Reverse Transcriptase-polymerase Chain Reaction Detection Research Articles

Epidemiological Parameters of Dengue Infections in Pakistan

Objective: The aim of this research work was to find out epidemiological features associated to age, nature of prevalence, serology and severity of the disease. Methodology: This study was conducted in Benazir Bhutto Hospital; Rawalpindi and the duration of this study was from June 2021 to November 2021. We collected total 149 specimens of sera of the suspected patients appearing with the symptoms of dengue. We analyzed the gathered samples with the help of serological detection of IgM (Immunoglobulin-M), IgG (Immunoglobulin-G), RT-PCR (Reverse transcriptase-Polymerase chain reaction) and culture of virus in CelllineC36/36. Results: Findings of this research work showed that out of total one hundred and forty-nine specimens, 32.20% (n: 48) were identified with dengue virus with the use of RT-PCR & cell culture and 72.50% (n: 108) samples were found positive by serology. Of one hundred and eight cases, 26.80% (n: 48) patients were IgM positive, 41.0% (n: 61) were both IgM & IgG positive and 4.70% (n: 07) patients were IgG positive only. There was very high prevalence of dengue in age-group of patients having 20-29 years of age and the incidence of the infection was much high in male patients as compared to females. There was highest epidemic intensity in month of June as compared to other months. Practical Implication: This current research makes it possible to classify the serological results of dengue by mixed primary-secondary, primary and secondary dengue infections. There is also relation of dengue infection with the season as there is high activity of this infection after season of monsoon Conclusion: The prevalence of dengue is very endemic in our country Pakistan and the most vulnerable age group was 20-29 years for this infection. There is strong need of the effective prevention as well as control measures for the reduction of this infection. Keywords: Infection, serology, dengue, RT-PCR, IgM, epidemiology, IgG, prevention, prevalence, incidence, endemic.

Evaluation of the SARS-CoV-2 positivity ratio and upper respiratory tract viral load among asymptomatic individuals screened before hospitalization or surgery in Flanders, Belgium.

IntroductionThe incidence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections in the Belgian community is mainly estimated based on test results of patients with coronavirus disease (COVID-19)-like symptoms. The aim of this study was to investigate the evolution of the SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) positivity ratio and distribution of viral loads within a cohort of asymptomatic patients screened prior hospitalization or surgery, stratified by age category.Materials/MethodsWe retrospectively studied data on SARS-CoV-2 real-time RT-PCR detection in respiratory tract samples of asymptomatic patients screened pre-hospitalization or pre-surgery in nine Belgian hospitals located in Flanders over a 12-month period (1 April 2020–31 March 2021).ResultsIn total, 255925 SARS-CoV-2 RT-PCR test results and 2421 positive results for which a viral load was reported, were included in this study. An unweighted overall SARS-CoV-2 real-time RT-PCR positivity ratio of 1.27% was observed with strong spatiotemporal differences. SARS-CoV-2 circulated predominantly in 80+ year old individuals across all time periods except between the first and second COVID-19 wave and in 20–30 year old individuals before the second COVID-19 wave. In contrast to the first wave, a significantly higher positivity ratio was observed for the 20–40 age group in addition to the 80+ age group compared to the other age groups during the second wave. The median viral load follows a similar temporal evolution as the positivity rate with an increase ahead of the second wave and highest viral loads observed for 80+ year old individuals.ConclusionThere was a high SARS-CoV-2 circulation among asymptomatic patients with a predominance and highest viral loads observed in the elderly. Moreover, ahead of the second COVID-19 wave an increase in median viral load was noted with the highest overall positivity ratio observed in 20–30 year old individuals, indicating they could have been the hidden drivers of this wave.

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.

Impact of DNA Demethylases on the DNA Methylation and Transcription of Arabidopsis NLR Genes.

Active DNA demethylation is an important epigenetic process that plays a key role in maintaining normal gene expression. In plants, active DNA demethylation is mediated by DNA demethylases, including ROS1, DME, DML2, and DML3. In this study, the available bisulfite sequencing and mRNA sequencing data from ros1 and rdd mutants were analyzed to reveal how the active DNA demethylation process shapes the DNA methylation patterns of Arabidopsis nucleotide-binding leucine-rich repeat (NLR) genes, a class of important plant disease resistance genes. We demonstrate that the CG methylation levels of three NLR genes (AT5G49140, AT5G35450, and AT5G36930) are increased in the ros1 mutants relative to the wild-type plants, whereas the CG methylation level of AT2G17050 is decreased. We also observed increased CG methylation levels of AT4G11170 and AT5G47260 and decreased CG methylation levels of AT5G38350 in rdd mutants. We further found that the expression of three NLR genes (AT1G12280, AT1G61180, and AT4G19520) was activated in both ros1 and rdd mutants, whereas the expression of another three NLR genes (AT1G58602, AT1G59620, and AT1G62630) was repressed in these two mutants. Quantitative reverse transcriptase–polymerase chain reaction detection showed that the expression levels of AT1G58602.1, AT4G19520.3, AT4G19520.4, and AT4G19520.5 were decreased in the ros1 mutant; AT3G50950.1 and AT3G50950.2 in the rdd mutant were also decreased in expression compared to Col-0, whereas AT1G57630.1, AT1G58602.2, and AT5G45510.1 were upregulated in the rdd mutant relative to Col-0. These results indicate that some NLR genes are regulated by DNA demethylases. Our study demonstrates that each DNA demethylase (ROS1, DML2, and DML3) exerts a specific effect on the DNA methylation of the NLR genes, and active DNA demethylation is part of the regulation of DNA methylation and transcriptional activity of some Arabidopsis NLR genes.

One-Step reverse transcriptase PCR for detection of arboviruses in serum samples of patients assisted in Basic health Units in the State of Maranhão, Brazil

One-Step reverse transcriptase PCR for detection of arboviruses in serum samples of patients assisted in Basic health Units in the State of Maranhão, Brazil

Entomological surveillance of Zika virus in Sardinia, Italy, 2016.

Zika Virus (ZIKV) is a RNA virus belonging to the genus Flavivirus, family Flaviviridae. This virus is transmitted through bite of Aedes mosquitoes, in particular Ae. aegypti. On February 1st 2016, the World Health Organization (WHO) has declared ZIKV a Public Health Emergency of International Concern. Successively, considering the establishment of Ae. albopictus, WHO has classified Italy as having a moderate likelihood of local transmission of ZIKV, preceded in Europe only by France. For this reason an entomological surveillance plan was been activated in Sardinia in 2016. BG Sentinel Mosquito Traps have been positioned in 29 sites, comprising urban areas and points of entry, as ports and airports. Mosquitoes were collected fortnightly from April to December. A total of 3,089 mosquitoes were collected belonging to 10 species. The most numerous species have been Cx. pipiens s.l. and Ae. albopictus. All mosquitoes sampled have been assayed by real time reverse transcriptase PCR for detection of ZIKV RNA. A total of 584 pool have been analyzed and have been reported no evidence of ZIKV. A permanent entomological surveillance should be implemented principally in the urban areas and points of entry, as ports and airports, because Ae. albopictus, susceptible to ZIKV, is established in Sardinia and also know the recent introduction of invasive mosquitoes species Ae. koericus and Ae. japonicus in Italy.

AR-V7 Transcripts in Whole Blood RNA of Patients with Metastatic Castration Resistant Prostate Cancer Correlate with Response to Abiraterone Acetate

The expression of AR-V7 (androgen receptor splice variant) 7 in circulating tumor cells has been associated with resistance to abiraterone and enzalutamide in patients with metastatic castration resistant prostate cancer. We used a sensitive, whole blood reverse transcriptase-polymerase chain reaction assay that does not require circulating tumor cell enrichment to correlate outcomes of abiraterone with whole blood expression of AR-V7 and other prostate cancer associated transcripts. We assessed the expression of AR-V7, FOXA1, GRHL2, HOXB13, KLK2, KLK3 and TMPRSS2:ERG mRNA in 2.5 ml whole blood from each of 27 patients with metastatic castration resistant prostate cancer and 33 controls without cancer as the discovery cohort. Cycle threshold values of controls with the highest gene expression were set as the threshold for a positive test. Thresholds were then applied to a validation cohort of 37 patients with metastatic castration resistant prostate cancer who were commencing abiraterone. Gene expression was correlated with the prostate specific antigen response rate using the chi-square test, and with time to prostate specific antigen progression and overall survival using the log rank test. In the discovery cohort 3 of 27 patients (11.1%) with metastatic castration resistant prostate cancer were AR-V7 positive vs 4 of 37 (10.8%) in the validation cohort. In the validation cohort patients with a positive AR-V7 test had a lower prostate specific antigen response rate (0% vs 42%, p = 0.27) together with shorter median prostate specific antigen progression (0.7 vs 4.0months, p <0.001) and median overall survival (5.5 vs 22.1 months, p<0.001). Reverse transcriptase-polymerase chain reaction detection of AR-V7 transcripts in whole blood was associated with inferior outcomes in patients treated with abiraterone. These results reinforce the potential usefulness of AR-V7 as a prognostic and predictive biomarker for metastatic castration resistant prostate cancer.

Comparison of RNA extraction methods for RT-PCR detection of Coconut cadang-cadang viroid variant in orange spotting oil palm leaves

Coconut cadang-cadang viroid (CCCVd) is a lethal disease that has devastated the coconut industry in the Philippines. Recently, a CCCVd variant associated with orange spotting of foliage, which is a phenomenon reported in the Malaysian oil palm industry, occurring as an isolated palm with no similar symptoms on the adjacent neighbouring palms. The presence of this viroid in oil palms is difficult to detect as it is found in very low concentrations. Thus, an efficient RNA extraction method for isolating the CCCVd variant and to detect it through reverse transcriptase polymerase chain reaction (RT-PCR) using specific primers for CCCVd was required. Leaf samples from symptomatic and asymptomatic oil palms were collected from various locations in Malaysia, namely, Negeri Sembilan, Selangor and Perak. The samples were subjected to three RNA extraction methods – natrium chloride EDTA Tris-HCl mercaptoethanol extraction (NETME), polyethylene glycol extraction (PEG) and cetyltrimethylammonium bromide extraction (CTAB). The quantification of the RNA, based on optical density (OD) and concentration, showed that the CTAB extraction was the best, followed by the NETME extraction. The RNA extraction using PEG resulted in poor yields and had the lowest purity. The viroid was detected in all of the samples extracted via CTAB and NETME by RT-PCR with reduced starting material compared with PEG extracted RNA. A BLAST analysis indicated that the viroid sequences are highly conserved and shared a 93% sequence identity with the CCCVd246 variant from oil palm. Phylogenetic analysis showed that the variant belonged to an outgroup from other cocadviroids found in monocots.

Upregulation of microRNA-31 targeting integrin α5 suppresses tumor cell invasion and metastasis by indirectly regulating PI3K/AKT pathway in human gastric cancer SGC7901 cells.

To verify the hypothesis that upregulation of microRNA-31 (miR-31) targeting integrin α5 (ITGA5) suppresses tumor cell invasion and metastasis by indirectly regulating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human SGC7901 gastric cancer (GC) cells. The miRTarBase was used to predict whether ITGA5 is the target gene of miR-31, which was further confirmed by luciferase reporter gene assay. The SGC7901 GC cells were divided into five groups including the blank, miR-31 mimic, miR-31 mimic control, miR-31 inhibitor, and miR-31 inhibitor control groups. Reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, cell scratch test, and transwell assays were respectively performed in our study. TGA5 was found as the target gene of miR-31. The RT-PCR detection revealed that, compared with the blank group, ITGA5 messenger RNA (mRNA) expression decreased in the miR-31 mimic group, but increased in the miR-31 inhibitor group. The western blotting examination suggested that the expressions of ITGA5, PI3K, and AKT proteins reduced in the miR-31 mimic group, but enhanced in the miR-31 inhibitor group when compared to the blank group, respectively. The cell scratch and transwell assays indicated that the miR-31 expressions were negatively associated with GC cell migration and invasion. Besides, RT-PCR combined with western blotting demonstrated that the miR-31 expressions were higher in the normal tissues than those in the GC tissues, while the ITGA5 mRNA and protein showed lower expression in the normal tissues than they did in the GC tissues. Our study concluded that upregulation of miR-31 targeting ITGA5 may suppress tumor cell invasion and metastasis by indirectly regulating PI3K/AKT signaling pathway in human SGC7901 GC cells.

Design and Evaluation of the Primers for Rift Valley Fever (RVF) Virus RT-PCR Detection

Rift Valley Fever (RVF) is a notifiable multiple species diseases in the OIE list, and causes human and agricultural losses in endemic regions. To develop the rapid method for detecting of RVF, 2 specific primers for reverse transcriptase polymerase chain reaction (RT-PCR) and 7 overlapping oligo primers were designed according to the nucleotide sequence information of RVFV published in GenBank, and a DNA fragment about 318 bp of the segment S was synthesized in vitro by overlap extension PCR to construct the recombinant plasmid pMD19-T-RVFVS. Then, the 2 specific primers were evaluated via a serial of tests, including reaction temperature optimization test, sensitivity and specificity tests. The results showed that the 2 designed primers are suitable for RVFV RT-PCR detection which is a rapid method with good specificity and sensitivity, the detection limit was approximately 85 copies of the cloned viral genomic fragments (pMD19-T-RVFVS) as well as resulted in no cross-reaction for peste des petits ruminants virus (PPRV), Epidemic encephalitis B virus, E.coli , Salmonella and Pasteurella multocida etc common pathogens isolated from ruminants detection.

Human Parechovirus Central Nervous System Infections in Southern California Children

Human parechoviruses (hPeV) are increasingly recognized as significant etiological agents for meningoencephalitis especially in young children, but testing of cerebrospinal fluid (CSF) for hPeV by PCR is not routinely performed. We used real-time reverse transcriptase PCR for detection of serotypes 1-6 in CSF samples of 440 children who underwent a lumbar puncture to exclude an infectious etiology of their clinical presentation. We then compared the prevalence and clinical presentation of children with hPeV-positive CSF with that of children with enterovirus (EV)-positive CSF. HPeV was detected in 2.7% and EV in 10.7% of CSF samples. Many hPeV-positive patients were <3 months of age and usually had CSF parameters within the age-adjusted normal range. However, children with hPeV-positive CSF presented with neurologic symptoms more frequently than those with EV-positive CSF. HPeV infections of the central nervous system occurred mainly in young infants and were more commonly associated with neurologic symptoms at presentation, despite the fact that CSF findings were within the normal range in the vast majority of these cases. HPeV should be included in the differential diagnosis of young children with central nervous system symptoms and sepsis-like illness, even in the presence of normal CSF parameters.

Determination of RT-PCR detection limit of live and dead Salmonella cells in raw and sterilized milk

Summary The objective of the current study was to evaluate the reproducibility of a reverse transcriptase PCR (RT-PCR)-based technique to differentiate viable and dead Salmonella cells in raw and sterilized milk. The microorganism was initially inoculated into the milk samples followed by incubating at 37°C for 4 h prior to inactivation by heat at 80°C for 10 min. The treated and non-treated samples were subsequently monitored using both PCR and RT-PCR, in vitro . Following 4 h incubation, the invA gene of Salmonella was clearly amplified by RT-PCR, while no band was detected in the heated samples. On the other hand, using the conventional PCR, it was possible to amplify the gene in both samples. Our results may suggest an important application of the RT-PCR technique, especially when the number of live organisms is an imperative factor to produce food borne infections and to establish a detection limit of the test.

English

This study was carried out to evaluate the diagnostic value of nested reverse transcriptase-polymerase chain reaction (RT-PCR) for HEV (hepatitis E virus) RNA detection relative to anti-HEV immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays. One hundred and twenty six (126) patients with sporadic acute hepatitis E were included in this study. We focused on the popular genotype I and IV of HEV-positive patients in China, and selected the conserved region located in ORF2 for designing a set of nested RT-PCR primers. HEV RNA was detected in 53.2% (67/126) of patients, while all control subjects were negative for HEV RNA. The total agreement between IgM and nested RT-PCR detection was 80.9%, showing a fine coincidence. The results further suggested that there was a significant difference between nested RT-PCR detection and IgM ELISA: 3 cases with positive results for HEV RNA showed negative anti-HEV IgM at the early phrase, and presented positive IgM reaction in succession after the trail of detection. HEV RNA was detected in serum samples from sporadic acute hepatitis patient usually by day 1 to 12 after the onset of symptoms, but showed a decreasing sensitivity with the increasing disease course. From these experiments, we can conclude that HEV RNA detection is of great clinical significance, which has an obvious advantage in diagnosis of early infection of HEV. Key words: Hepatitis E virus, nested reverse transcriptase-polymerase chain reaction (RT-PCR), RNA, antibody.

Further evidence for the widespread co-circulation of lineages 4b and 7 velogenic Newcastle disease viruses in rural Nigeria

Newcastle disease (ND) is an endemic disease in rural poultry of Western Africa. It may cause severe economic losses in the poultry sector and, as such, is listed as a notifiable disease by the World Organisation for Animal Health (OIE). Recently, a new genetic lineage of ND viruses was discovered in Western Africa. We determined the complete fusion (F) gene coding sequence of 12 ND viruses isolated from pigeons and rural chickens in six Nigerian states in 2007 and 2008. Phylogenetic analysis of the complete F coding sequence confirmed the circulation of genetically diverse ND isolates in a large geographic area in Nigeria. Next to isolates belonging to lineage 4b, viruses of the recently discovered lineage 7 (some of which were previously reported to escape routine real-time reverse transcriptase-polymerase chain reaction detection) were isolated in six states during the two-year period. The documented genetic variants occurred over a large geographic area, indicating an endemic circulation of these viruses. Three different velogenic fusion gene cleavage site motifs were observed. These findings confirm the endemic circulation and diversification of ND isolates in rural poultry and pigeons in Nigeria and highlight the importance of surveillance in developing countries to monitor the validity of rapid molecular diagnostic tools and of vaccination regimes.

Sensitivity and Specificity of a Competitive Enzyme-Linked Immunosorbent Assay Relative to Quantitative Reverse Transcriptase PCR for Detection of Anaplasma marginale and A. phagocytophilum

[Reinbold et al.][1] ([2][2]) reported performance results from a study comparing a commercial competitive enzyme-linked immunosorbent assay (cELISA) against a duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) developed for the detection of Anaplasma marginale and A. phagocytophilum

Reply to “Sensitivity and Specificity of a Competitive Enzyme-Linked Immunosorbent Assay Relative to Quantitative Reverse Transcriptase PCR for Detection of Anaplasma marginale and A. phagocytophilum”

We thank Pascale Aubrey and Julie Pare for their thoughtful consideration ([1][1]) of the information presented in our article ([2][2]). Many points of consideration have been presented, as well as the identification of the need for authors' corrections. Therefore, we will respond to each as they

SURVEILLANCE OF AVIAN INFLUENZA VIRUSES IN MIGRATORY BIRDS IN EGYPT, 2003–09

Migratory (particularly aquatic) birds are the major natural reservoirs for type A influenza viruses. However, their role in transmitting highly pathogenic avian influenza (HPAI) viruses is unclear. Egypt is a "funnel" zone of wild bird migration pathways from Central Asia and Europe to Eastern and Central Africa ending in South Africa. We sought to detect and isolate avian influenza viruses in migratory birds in Egypt. During September 2003-February 2009, the US Naval Medical Research Unit Number 3, Cairo, Egypt, in collaboration with the Egyptian Ministry of Environment, obtained cloacal swabs from 7,894 migratory birds captured or shot by hunters in different geographic areas in Egypt. Samples were processed by real-time reverse transcriptase PCR for detection of the influenza A matrix gene. Positive samples were processed for virus isolation in specific-pathogen-free embryonated eggs and isolates were subtyped by PCR and partial sequencing. Ninety-five species of birds were collected. Predominant species were Green-Winged Teal (Anas carolinensis; 32.0%, n=2,528), Northern Shoveler (Anas clypeata; 21.4%, n=1,686), and Northern Pintail (Anas acuta; 11.1%, n=877). Of the 7,894 samples, 745 (9.4%) were positive for the influenza A matrix gene (mainly from the above predominant species). Thirteen of the 745 (1.7%) were H5-positive by PCR (11 were low-pathogenic avian influenza and two were HPAI H5N1). The prevalences of influenza A was among regions were 10-15%, except in Middle Egypt (4%). Thirty-nine influenza isolates were obtained from PCR-positive samples. Seventeen subtypes of avian influenza viruses (including H5N1 and H7N7) were classified from 39 isolates using PCR and partial sequencing. Only one HPAI H5N1 was isolated in February 2006, from a wild resident Great Egret (Ardea alba). No major die-offs or sick migratory birds were detected during the study. We identified avian influenza virus subtypes not previously reported in Egypt. The HPAI H5N1 isolated or detected indicates that migratory birds may play a role in the dispersal of HPAI virus, but a detailed mechanism of this role needs to be elucidated.

Synovial sarcoma of the cauda equina

Primary synovial sarcoma originating from the cauda equina is extremely rare. Only one case, involving an 11-year-old girl, has been reported. The authors describe the case of a 23-year-old woman with a primary synovial sarcoma of the cauda equina. The patient visited a local hospital and described a 2-month history of low-back pain. She was referred to the authors' hospital for further evaluation. On physical examination, she had a straight-leg raising result of 70° bilaterally. Motor examination revealed Grade 4/5 strength in the bilateral extensor hallux longus muscles. There was normal sensation to light touch and vibration in the lower extremities. Sagittal Gd-enhanced T1-weighted MR imaging demonstrated an intradural, extramedullary, and uniformly enhancing mass that extended from L-3 to L-4. The mass was totally resected and adjuvant local radiation therapy was administered. Reverse transcriptase polymerase chain reaction (RT-PCR) of a paraffin-embedded tissue sample revealed SYT-SSX fusion transcripts, and the diagnosis of synovial sarcoma was confirmed. Five and a half years after surgery, the patient is free of local recurrence and metastatic disease. The RT-PCR detection of SYT-SSX fusion transcripts played a key role in establishing the diagnosis of synovial sarcoma of the cauda equina. Complete resection of the mass with adjuvant local radiation therapy proved to be effective.

Biological and Molecular Characterization of Two Distinct Tomato Strains of Cucumber mosaic virus Based on Complete RNA-3 Genome and Subgroup Specific Diagnosis.

Cucumber mosaic virus (CMV) is one of the most important viral pathogen infecting several plant species in India. Five isolates of CMV obtained from cucumber, muskmelon, tobacco and tomato from distinct geographical locations in India were analysed based on host-reactions and genome sequence. The majority of the isolates were very similar and only two isolates, Tfr-In and Tss-In showed distinct symptoms in tomato and high sequence diversity (77.8%) in coat protein (CP) gene. Tfr-In was isolated from tomato fruit showing grey patches in Aurangabad and Tss-In from tomato plant showing shoe-string symptoms in New Delhi. The RNA-3 genomes of Tfr-In (2,214 nt; JF279606), shared only 70.3% nucleotide sequence identity with Tss-In (2,178 nt; JF279605. The complete RNA-3 genome of Tss-In and Tfr-In were compared with that of 65 CMV isolates reported from various plants of the world, which formed four distinct subclades-IA, -IB, -IC and -II. The Tfr-In isolate clustered with the CMV subgroup-IB and Tss-In with the subgroup-II. The comparison of the RNA-3 sequence of both the isolates revealed maximum heterogeneity in the intergenic region (IR). Reverse transcriptase polymerase chain reaction (RT-PCR) based detection of CMV subgroup-I and -II was developed designing primers from flanking IR region. The specificity of the RT-PCR detection was confirmed using Tfr-In and Tss-In representing subgroup-I and -II and validated with field samples of tomato, cucurbits and chilli. This is the first report of complete RNA-3 of subgroup-IB CMV causing grey patches in tomato fruit and subgroup-II CMV causing shoe-string symptoms in tomato in India. The present and previous studies together showed that tomato in India was affected by multiple strains of CMV.

Construction and Clinical Application of a Human Tissue-Engineered Epidermal Membrane

Prolonged healing times and hypertrophic scarring of the donor site for split-thickness-skin grafts thicker than 0.3 mm are common problems that continue to challenge plastic surgeons in the clinic. As such, a human tissue-engineered epidermal membrane was constructed to promote wound healing and reduce scar hypertrophy. An artificial allogenic epidermis was created in vitro using human keratinocytes and chitosan-gelatin membrane. Split-thickness skin graft donor sites were divided into three treatment groups: those covered with the combined keratinocyte/chitosan-gelatin membrane, those covered with chitosan-gelatin membrane only (control group), and those covered with traditional petroleum jelly gauze (blank group). The degree of wound healing was assessed at various time points after the operation by gross observation, hematoxylin and eosin staining, immunohistochemistry, and an assay of type I collagen using the picrosirius polarization method. Reverse-transcriptase polymerase chain reaction detection of the Y chromosome was also performed to distinguish between sexes. Over a 6-month observation period, treatment with the human tissue-engineered epidermal membrane (keratinocyte/chitosan-gelatin) appeared to decrease donor-site healing time (48 wounds in 24 cases). Average healing time was 8.1 +/- 1.3 days for the keratinocyte/chitosan-gelatin group, 16.4 +/- 1.7 days for the chitosan-gelatin group, and 22.9 +/- 4.2 days for the blank group. The artificial epidermis survived well and maintained a normal structure. Furthermore, hypertrophic scar formation was less severe for these wounds. Keratinocyte/chitosan-gelatin membranes can be constructed in vitro and survive temporarily in vivo. They can be used to promote wound healing and reduce the severity of hypertrophic scarring of skin graft donor sites.