Reverse Transcriptase/polymerase Chain Reaction-restriction Fragment Length Polymorphism Research Articles

Detection and differentiation of infectious bursal disease virus from the outbreaks in two layer farms by PCR-RFLP in Jos, Nigeria

Aim: Characterization of Infectious bursal disease viruses (IBDV) from the two outbreaks in Jos Nigeria, using reverse transcriptase polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) technique. Materials and Methods: A total of 40 bursa samples were collected from two outbreaks in November 2011 from two farms of 6-8 weeks old pullets within Jos South Local Government Area, with mortality between 60 – 74.2 % in commercially reared layer chicken flocks experiencing signs typical of infectious bursal disease (IBD). All the samples were found to contain IBDV genome by One Step RT-PCR using VP2 gene specific primers. Result: The assay amplified a 743bp fragment from 701-1444 nucleotides. RT-PCR product was further subjected to restriction digestion using TaqI, MvaI and SacI Restriction enzymes to differentiate classical from very virulent phenotypes. The RFLP profile was found similar for all eight isolates with TaqI and MvaI enzyme but different for SacI. All eight TaqI positive Viruses were further found positive for MvaI digestion and yielded RFLP profile similar to vvIBDV in Europe whereas one isolate was SacI positive and had a RFLP profile similar to classic IBDV strains. Conclusion: The clinical history of high mortality and TaqI and MvaI restriction enzyme positivity revealed that vvIBDV strains still exist in Jos, North central Nigeria.

Molecular Typing of Field Isolates from two outbreaks of Infectious Bursal Disease Virus from Pakistan

A reverse transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR/RFLP) technique was used for the identification and characterization of Pakistani field isolates of infectious bursal disease virus (IBDV). A total of 8 bursa samples were collected from two outbreaks during September and October 2003 from Tehsil Sumandri, Dist. Faisalabad with 40-50% mortality in commercially reared broiler chicken flocks experiencing signs typical of infectious bursal disease (IBD). Four samples were found to contain IBDV genome by One Step RTPCR using VP2 gene specific primers. The assay amplified a 743 bp fragment from 701-1444 nucleotides. RT-PCR product was further subjected to restriction digestion using MboI and MvaI restriction enzymes. A third enzyme SspI was used to identify the very virulent phenotype. The RFLP profile was found similar for all four isolates with MvaI enzyme but different for one isolate when digested with MboI. All three MvaI-positive viruses were further found positive for SspI digestion and yielded RFLP profile similar to vvIBDV in Europe whereas one isolate was SspI negative and had a RFLP profile similar to classic IBDV strains. The clinical history of high mortality and SspI restriction enzyme positivity revealed that vvIBDV strains exist in Pakistan.

Identification of Japanese strains of Barley yellow mosaic virus by RT-PCR/RFLP analysis of the coat protein-coding region

Reverse transcriptase-polymerase chain reaction–restriction fragment length polymorphism (RT-PCR/RFLP) was used to detect and identify Japanese strains of Barley yellow mosaic virus (BaYMV). A primer pair, amplifying a 765-bp fragment of the coat protein (CP)-coding region, was used for the RT-PCR, and RFLP profiles of the RT-PCR products were compared after digestion with the restriction endonucleases MseI, RsaI and SphI. These enzymes were selected based on the nucleotide sequence of BaYMV strains I, II, III and IV in the CP-coding region. These four strains, as well as the isolates from naturally infected roots of barley cultivars, were differentiated by RT-PCR/RFLP analysis. The results coincided with strain identification using the phenotypes of symptoms on the differential variety.

Identification of Soybean mosaic virus Strains by RT-PCR/RFLP Analysis of Cylindrical Inclusion Coding Region.

A reverse-transcriptase polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) was employed successfully for detection and identification of Soybean mosaic virus (SMV) strains. A primer pair amplifying a 1,385-bp fragment of the cylindrical inclusion (CI) coding region was used for RT-PCR and the RFLP profiles of the RT-PCR products were compared after restriction digestion with RsaI, EcoRI, or AccI restriction endonucleases. These enzymes were chosen based on the nucleotide sequences of SMV strains G2, G5, G5H, G7, and G7H in the CI coding region. These five strains, as well as seedborne SMV isolates from local soybean cultivars, could be differentiated by RT-PCR/RFLP analysis. The results correlated well with strain identification by symptom phenotypes produced on differential cultivars inoculated with strains and isolates. The sensitivity of RT-PCR enabled detection of SMV from plants with necrotic symptoms in which the number of virus particles was too low to be detected by enzyme-linked immunosorbent assay.

Characterization of Egyptian field strains of infectious bursal disease virus.

The reverse transcriptase-polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) technique was used for identification and characterization of Egyptian field strains of infectious bursal disease viruses (IBDVs) that caused severe outbreaks with (30%-60%) mortality rate. Twenty-four bursal samples collected from 24 field outbreaks in commercially reared chicken flocks experiencing signs typical of infectious bursal disease (IBD) were used. Ten of the bursal samples examined were determined to contain IBDV as evidenced by amplification of a 743-bp region of the VP2 gene of IBDV by RT-PCR. The RT-PCR products of the detected viruses were characterized by digestion with three restriction enzymes, BstN, MboI, and SpI. Three different RT-PCR/RFLP profiles were observed. Seven of the detected viruses had RFLP profiles identical to the very virulent European strains of IBDV (vvIBDVs). One virus had a RFLP profile identical to the U.S. classic vaccine strain, and one virus had a unique RFLP profile. The clinical history of the outbreaks and the presence of the SspI site in the 743-bp RT-PCR fragment were the criteria for designating the viruses as belonging to the very virulent (vv) phenotype.

Preservation of rabies virus RNA from brain tissue using glycerine

The challenge virus standard (CVS) strain and a wild isolate from a Mexican child who died of hematophagous bat (Desmodus rotundus) -transmitted rabies were injected intracerebrally into BALB/c mice. Brains obtained from infected mice were immersed in 80%, 50%, and 40% glycerine/phosphate-buffered saline (PBS). RNA was extracted from brains on days 1, 2, 3, 7, 21, and 60, and reverse transcriptase polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) tests were performed for rabies virus characterization. Storage temperature variation was recorded during the preservation period. The RT-PCR-RFLP tests were successfully performed on brain samples preserved in 50% glycerine/PBS, but not in those preserved in 80% or 40% glycerine/PBS. Temperatures ranged from 12 to 33°C and were not harmful, provided that 50% glycerine/PBS was used. We concluded that brain samples obtained and stored under field conditions (i.e. without refrigeration) for up to 60 d can arrive at a reference laboratory in an adequate condition for viral RNA analysis.

Restriction fragment length polymorphism of porcine reproductive and respiratory syndrome viruses recovered from Ontario farms, 1998-2000.

From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tested for porcine reproductive and respiratory syndrome viruses (PRRSV) using reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR product restriction fragment length polymorphism (RFLP) analysis. A total of 516 samples from 284 cases were RT-PCR positive for the PRRSV open reading frame (ORF) 7 sequence. The RT-PCR RFLP typing assay was performed using 2 different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and 6 of PRRSV. Samples from 254 cases were typeable, yielding 34 different RFLP types. Of these, 164 cases had 32 different RFLP types of field or intermediate strains, 86 had a pattern similar to a commercial PRRSV vaccine or VR 2332 strain of the virus, 4 had a RFLP type shared by another commercial vaccine and a field strain. In 4 cases, 2 different RFLP types were identified from tissues from different pigs that were submitted at the same time from the same farm. Of the 195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than 1 occasion during the specified time frame. In 23 of those 48 farms, RFLP patterns of PRRSV differed between submissions, whereas in the other 25 farms, the RFLP pattern remained unchanged. There were 34 different PRRSV patterns identified from 236 cases using the primer set amplifying 716 base pairs of PRRSV. There were 18 cases, consisting of 9 different patterns, typeable only by using the primers amplifying a 933-base pair fragment of the virus.

Use of reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism to examine the interaction between infectious bronchitis virus strains Massachusetts 41 and JMK in ovo

The reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism (RTPCR-RFLP) technique has been developed and used in the serotype diagnosis of infectious bronchitis virus (IBV) infection. In this report, we first demonstrate that the RT-PCR-RFLP was sensitive in detecting both Massachusetts 41 (Mass 41) and JMK strains of IBV singly; the detection limit for both was approximately 0.15ng viral RNA using gel electrophoresis. Subsequently, the ability of the technique to semi-quantify the relative amounts of Mass 41 and JMK RNA in their mixture was shown by performing RT-PCR-RFLP on various combinations of serially diluted known amounts of Mass 41 and JMK RNA. Results indicated that both Mass 41 and JMK can be detected and semi-quantified when there is less than 103-fold difference between the two viral RNA template inputs, which are above the lower detection limit. Based on this result, the interaction between Mass 41 and JMK in ovo was examined by inoculating 10-day-old embryos with various combinations of different EID50 titers of Mass 41 and JMK. RT-PCR-RFLP results showed that Mass 41 dominates over JMK in an EID50 titerdependent fashion, but not the reverse. To further investigate the underlying mechanism, replication efficiency of Mass 41 and JMK was compared at 6, 12, 18, 24, 36 and 48h after inoculation of embryos with 105.3 EID50 of individual Mass 41 and JMK. No significant difference (P > 0.05) on replication efficiency between these two serotypes was found based on the results of total RNA concentration and RT-PCR results of 10-fold dilution of RNA at the indicated time points. By inoculating heat-inactivated Mass 41 with live JMK into embryos, only JMK was detected by RT-PCR-RFLP and no dominating interference was observed. Data suggest that the dominance is not due to the replication efficiency of Mass 41 and JMK; a receptor-mediated mechanism might be responsible for it.

Restriction Fragment Length Polymorphism Analysis of Highly Virulent Strains of Infectious Bursal Disease Viruses from Holland, Turkey, and Taiwan

Strains of highly virulent infectious bursal disease virus (HVIBDV) from Turkey (OA, OE), Holland (HOL), and Taiwan (PT, IL) were characterized by reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) analysis and compared with the United States of America (USA) classic (STC) and variant (MD, IN) serotype 1 viruses and serotype 2 OH strain. A primer pair that amplified a 743-bp fragment of the VP2 gene was used. The RFLP profiles of the RT/PCR products were determined with four restriction enzymes (SspI, BstNI, MboI, and StyI). The SspI enzyme distinguished the HVIBDVs from the USA classic and variant viruses and the serotype 2 virus. The USA serotype 1 viruses used in this study did not have restriction sites for SspI in the 743-bp VP2 fragment. The RFLP profiles of the five HVIBDVs when digested with BstNI were different from the USA classic STC virus but were similar to the variant viruses (MD, IN). The RFLP profiles for isolates from Turkey and Holland were similar to the classic STC strain when digested with MboI but were different from the variant USA viruses (MD, IN). The RFLP profiles of the two Taiwan isolates were different from the Holland, Turkey, and USA classic and variant viruses when digested with MboI. When the five HVIBDVs were digested with StyI enzyme, their RFLP patterns were similar to those of the USA classic STC strain but were different from the USA variant viruses. None of the strains tested had patterns similar to serotype 2 OH virus.

Restriction Fragment Length Polymorphisms in the VP2 Gene of Infectious Bursal Disease Viruses

Infectious bursal disease viruses (IBDVs) were examined for restriction fragment length polymorphisms in a fraction of the VP2 gene with the use of the reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay. The restriction enzymes BstNI and Mbol were used to obtain RFLP results. A third enzyme, StyI, was tested, but its utility for differentiation of IBDV strains was limited. Thirteen vaccine viruses and five IBDV strains that were previously characterized were placed into five molecular groups. Two groups contained viruses described as being classic strains, and two groups contained viruses described as being variant strains. The fifth group contained both classic and variant strains. The RFLP observed for the serotype 2 IBDV strain OH was unlike any of the RFLPs observed in viruses in the five molecular groups. Seven IBDV strains from commercially reared chickens in the United States, Mexico, Puerto Rico, and Thailand were tested in the RT/PCR-RFLP assay to determine if they were similar to the commercial IBDV vaccine strains tested. These viruses were selected because they were associated with lesions in the bursa of chickens that should have been protected by maternal antibodies or active immunity. Each of the viruses tested contained a unique RFLP compared with the IBDV strains and vaccine viruses examined in this study and, thus, did not fit into any of the five molecular groups. These viruses also were distinguishable from each other.

Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains. Correlation with antigenicity and pathogenicity.

Transmissible gastroenteritis virus (TGEV) causes an economically important enteric disease of swine. Differences in the pathogenicity, antigenicity and tissue tropism have been observed among porcine coronaviruses. Although porcine respiratory coronavirus (PRCV) is antigenically similar but not identical to TGEV isolates, these respiratory coronaviruses differ markedly in pathogenicity and tissue tropism compared to TGEV isolates. Using a reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay, TGEV and PRCV isolates were assigned to several distinct groups. By RFLP analysis of the 5' region of the S gene, TGEV strains were differentiated into 4 groups using the restriction enzyme Sau3AI. A fifth Sau3AI group contained the PRCV isolates. These 5 groups correlated with antigenic groups previously defined using monoclonal antibodies in our laboratory. Several restriction enzymes could be used to differentiate the TGEV strains into Miller and Purdue types. Analysis of a PCR amplified product in the 3 and 3-1 genes indicated the RT/PCR-RFLP assay results for TGEV Miller strains could be correlated with lower virulence created by passage in cell culture.

Differential expression of angiotensin receptor 1A and 1B in mouse.

At least two distinct genes (AT1A and AT1B) encode type 1 angiotensin II (AT1) receptors in rodents. Receptor binding and Northern blot analysis have clearly demonstrated the presence of AT1 receptors and AT1-receptor mRNA in many tissues but fail to differentiate which type 1 receptor subtype is expressed. A reverse-transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) assay was developed to differentiate the expressed mRNA by subtype. Expression of AT1A was clearly evident in kidney, liver, adrenal gland, ovary, brain, testes, adipose tissue, lung, and heart of adult mice. AT1B was absent from most of these tissues but was detectable in brain, testes, and adrenal gland. No significant differences in expression were evident in kidney, liver, brain, lung, or heart from 16.5- or 18.5-gestation-day fetuses, and only AT1A was evident in placenta. Expression of AT1B was confirmed in adrenal gland, brain, and testes, using a primer set that specifically amplifies only AT1B mRNA. Expression of AT1A and AT1B was also examined in As4.1 cells, a renin-expressing mouse kidney tumoral cell line. Receptor binding and competition assays using AT1- and AT2-receptor antagonists revealed that only AT1 receptors are present on the cell surface. Extremely low levels of AT1-receptor mRNA was detected by Northern blot, and RT-PCR-RFLP analysis revealed that only the AT1A subtype is expressed in this cell line. Despite the high homology between the coding sequence of the AT1A and AT1B genes, they exhibit disparate tissue-specific expression profiles.