Polymorphism of prion protein gene in sheep of Inner Mongolian,China

Susceptibility to natural scrapie in sheep is associated with polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene. To assess the risk of scrapie in sheep raised in China, DNA from 30 sheep of two breeds was isolated, amplified and sequenced for the PrP gene. The ovine PrP gene was found to be highly homogenous. The genotype associated with high susceptibility to scrapie (VRQ) was absent, whereas that associated with the resistance (ARR) was present in 6.7% of sheep examined. ARK was also rare (6.7%). ARQ that is associated with an intermediate susceptibility was the genotype observed in the most of sheep examined (86.6%). These data suggest that Chinese sheep of Mongolian sheep breed are susceptible to scrapie.     

Enhancement of detection and quantification of rotavirus in stool using a modified real-time RT-PCR assay

A sensitive and specific real-time RT-PCR assay to detect rotavirus in stool samples was optimized and validated using a wide range of rotavirus genotypes. The target of the original TaqMan(R) assay is an 87 bp fragment of the highly conserved non-structural protein 3 (NSP3) gene. Here we modified the original assay by introducing degeneracy into the forward primer to account for sequence variation between rotavirus genotypes, added four nucleotides at the 3' end of the reverse primer to reduce its stability, and modified the probe label. Amplification and detection conditions were optimized using purified dsRNA from two cultivated strains. The limit of detection of the modified assay was calculated to be approximately 44 genome copies per reaction. To validate the reactivity of the assay, 103 archived RNAs that had been extracted from stools and genotyped during routine U.S. surveillance were tested. Samples were selected to represent both rare and common genotypes that have been detected in U.S. children. Nine genotypes known to be circulating in the United States were detected by the real-time assay demonstrating broad reactivity. In addition, other enteric viruses were not detected demonstrating that the assay is specific for rotavirus and does not cross-react with other viruses potentially present in stool samples. This real-time assay is an important addition to the arsenal of molecular tools available to quickly identify rotavirus in stool samples during routine surveillance.     

Polymorphisms of the prion protein gene in sheep of Inner Mongolia,China

Polymorphisms of the prion protein gene (Prnp), especially the amino acid residue alterations at codons 136, 154, and 174, in sheep have been found to be associated with susceptibility to scrapie disease. We investigated Prnp polymorphisms in three local sheep breeds in Inner Mongolia, China. Blood samples were collected from 46 Ujumqin, 34 Sunite, and 22 Mongolian sheep. The genetic DNA of blood samples was extracted, amplified and sequenced, and amino acid alignment was determined. Polymorphisms were detected at 8 codons, among which M157I, Q220H, and R223K have not been previously reported. The frequency of the amino acid residues ARQ/ARQ at codons 136, 154, and 171, respectively, which is associated with medium-high susceptibility to scrapie, was 74.5%, and the frequency of scrapie-resistant genotype ARR/ARR was 7.9%. The highly susceptible genotype VRQ/VRQ at these codons as not detected from the tested sheep. Of the three sheep breeds, Ujumqin sheep had the highest frequency (15.2%) of scrapie-resistant amino acid sequence, ARR/ARR at codons 136, 154, and 171, respectively, accounting for 87.5% sheep that carry these polymorphisms. Our findings are of special importance for both live sheep export and sheep breeding.     

Influence of the prion protein gene,Prnp, on scrapie susceptibility in sheep

Natural scrapie in sheep occurs through a complex interplay between host genetic elements and various strains of the infectious scrapie agent. Scrapie-related polymorphisms in the coding region of the prion protein (PrP) gene, Prnp, have been studied in a number of breeds. The disease-promoting V136 allele, and the susceptibility-reducing R171 allele, have proved to be most important. However, variation in the coding region of Prnp cannot alone explain the diverse patterns of scrapie susceptibility in various breeds. For instance, in many breeds plagued with scrapie, the V136 allele appears to be a rarity. The R171 allele greatly reduces scrapie susceptibility This lays the molecular foundation for marker-assisted breeding for reduced scrapie susceptibility now underway in many countries. Although potentially important, and still under investigation, variable expression level and pattern of the ovine Prnp appears to be of little importance for the occurrence of natural scrapie. Studies of scrapie in mice also indicate that genetic elements other than Prnp may have a strong influence on scrapie incubation time, and hence susceptibility. Narrowing down the search to focus on these elements and identification of candidate genes are important tasks for future research in sheep scrapie.     

Detection and quantification of hepatitis A virus in seawater via real-time RT-PCR

A real-time RT-PCR method utilizing SYBR Green chemistry was developed to detect and enumerate hepatitis A virus (HAV) in ocean water. Ocean water samples were taken at the Tijuana River mouth (Tijuana, Mexico) and Imperial Beach pier (1.4 km north of the Tijuana River mouth in San Diego, California) following four separate rain events. A total of eight samples were collected, one from each location, each consisting of 4 l of ocean water. Using conventional RT-PCR and primers based on the conserved sequence at the VP3-VP1 genes of HAV, a 247 bp cDNA was amplified from six out of eight rain event water samples. HAV cDNA (confirmed by sequence analysis) was cloned into a TOPO vector (Invitrogen, Carlsbad, CA), and four primer sets were designed for application in SYBR Green real-time RT-PCR. The water samples were shown to contain inhibitors that affected real-time RT-PCR amplifications, however diluting the cDNA solution enabled successful amplification. Using real-time RT-PCR, HAV could be detected in all eight samples. Depending on the rain event, the viral load in these samples varied from 90 to 3523 copies of HAV/L of ocean water near the mouth of the Tijuana River, and 347 to 2656 copies/l near the Imperial Beach pier. The sensitivity, quantitative ability and the high throughput nature of SYBR Green real-time RT-PCR will be useful in monitoring HAV contamination in seawater.     

Comparison of real-time fluorescent RT-PCR and conventional RT-PCR for the detection of hepatitis E virus genotypes prevalent in China

To compare the specificity and sensitivity of a real-time fluorescent RT-PCR assay with conventional RT-PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non-A-C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real-time RT-PCR could detect the same final dilution of genotype 1 as conventional RT-PCR but could detect a 10-fold lower concentration of genotype 4 than conventional RT-PCR. Of 416 samples from patients with a clinical diagnosis of non-A-C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real-time and conventional RT-PCR, respectively. The concordance of real-time and conventional RT-PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti-HEV IgM by real-time and conventional RT-PCR, respectively, and 31 and 26 of 245 samples negative for anti-HEV IgM, were positive by real-time and conventional RT-PCR, respectively. All amplicons positive by conventional RT-PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real-time RT-PCR assay is more sensitive than conventional RT-PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China.     

Quantification of human astroviruses in sewage using real-time RT-PCR

Human astroviruses constitute a significant cause of acute diarrhea in children. Viral transmission occurs via the fecal-oral route, predominantly person-to-person, but the consumption of fecally contaminated water and shellfish has also been implicated. Viral pathogen detection in water, especially, in wastewater, is difficult and PCR is widely used to detect these viruses. Despite the recent development of real-time quantitative PCR, quantification of astroviruses in sewage had not been available up to now. We have developed a method to quantify astroviruses in sewage. For this purpose, we designed a set of primers and a fluorogenic probe located at the 3' -end of the genome of human astroviruses. The amplified region was cloned and the plasmid was transcribed to generate calibration standards for quantification. After validation of the standards, the method was evaluated in artificially contaminated samples. To validate the method on naturally contaminated samples, raw and treated wastewater samples were collected monthly for one year in a sewage treatment plant. Astrovirus genomes were detected in all samples collected at the entrance to the sewage treatment plant, with a mean value of 4.1 x 10(6) astrovirus genomes for 100 ml. Effluents were less strongly contaminated, with a mean value of 1.01 x 10(4) astrovirus genomes. The high prevalence of astroviruses in sewage treatment plant effluents indicates that these plants are not efficiently eliminating the virus. This is a major public health concern and new techniques of depuration are needed. Our method could be effectively used in evaluating new treatment processes to reduce the viral load in the effluent of treatment plants.     

改进竞争性RT-PCR法在定量PER1基因表达中的应用

目的研究使用不同样品稀释液及变性高效液相色谱(DHPLC)梯度条件对定量检测PER1基因表达的影响。方法竞争性RT-PCR与DHPLC结合定量检测基因表达。结果样品稀释液中含有载体可极大提高实验的重复性、准确性;不同DHPLC梯度条件对定量结果无明显影响。结论建立定量PER1基因mRNA表达水平的竞争性RT-PCR系统有助于精确检测微量标本中节律基因表达的变化,探寻生物节律与疾病状态间的关系。     

A case of scrapie in a sheep carrying the lysine-171 allele of the prion protein gene

Summary. Susceptibility to scrapie in sheep depends on the host PrP genotype. No data about the linkage of the rare ARK allele to differential scrapie susceptibility are currently available. Several tissues isolated from sheep from an Italian scrapie outbreak and carrying the ARK allele were examined for the presence of the pathological prion protein. A weak positivity was detected only by Western blot in the brainstem of one ARK/ARH sheep. This result shows that the ARK allele does not confer full resistance against scrapie and that the allele needs to be studied further before it can be considered for breeding purposes.     

Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.     

Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)-molecular size and glycosylation profile-in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.