Polyacrylamide Gel Electrophoresis Of RNA Research Articles

Lambda carrageenan displays antiviral activity against the infectious pancreatic necrosis virus (IPNV) by inhibiting viral replication and enhancing innate immunity in salmonid cells

Sulfated polysaccharide lambda carrageenan (λ-CGN) was evaluated for its antiviral effect against IPNV using the in vitro infection model of CHSE-214 salmonid cells. A plaque reduction lysis assay revealed that λ-CGN has an IC50 of 0.9 μg⋅mL−1, CC50 > 128 μg⋅mL−1 and a Selectivity Index (SI) > 142. In comparison, iota, kappa carrageenans and lambda oligo-carrageenan (λ-OC) were less effective than λ-CGN against IPNV. λ-CGN showed no virucidal activity when applied directly to viral particles. Time of addition experiments showed that pre-treatment, co-treatment, and post-treatment with λ-CGN significantly reduced viral RNA copies in the cell supernatant. Additionally, a decrease in intracellular viral RNA was observed with pre-treatment and post-treatment, indicating an impact on different stages of viral replication. Polyacrylamide gel electrophoresis of IPNV genomic RNA in presence of λ-CGN showed a reduction in the level of viral genomic RNA. Confocal microscopy confirmed the intracellular localization of λ-CGN, suggesting that λ-CGN may inhibit the synthesis of IPNV genomic RNA. Moreover, cells pre-treated with λ-CGN showed an increased expression of innate immunity genes CXCL11, IL1β, IFNa, and IRF3. These findings highlight the need for further research to confirm the in vivo pharmacological potential of λ-CGN as a new antiviral agent in salmonids.

Emergence of equine-like G3 strains as the dominant rotavirus among children under five with diarrhea in Sabah, Malaysia during 2018-2019.

Rotavirus infection is a dilemma for developing countries, including Malaysia. Although commercial rotavirus vaccines are available, these are not included in Malaysia’s national immunization program. A scarcity of data about rotavirus genotype distribution could be partially to blame for this policy decision, because there are no data for rotavirus genotype distribution in Malaysia over the past 20 years. From January 2018 to March 2019, we conducted a study to elucidate the rotavirus burden and genotype distribution in the Kota Kinabalu and Kunak districts of the state of Sabah. Stool specimens were collected from children under 5 years of age, and rotavirus antigen in these samples was detected using commercially available kit. Electropherotypes were determined by polyacrylamide gel electrophoresis of genomic RNA. G and P genotypes were determined by RT-PCR using type specific primers. The nucleotide sequence of the amplicons was determined by Sanger sequencing and phylogenetic analysis was performed by neighbor-joining method. Rotavirus was identified in 43 (15.1%) children with watery diarrhea. The male:female ratio (1.9:1) of the rotavirus-infected children clearly showed that it affected predominantly boys, and children 12–23 months of age. The genotypes identified were G3P[8] (74% n = 31), followed by G1P[8] (14% n = 6), G12P[6](7% n = 3), G8P[8](3% n = 1), and GxP[8] (3% n = 1). The predominant rotavirus circulating among the children was the equine-like G3P[8] (59.5% n = 25) with a short electropherotype. Eleven electropherotypes were identified among 34 strains, indicating substantial diversity among the circulating strains. The circulating genotypes were also phylogenetically diverse and related to strains from several different countries. The antigenic epitopes present on VP7 and VP4 of Sabahan G3 and equine-like G3 differed considerably from that of the RotaTeq vaccine strain. Our results also indicate that considerable genetic exchange is occurring in Sabahan strains. Sabah is home to a number of different ethnic groups, some of which culturally are in close contact with animals, which might contribute to the evolution of diverse rotavirus strains. Sabah is also a popular tourist destination, and a large number of tourists from different countries possibly contributes to the diversity of circulating rotavirus genotypes. Considering all these factors which are contributing rotavirus genotype diversity, continuous surveillance of rotavirus strains is of utmost importance to monitor the pre- and post-vaccination efficacy of rotavirus vaccines in Sabah.

Investigation of a large waterborne acute gastroenteritis outbreak caused by group B rotavirus in Maharashtra state, India.

An acute gastroenteritis outbreak at Devli Karad village, Maharashtra, India with an attack rate of 22.6% affected mainly adolescent and adult population. The viral investigations conducted on fecal specimens of patients hospitalized indicated the presence of rotavirus B (RVB) using RNA polyacrylamide gel electrophoresis and reverse transcription polymerase chain reaction. The samples collected from the source of drinking water also showed the presence of the only RVB. Absence of other viral agents and identification of RVB of genotype G2 as the etiological agent of the acute gastroenteritis outbreak highlights, the necessity of monitoring RVB, the viral agent known for its large outbreak potential.

Occurrence of Neglected Picobirna Virus in Buffalo Calves Associated with Diarrhoea

Picobirna viruses (PBVs) thought to be neglected viruses, are emerging as causative agents of viral gastrointestinal infections in human and other animal species with enteritis. In the present study, a total of 408 fecal samples of diarrheic buffalo calves of under age of 2 months were collected from different locations in Haryana. These samples were preliminarily screened for PBV by RNApolyacrylamide gel electrophoresis (RNA-PAGE). The RNA-PAGE positive samples were further confirmed by RT-PCR using RdRp gene specific primer pair PicoB25 (+) and PicoB43 (-) for amplification of genogroup I PBV that generated an amplicon of 201 bp. Out of 408 buffalo calves fecal samples, 52 (13%) samples were found positive for genogroup 1(GGI) PBV using RT-PCR. Genogroup II (GGII) specific primer generating 369 bp product size did not show amplification in any of the samples. The results suggested occurrence of PBV in Haryana State with predominance of GGI PBV. To the best of our knowledge, this is the first report of detection as well as occurrence of GGI picobirna virus among diarrheic buffalo calves in Haryana State.

Detection of mammalian-like group a rotavirus in diarrhoeic poultry using RNA-PAGE In Kerala, India

The present work was conducted to determine the prevalence of rotavirus infection among poultry birds having diarrhoea. A total of 143 faecal samples were collected from different parts of Kerala and screened for the presence of rotavirus using RNA polyacrylamide gel electrophoresis (RNA-PAGE). Out of 143 samples, 5 (3.49%) were found to be positive in RNA PAGE with a migration pattern 4:2:3:2 of a mammalian-like electropherogroup A rotavirus. The study records the first evidence of rota virus detection from avian species in Kerala.

Identification of group B rotavirus as an etiological agent in the gastroenteritis outbreak in Maharashtra, India.

Acute gastroenteritis outbreak occurred at Pargaon, Maharashtra, India in 1789 cases with an attack rate of 32.5% between November to December 2015. The stool specimens (n = 32) were investigated for different enteric viral agents using conventional methods. Transmission electron microscopy and RNA polyacrylamide gel electrophoresis respectively identified morphologically distinct rotavirus particles in 28% and RNA migration pattern of Group B Rotavirus (GBR) in 72% of the specimens. Reverse transcription polymerase chain reaction and nucleotide sequencing confirmed presence of GBR in 97% of the samples analyzed. The predominance of GBR infections and absence or insignificant presence of other agents confirmed GBR as an etiological agent of the gastroenteritis outbreak occurred in Maharashtra, India.

Changes of haemogram and serum biochemistry in neonatal piglet diarrhoea associated with porcine rotavirus type A.

Porcine rotavirus type A (RVA) is a major cause of neonatal piglet mortality in India. The effect of the disease on haemogram and serum biochemical profile is not well established in piglets. Accordingly, we assessed the haemogram and serum biochemical profile in the neonatal piglet diarrhoea with RVA infection (n=17). The diagnosis of RVA was confirmed using RNA-polyacrylamide gel electrophoresis (RNA-PAGE), commercially available enzyme-linked immunosorbent assay (ELISA) kit and reverse transcription-polymerase chain reaction (RT-PCR). Non-infected healthy piglets (n=6) served as control. The concentrations of total protein, albumin, alanine amino transaminase (ALT), aspartate amino transaminase (AST), blood urea nitrogen (BUN) and creatinine in serum were measured by spectrophotometric method. Haemogram was done in the blood using sodium ethylenediaminetetraacetic acid (Na2 EDTA) as anticoagulant. The mean values of total protein, albumin and globulin concentrations were significantly (P<0.001) decreased and concentrations of ALT, AST, BUN and creatinine were significantly increased (P<0.001) in the RVA-infected piglets. Haemogram showed marked haemoconcentration (P<0.001), leukopenia (P<0.01) and neutropenia (P<0.01) in the presence of RVA infection than healthy piglets. The results indicated a possible extra-intestinal spread of RVA in piglets during neonatal diarrhoea. The finding might be helpful to clinicians and while treating such type of clinical cases, incorporation of organ protective drugs will be helpful for better response in the treatment schedule.

Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species.

We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings.

Molecular characterization of avian rotaviruses circulating in Italian poultry flocks

Avian rotaviruses are still largely undefined despite being widespread in several avian species and despite the economic impact of rotavirus (RV) enteritis in poultry flocks. In this study, the presence of different avian RV groups was investigated in commercial poultry flocks reared in Northern and Central Italy and with a history of enteric diseases. Faeces or intestinal contents from different avian species previously found to contain RV particles by electron microscopy (EM) were analysed by both RNA-polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction specific for groups A, D, F and G RVs. Group D avian RV was detected in 107 of 117 samples tested (91.5%), whereas groups A, F and G avian RVs were present in 70 (59%), 61 (52.1%) and 31 (26.5%) samples, respectively. Multiple presence of different RV groups was detected in 83% of samples. This study provides novel data on the prevalence of genetically different avian RVs in Italian poultry flocks. This information is useful to elucidate the epidemiology of avian RVs circulating in Italy.

Concurrent Infection of Rotavirus and Shiga ToxigenicEscherichia coli(STEC) in Buffalo Calves in Mumbai Metropolis, Western India

Faecal samples from 113 diarrhoeic cattle and buffalo calves less than one year of age, from different farms in Mumbai metropolis in India, were screened for the presence of Rotavirus and Shiga toxin-producing Escherichia coli (STEC). RNA-Poly acrylamide gel electrophoresis (RNA-PAGE) analysis of these samples revealed 11 (9.73%) as positive for group A rotavirus (GARV). Out of 11 samples found positive by RNA-PAGE, which were subjected to amplification of VP7 gene of GARV, 10 samples yielded a specific product of 1, 013 bp. The incidence of Shiga toxigenic E. coliin diarrhoeic buffalo calves was 13.23% (9 out of 68) and 6.66% in the cattle calves (3 out of 45) as demonstrated by multiplex polymerase chain reaction (PCR) of the shiga toxin (stx) genes. Eight (66.66%) isolates carried stx2 gene and four (33.33%) isolates had stx1 gene. To the best of our knowledge, we report for the first time the concurrent infection of Shiga toxigenic E. coli and GARV (6 cases; 8.82%) in diarrhoeic faecal samples of buffalo calves from India.

Isolation of bluetongue virus serotype 1 from aborted goat fetuses.

Abortions and stillbirths were noticed in pregnant goats on a farm in the state of Gujarat, India. About 50% of the pregnant goats aborted or gave birth to dead kids. Bluetongue virus (BTV) antibody in the sera of affected goats was detected using a competitive enzyme-linked immunosorbent assay (ELISA). Viral antigen in the blood of these goats and in the aborted fetal spleens was detected using a sandwich ELISA. Two viruses (SKN-9, SKN-10) were isolated in cell culture from aborted fetal spleens and were confirmed as Orbivirus by demonstration of ten bands in RNA polyacrylamide gel electrophoresis and identified as BTV-1 by sequencing of the VP2 gene. Sequence analyses revealed thatthese isolates were very closely related to a BTV-1 (strain SKN-8) isolated from Culicoides vectors captured on the same farm one month after the occurrence of abortion. Isolation of BTV-1 from fetuses is probably evidence of transplacental transmission of the wild-type strain, because attenuated or laboratory-adapted BTV-1 strains have never been used in this region. This may have important implications in the epidemiology of bluetongue, considering the presence of many BTV serotypes in India.

Pseudo-outbreak of rotavirus infection in a neonatal intensive care unit.

A rotavirus outbreak in a neonatal intensive care unit (NICU) may have catastrophic consequences for young infants receiving critical care. From May 13, 2011 to July 11, 2011, a significant increase in stool samples testing positive for rotavirus antigens in the NICU of a university affiliated hospital was observed. Due to lack of clinical presentations suggestive of rotavirus infection in the patients and the rarity of rotavirus infection in the NICU in the past, a pseudo-outbreak was suspected. Infection control measures were reinforced initially. To investigate the outbreak, a prospective laboratory-based active surveillance of all infants in the NICU was conducted right after the cluster was identified. Repeated testing using a modified enzyme immunoassay (EIA) kit, rotavirus RNA polyacrylamide gel electrophoresis (PAGE), reverse transcription polymerase chain reaction (RT-PCR), and retrospective chart review methods were used to confirm the pseudo-outbreak. Seven infants in the NICU, with or without gastrointestinal symptoms, tested positive for the rotavirus antigen using the old version of an EIA kit, which indicated a possible outbreak. Active surveillance with repeated tests for recollected stool samples using a modified EIA kit showed negative results in all 24 infants in the NICU. Seven stored stool samples from four infants, which previously tested positive for the rotavirus antigen, tested negative for rotavirus using the modified EIA kit, PAGE, and RT-PCR. Chart reviews showed no clinical difference between index cases and controls. False positivity might arise from unsatisfactory specificity of the old EIA kit. After the introduction of the modified EIA kit, no rotavirus was detected in the NICU for at least 7 months. This cluster of patients who tested positive for the rotavirus antigen in stools was confirmed to be a pseudo-outbreak. Interpretation of the old EIA for rotavirus in an NICU setting should be done with caution until the mechanism of the false-positive reaction is elucidated.

RNA-RNA SELEX.

Systematic evolution of ligands by exponential enrichment (SELEX) protocol is a valuable technique to identify RNA aptamers interacting with RNA structural motifs. RNA aptamers are mainly resolved with affinity column chromatography and electrophoretic mobility shift assay (EMSA). Here, we describe the separation of the RNA aptamers binding to an RNA stem-loop target with affinity chromatography using the column attached the target RNA and nondenaturing polyacrylamide gel electrophoresis to obtain a single predominant RNA aptamer family.

English

virus. Bluetongue virus is a member of the genus Orbivirus in the family Reoviridae. Its genome consists of ten double-stranded (ds) RNA segments coding for seven structural proteins (VP1-VP7) and four non-structural proteins (NS1-NS3 or NS3A, and NS4). At present, 26 serotypes have been reported throughout the world. Bluetongue can be diagnosed from several kinds of samples. For virus isolation, embryonated eggs, 9 to 12 days old, at intra­venous inoculation is best however, cell lines like KC line, the insect cell line C6-36 derived from Aedes spp., Baby hamster kidney (BHK-21) and African green monkey kidney (Vero) lines can be used. For antigen identification sandwich enzyme linked immunosorbent assay (ELISA), serotype specific reverse transcriptase Polymerase chain reaction (RT-PCR), Serogroup-specific RT-PCR, Real time quantitative PCR, Sequencing and phylogenetic analysis (targeting conserved genome segments), Restriction enzyme profile analysis (REPA), Molecular probes, RNA polyacrylamide gel electrophoresis (RNA-PAGE) are now available in an increasing number of laboratories for the identification of Bluetongue virus (BTV). For antibody identification, it is possible to use blocking ELISA, competition ELISA, indirect ELISA, agar-gel immunodiffusion test (AGID), Complement-fixation test (CFT) and Haemagglutination inhibition test (HI), serum or virus neutralisation test (VNT/SNT), Immunofluorescence, Immunoperoxidase, and Dot immunobinding assays (DIA). Key words: Antibody, antigen, bluetongue, bluetongue virus, diagnosis, structure, molecular technique.

Detection of picobirnavirus and rotavirus in diarrhoeic faecal samples of cattle and buffalo calves in Mumbai metropolis, Western India.

In this study 113 diarrhoeic faecal samples obtained from buffalo (n = 68) and cattle (n = 45) calves under 1 years of age were analysed in order to determine the presence of rotavirus infection and the frequency of picobirnavirus excretion. Eleven (9.73%) samples positive for group A rotavirus were identified through RNA-polyacrylamide gel electrophoresis (RNA-PAGE), while 4 (3.53%) samples showed a bisegmented genome with a typical picobirnavirus pattern. This is the first report of picobirnavirus in cattle and buffalo calves from Western India.

Genetic Characterisation of Group-A Rotavirus Detected in Diarrhoeic Bovine Calves in Madhya Pradesh

Diarrhoeal disease continues to be an important cause of morbidity and mortality among neonates of various animal species worldwide. Amongst several viral agents associated with diarrhoea, rotavirus (RV) has been recognised as the major aetiological agent. In the following study, 184 faecal samples from diarrhoeic bovine claves were collected from different dairy farms of Madhya Pradesh (M.P), central India. Of these, 25 samples (13.6%) were found to be positive for RV infection using RNA polyacrylamide gel electrophoresis and reverse-transcription-polymerase chain reaction. One of the isolates (cow/C-32/IND/2008/G10) of predominant genotype (G10) was selected for sequence and SimPlot analysis. The results suggested occurrence of group-A RV in bovine population of central India, with circulation of a G10 genotype, which showed sequence identity of 63-98% with other bovine, human and porcine G10 isolates from India and other parts of the world. The C-32 isolate showed maximum sequence identity (98%) with a RV strain isolated from water buffaloes of Punjab (BRV 73Acc. No. EU311194). Our isolate showed minimum sequence identity (6372%) with G10 bovine isolates from the eastern region of the country (RUBVM135, EF200544 and EF200543). The results confirm circulation of divergent strains of G10 genotypes in India and warrants further molecular studies to understand the evolution of divergent G10 virus population in nature.

Detection and Prevalence of Bovine Group A Rotavirus in the Mathura Region

Rotaviruses (RVs) are the leading cause of diarrhoea in cattle and buffalo calves worldwide. A total of 100 faecal samples were collected from the diarrhoeic cattle and buffalo calves from the organised dairy farms in the Mathura region. Extraction of viral RNA was done in 10% faecal suspension using phenol: chloroform/TRI reagent. The extracted RNA was subjected to RNA-polyacrylamide gel electrophoresis and silver staining. In total, 12 samples were positive for Group A RV, showing 11 segments of RNA genome of the virus in the pattern 4:2:3:2. Based on the migration of segments 10 and 11, all positive samples were characterised as long electropherotypes. Differences in the migration pattern of class I segments was observed. In type 1 pattern, all the four segments (1, 2, 3 and 4) migrated separately. In type 2 pattern, segments 2 and 3 co-migrated. In all the samples, class III segments (7, 8 and 9) moved as a single segment. One sample showed the presence of an additional band between gene segments 5 and 6. Multiple bands were present in two samples. A 12% prevalence of Group A bovine RV was found in the present study in the Mathura region. The samples were positive in the months of August-November and in calves aged below 1 month.

Predominance of rotavirus genotype G6P[11] in diarrhoeic lambs

Out of 500 faecal samples from lambs with diarrhoea in Jammu and Kashmir, India, 66 (13.2%) were positive for group A rotavirus (GARV) by the latex agglutination test (LAT). Electropherotyping by RNA-polyacrylamide gel electrophoresis revealed the typical GARV 4-2-3-2 migration pattern in 49/66 (74.2%) samples. Fifty-two samples (10.4%) were positive by reverse transcription-PCR. G6 was the predominant G genotype (25/52; 48.07%), followed by G10 (19/52; 36.54%) whereas, the predominant P genotype was P[11] (46/52; 88.46%). G6P[11] is the prevalent strain of group A rotavirus in sheep in Jammu and Kashmir, India.

Isolation of Bluetongue Virus Serotype 1 from Culicoides vector Captured in Livestock Farms and Sequence Analysis of the Viral Genome Segment-2

Bluetongue virus serotype-1 (BTV-1) was isolated from Culicoides oxystoma vectors captured on livestock farms in two places of Gujarat, India. The viruses were isolated on BHK-21 cells, which produced characteristic BTV-related cytopathic effects between 24 and 48 h post-infection. Virus antigen was demonstrated in infected cells at different passage by a BTV-specific sandwich ELISA. Further, polyacrylamide gel electrophoresis and silver staining of viral genomic RNA revealed ten double-stranded RNA segments characteristic of BTV. Serotype of the isolates was identified by virus neutralization and PCR coupled with sequencing. The isolates were designated as SKN-7 and SKN-8 and their genome segment-2 (VP2) were sequenced. Phylogenetic analyses revealed very close relationship between them although they are not identical. SKN-8 showed closer relationship with a recently isolated BTV-1 from goat. Bluetongue virus was earlier isolated from Culicoides in adjacent state more than 20 years ago, although the serotype of the virus was not determined.

Rotavirus Strain Diversity in the Centre Coast of Tunisia from 2000 through 2003

An epidemiological survey investigating rotavirus infection in children was undertaken in the coastal region of Tunisia from January 2000 through September 2003. A total of 309 fecal specimens were screened by enzyme-linked immunosorbent assay and latex agglutination assay for the presence of group A rotavirus antigen. The detection rate was 26.2%. Rotavirus outbreaks showed a temperature-dependant pattern (P = .026) but no significant association with rainfall. Rotavirus strains isolated were analyzed by RNA polyacrylamide gel electrophoresis and were characterized antigenically by monoclonal antibodies to the VP6 subgroup. Eight RNA electropherotypes were identified, with 3 long and 5 short different RNA profiles. Among VP6 typeable strains, all isolates with a long electrophoretic pattern carried the subgroup II specificity, whereas those with a short profile belonged to subgroup I. In total, 48 rotavirus-positive samples were analyzed for G and P typing by reverse-transcription polymerase chain reaction. A total of 8 different G and P combinations were found: G1P[8] (35.7%), G1P[6] (21.4%), G2P[4] (4.8%), G3P[4] (4.8%), G4P[6] (4.8%), G8P[8] (4.8%), G3P[8] (2.3%), and G4P[8] (2.3%). Mixed infections were detected in 19.1% of stool samples. The emergence in Tunisia of unconventional types, such as G8VP7 specificity, highlights the need for a continual survey of the uncommon strains in North Africa.