Analysis of NSP4 gene and its association with genotyping of rotavirus group a in stool samples

Background: Non-structural protein 4 [NSP4] is a critical protein for rotavirus [RV] replication and assembly. This protein has multiple domains and motifs that predispose its function and activity. NSP4 has a sequence divergence in human and animal RVs. Recently, 14 genotypes [E1-E14] of NSP4 have been identified, and E1 and E2 have been shown to be the most common genotypes in human

Methods: The gene and protein sequence of NSP4 in RV-positive samples were inspected with the aim of NSP4 genotyping and variation analysis in viroporin and other domains. P and G typings of RV samples were carried out by WHO primers using a semi-multiplex PCR method. Non-typeable RV samples were amplified by conserved primers and sequenced

Results: In viroporin and enterotoxin, conserved sequence was detected, and amino acids substitution with the same biochemical properties was found

Conclusion: Association of NSP4 genotype with P or G genotyping G1/G9 correlates with E1 genogroups. In electrophoretyping of RV, E2 genotype had a short pattern when compared to E1

Distribution of rotavirus genotypes circulating in Ahvaz, Iran in 2016

Background: Group A rotavirus [RVA] mainly causes acute gastroenteritis, exclusively in young children in developing countries. The prevalence and determination of the molecular epidemiology of rotavirus genotypes will determine the dominant rotavirus genotypes in the region and will provide a strategy for the development of appropriate vaccines

Methods: A total of 100 fecal samples were collected from children below five years with acute gastroenteritis who referred to Aboozar Children's Hospital of Ahvaz city during October 2015 to March 2016. All samples were screened by latex agglutination for the presence of rotavirus antigen. Rotavirus-positive samples were further analyzed by the semi-multiplex RT-PCR, and the sequencing was performed for G/P genotyping

Results: Findings showed that 32% of the specimens were RVA-positive. Among the 32 VP7 genotyped strains, the predominant G genotype was G9 [37.5%], followed by G2 [21.9%], G1 [12.5%], G12 [9.4%], G4 [9.4%], G2G9 [6.3%], and G3 [3.1%]. Among the 31 VP4 genotyped strains, P[8] genotype was the dominant [62.5%], followed by P[4] [31.3%] and P[4] P[8] [3.1%]. The genotypes for G and P were identified for 31 rotaviruses [96.87%], but only one strain, G9, remained non-typeable for the P genotype. The most prevalent G/P combination was G9P[8] [28.5%], followed by G2P[4] [18.8%], G1P[8] [9.4%], G12P[8] [9.4%], G4P[8] [9.4%], G2G9P[4] [6.3%], G9P[4] P[8] [3.1%], G3P[8] [3.1%], G9P[4] [3.1%], G2P[8] [3.1%], and G9P[nontypeable] [3.1%]

Conclusion: A novel rotavirus strain, G12, was detected, for the first time, in patients from the southwest of Iran. Comprehensive investigations are required to evaluate the emergence of this strain

E6-specific detection and typing of human papillomaviruses in oral cavity specimens from Iranian patients

Background: Detection and quantification of human Papillomavirus [HPV] genome in oral carcinoma play an important role in diagnosis, as well as implications for progression of disease

Methods: We evaluated tissues from 50 esopharyngeal cancers collected from different regions of Iran for HPV E6 using the two type-specific primers sets. E6 gene of HPV genotypes was amplified by specific primers. The sensitivity of PCR assay was analyzed and determined using HPV-DNA-containing plasmids. Real-time PCR was utilized to determine the prevalence and HPV viral load in patients with oral cavity squamous cell carcinoma

Results: Eighteen [36%] specimens were positive for HPV. Among the 18 positive specimens, 10 showed HPV-18 [55.55%], and 8 specimens were positive for HPV-11 [44.44%]. Of the 18 infected specimens, 6 [33.32%] and 12 [66.65%] were identified as high-titer and low-titer viral load, respectively

Conclusions: The PCR-based assay, developed in the current study, could be used for HPV detection, quantification, and genotyping in epidemiological and clinical studies

[Rearranged bovine rotavirus production through cultivation of virus by high multiplicity of infection [MOI] in cell culture and amplification of non-structural genes using RT-PCR]

Group A rotaviruses [GARV] are responsible for the vast majority of severe diarrhea worldwide that kills an estimated 600,000-870,000 children annually. Since infantile gastroenteritis is a main health problem, therefore diagnosis and treatment of this disease is crucial. Gene rearrangements have been detected in vitro during serial passages of the virus at a high multiplicity of infection [MOI] in cell culture, as well as in chronically infected immunodeficient individuals. In this study, we developed an RT-PCR method to detect and diagnose the standard and gene rearranged bovine rotavirus. Rotavirus RNA was extracted from confluent monolayers of infected MA-104 cells, stained with silver nitrate, and then electrophoresed in a 10% polyacrylamide gel. The full-length gene products that encoded the NSP1, 2, and 3 genes of the standard and rearranged rotavirus were amplified by RT-PCR using specific primers. We observed rearranged NSP1 and NSP3 genes that had different migration patterns seen with polyacrylamide gel electrophoresis. NSP1, 2, and 3 gene segments from standard and rearranged rotaviruses were amplified by RT-PCR, then the complete nucleotide sequence of each gene was subjected to sequencing. The results showed the generation of gene rearrangement through serial passages of the bovine rotavirus RF strain. Serial passage of rotavirus in cell culture at a high MOI and chronic infection in immunodeficient target groups might alter rotavirus evolution. The methods utilized for detection and characterization of rotaviruses are continually evolving and being refined. Data collection is necessary to understand the molecular and antigenic features of the rotavirus in order to have a successful implementation of rotavirus studies and the development of a rotavirus vaccine. This study shows the importance of genetic variation and can provide valuable information about the amplification, diversity, biology, and evolution of rotaviruses

Comparison of rotavirus rf strain and hsv-1 titration by CCID50% and plaque assays

Titration of viruses is important to determine the quantity of virus in vaccine development, master virus seed stock preparation, viral vector studies and virus replication. In this study, we compared the CCID50% and plaque assay as a standard titration method for rotavirus [RF] and HSV-1. The MA104 and Vero cells were inoculated by RF and HSV-1 in 6- and 96- well plates. Following infection and adsorption, the optimal time for the cytopathic effect caused by the viruses was noted and the results compared. The CPE[Cytopathic Effect] of RF was observed in less than 18 hours, which increased until 72 hours after inoculation. In HSV-1, the CPE was observed 24 and 72 hours after inoculation. The virus titration in the plaque assay was monitored at 96 hours post-infection for RF and at 72 hours post-infection for HSV-1. In both viruses the plaque titer method was lower than the CCID50 method, since the results indicated that 1 CCID50% was equal to 0.7 PFU. The plaque assay is one of the most accurate methods for viral titration. For the plaque assay, individual lesions may be isolated, which the plaques can be counted. The CCID 50% method is not applicable for purification of homogenous viruses, nor is this technique reproducible

Isolation and cloning of human papillomavirus 16 L1 gene from Iranian isolate

To isolate and construct cloning and expression vectors containing human papillomavirus [HPV16-L1] gene as target for application as recombinant vaccine. The study was performed in 2007 in Tarbiat Modares University, Tehran, Iran. Four genital specimens DNAs were amplified with the use of HPV type-specific primers based on HPV-16 L1, E6, and E7 genes. The polymerase chain reaction products were cloned into suitable cloning and expression vectors and were confirmed by restriction enzyme analysis and sequencing. The desired plasmids were sequenced and indicated 99% homology with those submitted full length L1 sequences in the GenBank. The results showed that there was 99% homology between our product and those mentioned in the GenBank. Nowadays empty viral capsids, termed virus-like particles, are synthesized with the use of microbial or cellular expression systems. Therefore, it can be concluded that the Iranian HPV16 full length L1 sequence is very similar to the other sequences in the GenBank and it can be used as a candidate gene in prophylactic vaccine for cervical cancer