Identification of hepatitis B virus surface antigen [HBsAg] genotypes and variations in chronic carriers from Isfahan province, Iran

Hepatitis B virus [HBV] gene and protein variations are frequently been seen in chronic patients. The aims of study were to determine the genotypes as well as the patterns of variations distribution in chronically-infected patients from the central part of Iran. The surface gene was amplified, sequenced and subsequently aligned using international and national Iranian database. All strains belonged to genotype D, subgenotype Dl and subtype ayw2. Of all 62 mutations occurred at 39 nucleotide positions, 31 [50%] were missense [amino acid altering] and 31 [50%] were silent [no amino acid changing]. At the amino acid level, 30 substitutions occurred, however, 3 were in positions 122 and 127, corresponded to subtypic determination. 22 [73%] out of 30 amino acid mutations occurred in different immune epitopes within surface protein, of which 12 [54.54%] in B cell epitopes in 10 residues; 5 [45.45%] in T helper epitopes in positions; 5 [22.73%] in inside CTL epitopes in 4 residues. The distribution of amino acid mutations as well as the ratio between silent and missense nucleotide mutations showed a narrowly focused immune pressure had already been on the surface protein in these patients, led to the emergence of escape mutants in these patients

Detection of pestivirus contamination in cellcultures by nested-PCR

In this study a nested-PCR assay was optimized for detection of two BVDVbiotype of NADL strain. Apart of 5' non-coding region of virus, 249 bp in size, was amplified in RT-PCR. PCR product was cloned in a pTZ57R/T vector and sequencing results confirmed the specificity of the test. Internal primers were designed and a 155 bp DNAfragment was amplified in nested-PCR. The 4 sensitivity of RT-PCR and nested-PCR for detection of virus in cell culture were found to be 10 2 TCID50 and 10 TCID50, respectively. Seven cell cultures were tested for BVDVcontamination using ELISA, RT-PCR and nested-PCR. Results indicate that sensitivity of molecular tests for detection of virus in cell culture samples is higher than ELISA

[ survery on frequency of foot-and-mouth disease virus carriers of slaughtered cattle in Zyaran abattoir]

To determine frequency of FMDV carriers in slaughtered cattle at Zyaran abattoir. Cross-sectional study. Three hundred and one tonsils of clinically normal cattle. All of tonsils were tested for determination of FMDV genome using RT-PCR. Then, 30 samples were cultured on MDBK. 20 samples had positive findings in RT-PCR test. Based on sex, age and breed of cattle, RT-PCR results compared with Chi-square and Fisher's Exact Test. McNemar test was used for comparing of virus culture results with RT-PCR. Ninety nine samples were positive in RT-PCR. Relative frequency of FMDV genome presence in tonsils of clinically normal cattle had a significant difference based on sex and breed. The Sistani and Holstein bred had the highest and the lowest relative frequency, respectively samples on cell serotype O of culture were FmDV. One positive sample [Asia 1] Two positive sample[Asia1] was shown by ELISA. Total relative frequency of positive FMDV genome [32.9%] indicates the extensive exposure to FMDV. Breed variation among Sistani cattle [as a variety of Bos indicus] and other bred should be studied in a controlled condition

Detection of avian infectious bronchitis virus and Massachusetts serotype by RT-PCR

The aim of the present study was using RT-PCR for the diagnosis of avian infectious bronchitis virus and Massachusetts serotype in tissue samples. Optimization of a molecular diagnostic method for the detection of avian bronchitis virus and identification of Massachusetts serotype was investigated. In order to detect infectious bronchitis virus [IBV] in tissue samples, an RT-PCR was optimized. Specific primers from conserved region of all known IBVserotypes were used in the first PCR assay. Specific primers for the identification of Massachusetts serotype were selected from S-1 gene of the virus in the second PCR reaction. The S-1 gene is a hypervariable region among IBV serotypes; therefore, the amplification of this region is important for serotype identification. Viral RNAwas extracted from vaccine and tissue samples from vaccinated and clinical tissue samples of suspected birds. After construction of cDNA, two PCR assays were performed. In the first RT-PCR, detection of virus in test samples was investigated and in the second PCR, Massachusetts serotype was identified. To identify the specificity of the test, PCR amplicons were sequenced. In the first reaction, the IBVwas detected in the samples and a 600 bp fragment was amplified. In the second PCR, a 355 bp fragment was amplified from S-1 gene, which confirmed the detection of Massachusetts serotype. In clinical samples, the IBVwas also detected. The specificity of the test was confirmed by sequencing of PCR products. Sequence data were submitted to the GenBank which could be accessible by AY954694 number. The RT-PCR is a specific assay for the detection of IBV. Detection of IBV and identification of genetic differences among IBV subtypes in Massachusetts serotype would be possible by sequencing of amplified products

Polymerase chain reaction for the detection and differentiation of Marek's disease virus strains MDV-1 and HVT

Marek's disease [MD] is a lymphoproliferative disease of chickens characterized by lymphocytic infiltration of various organs. The present study was an attempt to use polymerase chain reaction [PCR] to optimize a rapid and reliable assay for detection of MDV genome. Detection of serotype I of MDV [MDV-l] was confirmed by presence of a 200 bp DNA fragment as a PCR product. Differentiation of MDV- I and herpesvirus of turkeys [HVT] was also conducted using specific primers from the glycoprotein A [gA] gene and a 388 bp DNA fragment was amplified from HVT genome. The specificity of the test was confirmed by sequencing of PCR products. Results indicate that MDV-l can be diagnosed in clinical samples and inoculated cell cultures which is used for virus isolation. In addition, differentiation between MDV- I and HVT viruses was confirmed based on the size of PCR products. The test proved to be rapid and reliable and be performed as a robust diagnostic test in veterinary diagnostic laboratories