector stages of the parasite indicating its stage specificity (100%). The assay was found highly sensitive (100%), detecting all the infected pools as positive and specific detecting all uninfected pools as negative. The results of phase II showed inter-laboratory variation. Phase III evaluation from all the centres suggested that the infectivity rate determined for pooled mosquitoes by the RT-PCR assay (0.5%) was comparable to that by dissection method (1.2%) (95% confidence interval overlaps).Interpretation & conclusions:Overall, the results from three of the four participating centres indicated that the assay is at least as sensitive and stage specific as the conventional mosquito dissection technique, and hence, may be useful as a xenomonitoring tool for Transmission Assessment Survey in Mass Drug Administration programmes for LF.

Detection of SARS-CoV-2 by real time Reverse Transcriptase-Polymerase Chain Reaction assay in pleural effusion

SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) is a novel coronavirus identified for the first time in Wuhan (China) in 2019, responsible of the current pandemic infection known as Coronavirus-19 disease (COVID-19). Wide range of clinical presentation of COVD -19 has been observed, from asymptomatic carriers to ARDS. The common signs and symptoms of SARS-CoV-2 infection include fever, fatigue, dry cough, and dyspnoea; the severity of the disease is due to the impairment of the respiratory function. The radiological findings include a large variety of lesions; bilateral interstitial pneumonia is the most concerning presentation of COVID-19. Pleural involvement has been described in a minority of cases: pleural thickening had been observed in 32% of cases whereas pleural effusion is uncommon being described in only 5%. Furthermore, pleural involvement has been significantly associated with a worse prognosis. Coronavirus 2 (SARS-CoV-2), beyond the nasopharyngeal swab, has been detected in other samples; up to now, data about RT-PCR specific results in the pleural fluid of patients suffering from coronavirus disease 2019 5 (COVID-19) are very limited. The current gold standard for diagnosis is nucleic acid detection by real time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in nasopharyngeal swab. In this report, a case of a positive RT-PCR for Sars-Cov-2 in the pleura fluid and in the naso- pharyngeal swab of a patient affected by bilateral interstitial pneumonia and severe respiratory failure is described. As the presence of SARS-Cov-2 in the pleural fluid seems to be associated to a poor prognosis, physicians should carry out the specific RT-PCR assay both in the nasopharyngeal swab and in the pleural sample also when the fluid amount is very scarce and not recognizable in the chest X ray. Furthermore, the analysis of multiple samples allows to increase the test reliability.

Next-Generation Sequencing Combined With Conventional Sanger Sequencing Reveals High Molecular Diversity in Actinidia Virus 1 Populations From Kiwifruit Grown in China.

Kiwifruit (Actinidia spp.) is native to China. Viral disease–like symptoms are common on kiwifruit plants. In this study, six libraries prepared from total RNA of leaf samples from 69 kiwifruit plants were subjected to next-generation sequencing (NGS). Actinidia virus 1 (AcV-1), a tentative species in the family Closteroviridae, was discovered in the six libraries. Two full-length and two near-full genome sequences of AcV-1 variants were determined by Sanger sequencing. The genome structure of these Chinese AcV-1 variants was identical to that of isolate K75 and consisted of 12 open reading frames (ORFs). Analyses of these sequences together with the NGS-derived contig sequences revealed high molecular diversity in AcV-1 populations, with the highest sequence variation occurring at ORF1a, ORF2, and ORF3, and the available variants clustered into three phylogenetic clades. For the first time, our study revealed different domain compositions in the viral ORF1a and molecular recombination events among AcV-1 variants. Specific reverse transcriptase–polymerase chain reaction assays disclosed the presence of AcV-1 in plants of four kiwifruit species and unknown Actinidia spp. in seven provinces and one city.

Elective Surgery in the COVID-19 Era: A Screening Algorithm and Related Concerns

Surgical InfectionsVol. 22, No. 5 Letters to the EditorFree AccessElective Surgery in the COVID-19 Era: A Screening Algorithm and Related ConcernsFrancesco Taliente, Pietro Mascagni, Francesco Santullo, Francesco Ardito, and Fabio PacelliFrancesco TalienteAddress correspondence to: Dr. Francesco Taliente, Fondazione Policlinico Universitario Agostino Gemelli IRCCS—Rome, L. go Agostino Gemell, Rome 8-00168, Italy E-mail Address: talientef@gmail.comHepatobiliary Surgery Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.Search for more papers by this author, Pietro MascagniFondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy.Search for more papers by this author, Francesco SantulloSurgical Unit of Peritoneum and Retroperitoneum, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.Search for more papers by this author, Francesco ArditoHepatobiliary Surgery Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.Search for more papers by this author, and Fabio PacelliSurgical Unit of Peritoneum and Retroperitoneum, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.Search for more papers by this authorPublished Online:18 May 2021https://doi.org/10.1089/sur.2020.323AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail To the Editor:Surgery in the era of coronavirus disease 2019 (COVID-19) is uncharted territory. Non-urgent surgery was discouraged at the beginning of the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The need to reserve intensive care units for patients with COVID-19, the fear of spreading the infection through the hospital, and the possible worsening of surgical outcomes [1] were the main factors that motivated the postponement elective surgery. It has been estimated that as many as 28,404,603 operations were canceled during the first 12-week peak of the pandemic [2]. In the absence of scientific evidence on how best to resume surgical activity, we propose the algorithm used to screen potential surgical patients in a usually high-volume, tertiary referral university hospital in Rome, Italy (Fig. 1).FIG. 1. Flow chart of our screening algorithm. PCR = polymerase chain reaction; COVID-19 = coronavirus 2019.Patients are screened by telephone interview for symptoms and recent contacts. If negative, patients access the hospital the morning of the day before surgery. Both rapid enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 immunoglobulin G-immunoglobulin M (IgG-IgM) and reverse transcriptase-polymerase chain reaction (RT-PCR) for viral RNA in nasopharyngeal swabs are performed. The results of the serologic test are available within minutes. If negative, the patient is granted access to the ward with the RT-PCR result pending. Surgery is only confirmed for the next day once the swab result is negative, usually in the afternoon. If the ELISA rapid test is positive, the patient is sent to a special ward for suspected COVID-19 cases. In these circumstances, patients will only be confirmed for surgery if two consecutive RT-PCR swabs are negative. At any point if the RT-PCR swab is positive or symptoms develop, the patient is sent to a COVID-19 ward and the decision whether to perform surgery is then made by a multidisciplinary team.Recently, a female patient with a liver recurrence from cervical squamous cell carcinoma was scheduled for an explorative laparotomy. She was found to be IgM-positive and was isolated immediately. Two consecutive RT-PCR results were negative, hence the patient proceeded to surgery 48 hours after admission. A segment 6 liver resection was performed. The operation and post-operative course were uneventful. Four months after surgery the patient is well. No hospital staff developed COVID-19 symptoms.The viral RNA and ELISA rapid tests have different diagnostic windows, sensitivity, and specificity. The detection of viral RNA by RT-PCR is more likely from the onset of symptoms to three weeks afterward and has a specificity of 100% [3], whereas the detection of antibodies by ELISA likely becomes positive two weeks from onset of symptoms and has a high sensitivity [4]. These considerations, together with the rationale of limiting the number and duration of hospital access, call for the sequential combination of the two tests: the sensitive (and rapid) serology for screening and the specific (and slower) RT-PCR for diagnosis confirmation, as proposed in the algorithm presented above. However, conflicting results can occur, leading to complex decision-making. The case presented here may be interpreted either as a false-positive ELISA test or as an asymptomatic infection with positive IgM. Therefore, if the second hypothesis were true, questions arise. Are asymptomatic, IgM-positive patients at higher risk of post-operative complications? What types of precautions are needed when treating an IgM-positive patient?Research on these and other questions related to cost-effectiveness of systematic screening should be encouraged to resume surgical activities optimally and safely in the COVID-19 era. Awareness of the time and effort required to generate the much-needed data, early experiences, and doubts could be beneficial to physicians facing similar situations.

Hospital indoor air quality monitoring for the detection of SARS-CoV-2 (COVID-19) virus

On December 31, 2019, the novel human coronavirus (COVID-19) was identified in Wuhan, China and swiftly spread in all nations and territories around the globe. There is much debate about the major route of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmissions. So, more evidence is required to determine the potential pathway of transmission of SARS-CoV-2 including airborne transmission. Therefore, we examined the potential aerosol transmission of the virus through hospital wards indoor air by confirmed COVID-19 patients on May 7, 2020. In order to capture airborne SARS-CoV-2, the liquid impinger biosampler was used to take fourteen air samples in different wards of the indoor air of the hospital. The specific primer and probe real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were applied to detect viral genomes of the SARS-CoV-2 virus in positive air samples. Accordingly, we found two positive air samples (in the ICU) out of 14 ones taken from different wards with confirmed COVID-19 patients. The results revealed the possibility of airborne transmission of SARS-CoV-2 though more studies are required to determine the role of actual mechanisms such as cough, sneeze, normal breathing and speaking in the emission of airborne size carrier aerosols. Likewise, more quantitative analyses are needed to estimate airborne viability of SARS-CoV-2 in the carrier aerosols.

Detection of Novel Coronavirus on the Surface of Environmental Materials Contaminated by COVID-19 Patients in the Republic of Korea.

This study aimed to determine the presence of SARS-CoV-2 on surfaces frequently touched by COVID-19 patients, and assess the scope of contamination and transmissibility in facilities where the outbreaks occurred. In the course of this epidemiological investigation, a total of 80 environmental specimens were collected from 6 hospitals (68 specimens) and 2 “mass facilities” (6 specimens from a rehabilitation center and 6 specimens from an apartment building complex). Specific reverse transcriptase-polymerase chain reaction targeting of RNA-dependent RNA polymerase, and envelope genes, were used to identify the presence of this novel coronavirus. The 68 specimens from 6 hospitals (A, B, C, D, E, and G), where prior disinfection/cleaning had been performed before environmental sampling, tested negative for SARS-CoV-2. However, 2 out of 12 specimens (16.7%) from 2 “mass facilities” (F and H), where prior disinfection/cleaning had not taken place, were positive for SARS-CoV-2 RNA polymerase, and envelope genes. These results suggest that prompt disinfection and cleaning of potentially contaminated surfaces is an effective infection control measure. By inactivating SARS-CoV-2 with disinfection/cleaning the infectivity and transmission of the virus is blocked. This investigation of environmental sampling may help in the understanding of risk assessment of the COVID-19 outbreak in “mass facilities” and provide guidance in using effective disinfectants on contaminated surfaces.

First Complete Genome of Lake Sinai Virus Lineage 3 and Genetic Diversity of Lake Sinai Virus Strains From Honey Bees and Bumble Bees.

The complete genome of Lake Sinai virus 3 (LSV3) was sequenced by the Ion Torrent next-generation sequencing (NGS) technology from an archive sample of honey bees collected in 2010. This strain M92/2010 is the first complete genome sequence of LSV lineage 3. From October 2016 to December 2017, 56 honey bee samples from 32 different locations and 41 bumble bee samples from five different locations were collected. These samples were tested using a specific reverse transcriptase-polymerase chain reaction (RT-PCR) method; 75.92% of honey bee samples and 17.07% of bumble bee samples were LSV-positive with the RT-PCR method. Phylogenetic comparison of 557-base pair-long RNA-dependent RNA polymerase (RdRp) genome region of selected 23 positive samples of honey bees and three positive bumble bee samples identified three different LSV lineages: LSV1, LSV2, and LSV3. The LSV3 lineage was confirmed for the first time in Slovenia in 2010, and the same strain was later detected in several locations within the country. The LSV strains detected in bumble bees are from 98.6 to 99.4% identical to LSV strains detected among honey bees in the same territory.

Suggestions casted to the novel coronavirus nucleic acid amplification test from viral pneumonia pathogenesis/ 中华检验医学杂志

An outbreak of Novel Coronavirus (2019-nCoV), results in Coronavirus disease that began in Wuhan, China, has spread rapidly with cases now confirmed in multiple countries. Nucleic acid amplification test (NAAT), represent by reverse-transcriptase polymerase chain reaction (RT-PCR) plays an important role in disease diagnosis and treatment evaluation. The test results by RT-PCR have attracted much attention recently. As understanding to this novel pathogen is still limited, it would be much help to combine the knowledge about its pathogenesis to judge the test results, in addition to review the quality control in laboratory. This review will focus on understanding the specific RT-PCR performance of the 2019-nCoV, under the background of viral pneumonia. The purpose of this review is to add value to NAAT of 2019-nCoV, with combined knowledge of epidemiology, pathogenesis, clinical characteristics and pre-analysis quality control from viral pneumonia. Key words: Pneumonia, viral; Coronavirus; Nucleic acids; Clinical laboratory techniques; Quality control

Broilers of live bird markets at Mymensingh city carrying H9N2 Avian influenza virus in their respiratory tract

Avian influenza (AI) caused by Type A influenza virus is a global zoonosis, infecting vast majority of mammalian and avian species. Broilers are meat type birds and randomly reared and sold by the farmers in Bangladesh with poor biosecurity. This study was aimed to identify the Type and subtypes of AI viruses in the broilers of two live bird markets, Mymensingh. A total of 10 birds from each of the market were randomly selected, investigated by clinical, pathological, reverse transcriptase polymerase chain reactions (RT-PCR), sequencing and sequence analysis. Out of 20 birds investigated, 06 were sick, 02 were dead and 12 were apparently healthy. Clinically, the sick/dead birds did not reveal any changes typical to AI. During necropsy, the sick/dead birds showed congested lungs and moderate hemorrhages in the trachea. Such lesions was absent in the lungs of apparently healthy birds. Following histopathological examination interstitial pneumonia with bronchitis was seen in sick/dead birds. The RT-PCR protocol was adapted to identify matrix protein gene of Type A influenza virus and amplified 430bp fragment is even cases. To identify the sub types of AI viruses involved, hemagglutinin (HA) and neuraminidase (NA) gene specific RT-PCR was carried out. 1475bp and 1089bp amplicons specific to HA and NA genes of AI viruses were generated in 07 cases. The cDNAs of HA and NA genes were sequenced, edited and revealed that the AI virus circulated in the live bird market of Mymensingh city is H9N2 subtype. Two sick, one dead and four apparently healthy birds found to carry H9N2 AI virus. The H9N2 virus is naturally low pathogenic for poultry, has got public health significance, and may donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants of AI viruses; this was main concern for AI viral outbreak investigation in this study. It needs to examine large number of samples from wider sources to trace the rate of mutation and subsequent reemergence of pandemic AI viruses.Res. Agric., Livest. Fish.5(2): 225-233, August 2018

Distribution of foot and mouth disease virus serotypes in cattle of Bangladesh

Foot and mouth disease (FMD) is a highly contagious viral infection in cloven hoofed domestic and wild animals and endemic in many countries of the world including Bangladesh. Clinical investigation was carried out to identify natural cases of FMD and characteristics signs of FMD like salivation, ulceration in oral and pedal tissues and lameness was seen. The specific serotypes of FMD viruses involved in infected cattle were, therefore, identified using reverse transcriptase polymerase chain reaction (RT-PCR). Samples (N=97) from oral lesions was collected from infected cattle from seven divisions of Bangladesh during May to December, 2013. Viral RNA was extracted from the infected oral tissues and FMD virus specific uniplex RT-PCR was designed to detect FMD viruses. Multiplex RT-PCR was adapted to detect serotype specific amplicons. Out of 97 samples tested in uniplex and multiplex RT-PCR, 92 and 90 samples showed amplification reaction for FMD virus and viral serotypes respectively. Among the 90 FMD virus specific positive identification, single infectivity due to FMD viral Serotypes O, A and Asia 1 were seen in 56 (62.2%), 13 (14.4%) and 16 (17.8%) cases respectively. Three cattle (3.3%) were co-infected with FMD viral Serotypes O and Asia 1 and two (2.2%) with FMD viral Serotypes O and A. FMD viral serotype O was dominating all over the country followed by Asia1 and A. Cattle of Bangladesh were infected with FMD viral serotype O, A and Asia 1 alone or in combination. The RT-PCR protocols designed and adapted successfully detected FMD viruses and viral serotypes in a fraction of the time required for virus isolation and serological detection. These RT-PCR protocols can be used for rapid serotyping of FMD viruses from filed infectivity and selection of vaccine viruses.SAARC J. Agri., 15(1): 33-42 (2017)

Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses

Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV.

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.

Effects of imperfect test sensitivity and specificity on observational studies of influenza vaccine effectiveness

BackgroundThe recently developed test-negative design is now standard for observational studies of influenza vaccine effectiveness (VE). It is unclear how influenza test misclassification biases test-negative VE estimates relative to VE estimates from traditional cohort or case-control studies. MethodsWe simulated populations whose members may develop acute respiratory illness (ARI) due to influenza and to non-influenza pathogens. In these simulations, vaccination reduces the risk of influenza but not of non-influenza ARI. Influenza test sensitivity and specificity, risks of influenza and non-influenza ARI, and VE were varied across the simulations. In each simulation, we estimated influenza VE using a cohort design, a case-control design, and a test-negative design. ResultsIn the absence of influenza test misclassification, all three designs accurately estimated influenza VE. In the presence of misclassification, all three designs underestimated VE. Bias in VE estimates was slightly greater in the test-negative design than in cohort or case-control designs. Assuming the use of highly sensitive and specific reverse-transcriptase polymerase chain reaction tests for influenza, bias in the test-negative studies was trivial across a wide range of realistic values for VE. DiscussionAlthough influenza test misclassification causes more bias in test-negative studies than in traditional cohort or case-control studies, the difference is trivial for realistic combinations of attack rates, test sensitivity/specificity, and VE.

Isolation and Identification of bluetongue virus from goat

A total of 18 goat blood samples were processed for Bluetongue virus isolation in embryonated chicken eggs (ECE) and nine samples showed pathogenic changes on chicken embryo. All the nine ECE isolates adapted well to Baby Hamster Kidney 21 (BHK-21) cell culture and produced visible characteristic cytopathic effect (CPE) from third passage and onwards. Out of nine isolates showing CPE in BHK-21 cell line, three isolates produced 274 bp amplicons in NS1 gene specific Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) while in nested PCR, five isolates yielded a DNA band of 101 bp size. The NS1 gene based PCR confirmed the samples as BTV.

Downregulation of miRNAs during Delayed Wound Healing in Diabetes: Role of Dicer.

Delayed wound healing is a major complication associated with diabetes and is a result of a complex interplay among diverse deregulated cellular parameters. Although several genes and pathways have been identified to be mediating impaired wound closure, the role of microRNAs (miRNAs) in these events is not very well understood. Here, we identify an altered miRNA signature in the prolonged inflammatory phase in a wound during diabetes, with increased infiltration of inflammatory cells in the basal layer of the epidermis. Nineteen miRNAs were downregulated in diabetic rat wounds (as compared with normal rat wound, d 7 postwounding) together with inhibited levels of the central miRNA biosynthesis enzyme, Dicer, suggesting that in wounds of diabetic rats, the decreased levels of Dicer are presumably responsible for miRNA downregulation. Compared with unwounded skin, Dicer levels were significantly upregulated 12 d postwounding in normal rats, and this result was notably absent in diabetic rats that showed impaired wound closure. In a wound-healing specific quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) array, 10 genes were significantly altered in the diabetic rat wound and included growth factors and collagens. Network analyses demonstrated significant interactions and correlations between the miRNA predicted targets (regulators) and the 10 wound-healing specific genes, suggesting altered miRNAs might fine-tune the levels of these genes that determine wound closure. Dicer inhibition prevented HaCaT cell migration and affected wound closure. Altered levels of Dicer and miRNAs are critical during delayed wound closure and offer promising targets to address the issue of impaired wound healing.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV): a perpetual challenge.

Ann Saudi Med 2013 September-October www.annsaudimed.net 427 The emergence of a novel human coronavirus recently renamed the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the Arabian Peninsula has created global alarm because it is the causative agent of a severe and frequently fatal acute respiratory illness (SARI) resembling the illness caused by severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV).1-4 The case fatality rate (CFR) in patients infected with MERS-CoV is high—estimated at 43% in 147 patients reported so far by World Health Organization (WHO).3 This rate is higher than that of SARS—estimated at 15%, and is strongly ageand sex-dependent.4 Although the source of virus in patients with sporadic infection remains unknown, it appears likely to be some species of animal.4,5 Clear evidence of limited human-to-human transmission of MERS-CoV has now been documented in several case clusters, including particularly family members and patients in health care facilities,6-8 but all such clusters have, at least thus far, been limited in extent. However, a real concern persists that the virus will adapt to interhuman transmission and switch from an aborted epidemic to a pandemic similar to the SARS-CoV epidemic in 2003–2004. MERS-CoV is transmitted through droplets and contact. In the case of invasive respiratory procedures, MERS-CoV is transmitted through airborne route.2 Early diagnosis and strict implementation of the core components for infection prevention and control programs are crucial for preventing epidemic amplification.2 In the absence of an effective vaccine and a specific antiviral treatment, there is an urgent need to rapidly identify potential therapeutics.