The emergence of Zika-virus-associated congenital microcephaly has engendered renewed interest in the pathogenesis of microcephaly induced by infectious agents. Three of the original "TORCH" agents are associated with an appreciable incidence of congenital microcephaly: cytomegalovirus, rubella virus, and Toxoplasma gondii. The pathology of congenital microcephaly is characterized by neurotropic infectious agents that involve the fetal nervous system, leading to brain destruction with calcifications, microcephaly, sensorineural hearing loss, and ophthalmologic abnormalities. The inflammatory reaction induced by these four agents has an important role in pathogenesis. The potential role of "strain differences" in pathogenesis of microcephaly by these four pathogens is examined. Specific epidemiologic factors, such as first and early second trimester maternal infection, and the manifestations of congenital infection in the infant, shed some light on the pathogenesis. Immune aspects of normal pregnancy and their role in congenital infections is examined. In this review, we integrate all these findings to create a unified hypothesis of the pathogenesis of congenital microcephaly induced by these infectious agents.

Introduction: Hepatitis C virus (HCV) is one of the major medical problems. Human and chimpanzees are the only specific hosts which are naturally susceptible to HCV infection. Mice and other common laboratory animals are resistant to the virus, hence HCV prophylactic and therapeutic researches are very difficult and challenging. HCV nonstructural protein 3 (NS3) is one of the most attractive targets for developing novel anti-HCV therapies as it is essential for the viral replication. This study was designed to produce stable SP2/0 tumor cell lines expressing NS3 of HCV for future basic and vaccine studies. Methods: A lentivector expressing NS3, named PCDH-NS3, was constructed by cloning of NS3 cDNA into downstream of CMV promoter of pCDH-CMV-MCS-EF1-Puro-GFP. The constructed plasmid was co-transfected with pMD2.G plasmid which encodes envelope VSV G protein and psPAX2 packaging plasmid into HEK-293T cells. The lentivector-containing supernatant was collected every 12 h for 72 h and NS3-Lentivector was concentrated by ultracentrifugation. Titers of the NS3 lentivector were estimated using flow cytometry. The SP2/0 cells were then infected by NS3 lentivector. Puromycin as a selective antibiotic was added to the culture for 2 weeks to select NS3 positive cells. A single transfected clone was obtained using limiting dilution. The 1st and 6th passages of the cells cultured in vitro were harvested and NS3 mRNA was detected for by RT-PC. Results: The results showed that NS3 expressing lentivector plasmids and the two other helper plasmids could be transfected into HEK293T efficiently and packaged successfully as a pseudo-lentivector. Finally, the detection of NS3 mRNA in the 1st and 6th passages of SP2/NS3 cells was confirmed by establishment of a stable cell line. Conclusion: SP2/0 Cell line with stable expression of NS3 can be used as a suitable tumor model to facilitate research on HCV vaccine in vitro and in mice model and it could be served as a valuable tool for pharmaceutical HCV research to pave the way for further research on NS3 vaccine function.

The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors.The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression.However, the possible correlation between miR-122 levels and fibrosis state has been less investigated.Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line.Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1).Moreover, the transforming growth factor beta (TGF-β) levels in the supernatants of transfected cells were evaluated by ELISA.The gene expression analysis of fibrotic genes and TGF-β cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001).The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function.These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

Background: Human cytomegalovirus (HCMV) may play a role in the development of glioma disease that is one of the most common brain tumors. Objective: The aim of this study was to detect human CMV in patients with glioma in Imam Khomeini hospital, Tehran. Methods: This experimental study was conducted on paraffin-embedded tumor samples of 18 patients referred to Imam Khomeini hospital in 2012. Immunohistochemistry (IHC) was performed with monoclonal antibody specific for HCMV PP65 protein and the samples were assessed using a light microscope. Findings: Of 18 patients, 13 (72.2%) were positive for HCMV PP65 protein and four of them expired. Conclusion: With regards to the results, more comprehensive studies are recommended for detection of HCMV in patients with glioma using different diagnostic methods.

Background and Aims: DNA constructs containing HCV antigens have become one of the vaccine candidates for induction of anti-HCV cellular and humoral immunity. In this study, we constructed a novel expressing vector harboring a fusion sequence derived from an overlapping fragment in the middle of NS3 and a truncated core fragment to avoid troubles reported to be associated with full gene expression. Methods: The partial NS3 (pNS3) and core genes were amplified by RT-PCR method using serum of HCV infected patient harboring genotype 1a of virus. After purification and cloning the genes into TA-cloning vector, they were evaluated by sequencing and restriction digestion analysis. The resultant pNS3 and core gene subcloned into expression vector separately followed by expression evaluation using RT-PCR and western blotting. The core expressing vector exploited for amplification of a new truncated core (50-160aa) sequence using PCR. Truncated core fragment was first cloned into TA vector at a natural restriction site downstream of pNS3 fragment. The resulting fused sequence was cut and subcloned into expression vector. The integrity and ability of expression of this fused sequence was evaluated by sequencing followed by RT-PCR analysis after DNA transfection into 293 cells. Results: The repeated sequencing data showed sequence integrity among the gene fragments as well as homology among them and reference 1a sequences. The colony-PCR, RT-PCR and western blotting confirmed insertion of genes into expressing vector, expression of genes in 293 cell line and production of protein in 293 respectively. Conclusion: This new expressing vector harboring a novel fused fragments of NS3 and core genes may overcome shortcomings in vaccine design in the setting of HCV disease.

Background:The reasons for the viral persistence of hepatitis B virus infection (HBV) are unknown, but are probably related to host immune factors.Cytokines such as interleukin-1 (IL-1) play significant roles in inflammatory and immune defense.This study was undertaken to investigate the genetic polymorphisms for persistent HBV infection and development of hepatocellular carcinoma (HCC) by using single nucleotide polymorphism (SNP) chip.Methods: Two hundred ninety two patients with HBV infection (111 with chronic hepatitis, 95 with liver cirrhosis, 86 with HCC) and 107 healthy individuals who recovered from HBV infection were enrolled.We assessed a total of 1,536 SNPs in 114 genes by using IIlumina SNP chips.Results: Four SNPs of IL-1b (-581T>C, -2023G>C, 3340A>G, 289T>C) and six SNPs of IL-1 receptor associated protein (IL-1RAP) (-51668T>A, -8261T>C, -8183A>G, -256C>T, 59264G>A, 65445A>G) showed biallelic polymorphism.The -2023 C allele of IL-1b was found to be significantly associated with HBV persistence (OR = 1.63, p = 0.03, dominant model).IL-1RAP -8261 T allele and -8183 A allele were also significantly related to HBV persistence (OR = 0.64, p < 0.01, co-dominant model and OR = 0.20, p = 0.01, recessive model, respectively).In haplotype analysis, IL-1b 289C/-581C/-2023C haplotype and IL-1RAP -8261T/-8183A haplotype were associated with HBV persistence (OR = 0.61, p = 0.03, recessive model and OR = 0.64, p < 0.01, co-dominant model, respectively).There are no significant association between genotype or haplotype of IL-1b/IL-1RAP and HCC development. Conclusions:The genetic polymorphisms of IL-1b -2023 C allele, IL-1RAP -8261 T allele and -8183 A allele are probable host factors for HBV persistence.There is no association between genotype or haplotype of IL-1b/IL-1RAP and HCC development.

Human cytomegalovirus (HCMV) is a ubiquitous virus whose sole host is humans. Since HCMV can contagion from person to person through numerous ways, vast populations of humans are infected. HCMV infections can potentially have a range from asymptomatic infection in immuno-compet ent hosts to life-threatening diseases in organ recipients and patients with AIDS. The present article reviews the occurrence of HCMV infections and diseases in humans with different physiological and immunological status, and evaluates the existing laboratory methods for diagnosis of the disease. Iran J Med Sci 2008; 33(3): 127-132.

Hepatitis B virus (HBV) infection is a major cause of liver disease worldwide. Eight genotypes and 24 subgenotypes of HBV have been identified. The aim of this study was to determine the distribution of HBV genotypes, subgenotypes and subtypes, and to understand HBV genetic variability in the HBV genome circulating in Iranian provinces. Two hundred and forty-nine sera from HBV-infected patients living in 25 provinces of Iran were collected (2004-2007). A part of the HBV S/pol and whole BCP/C genes were amplified, sequenced and then subjected to phylogenetic, recombination and genetic variability analysis. Results revealed genotype D of HBV in all samples and subgenotypes D1 (98.52%), D2 (0.74%) and D3 (0.74%) among Iranian patients living in different provinces of Iran. Subtypes ayw2 (94.4%), ayw1 (2.8%), ayw3 (2%) and ayw4 (0.4%) were deduced, on the basis of HBV small surface antigen (HBsAg) amino acid sequences. The mean percentage intra-genotypic distance of S plus core regions was 2.8%; the mean percentage inter-genotypic distance of this region between Iranian strains and genotype D isolates was 3.1%; and this rate for other genotypes was 5.2-11.4%. Various rates of point mutations have been found within different HBV genes, e.g. HBsAg (17.2%), precore-G1896A (59.5%) and Basal core promoter (BCP) double mutations (49.2%), whereas no recombination was found. In conclusion, these results indicate that the only genotype circulating in the provinces of Iran is genotype D. There exist high genetic variabilities in the S/pol and BCP/C regions among the Iranian HBV isolates.

Limited information is available concerning the role of measles-specific cell mediated immunity as a correlate of long-term protection from measles infection. Although serological responses are determined in epidemiological studies and high antibody titer is a good indicator of protection, the role of Cell-Mediated Immunity (CMI) has to be defined more clearly. In this study, Lymphocyte Proliferation (LP) and Viral Neutralization Test (VNT) were used in order to measure measles-specific cellular and humoral immune responses of 100 high school students in Tehran. From total number of subjects studied, 33 were girls and 67 were boys and all were in good health. Of these, 77 had protective neutralizing measles antibody titers and 23 did not have such titer. The results of LP showed that 89 subjects had protective cellular immune responses and 11 did not. A quantitative relationship between humoral and cellular immune responses was not observed. These findings suggest that measles-specific protective CMI is measurable for longer time in comparison to humoral immunity. These data suggest that LP responses may be better sustained than antibody titers in some children.

To define the importantcis-elements in hepatitis delta virus (HDV) RNA, the viral genome was mutated by a linker-scanning mutagenesis strategy that maintained the native rod-like structure of HDV RNA. Mutant HDV cDNAs or their corresponding RNA transcripts were transfected into a Huh-7-derived cell line which continuously expressed small hepatitis delta antigen to study the viral replication and transcription. Here we report the following findings. (i) Although most of the mutant RNAs could self-process to generate the 1.7-kb genomic RNA and all their stabilities were similar, positions which surround the genomic ribozyme domain were found to be important for the self-processing of the dimeric RNA. (ii) The replication of viral RNA was greatly diminished in many mutants, suggesting that multiple regions in HDV RNA were required for replication. (iii) In certain mutants, replication of the HDV antigenomic RNA was selectively abolished but that of the genomic RNA was not. Therefore, this was the first report to show that thecis-elements needed for the replication of genomic or antigenomic HDV RNA could be different. (iv) A continuous region (nt 1625 to nt 431), spanning the HDAg mRNA initiation site and containing thein vitroidentified RNA promoter, was found to be important for mRNA productionin vivo.(v) The HDV RNA replication and transcription was previously proposed to be governed by a single “double-acting promoter.” However, two mutants which were deficient in mRNA synthesis still retained active viral RNA replication. It suggested that the HDV replication could initiate from sites other than this single promoter. This study therefore provided an insight into thecis-elements required for HDV RNA replication and transcription and further contributed to our understanding of HDV life cycle.

La sepsis postanginal o síndrome de Lemierre es una enfermedad rara en nuestros tiempos que ocurre después de una infección orofaríngea y se manifiesta por una tromboflebitis séptica de la vena yugular interna e infecciones metastásicas. Es producida por microorganismos anaerobios, siendo el Fusobacterium necrophorum el más frecuente. Para su diagnóstico, a parte del aislamiento microbiológico del germen, son fundamentales las pruebas de imagen como la ecografía doppler y la tomografía axial computarizada (TAC). El tratamiento consiste en antibioterapia precoz, prolongada, a dosis altas y en ocasiones la excisión quirúrgica de las venas yugulares.Presentamos el caso de una adolescente que tras ser diagnosticada de este síndrome presenta shock séptico grave que evoluciona a fracaso multiorgánico. Como hallazgo particular encontramos la presencia de trombosis a nivel de la vena yugular externa izquierda.Postanginal sepsis or Lemierre syndrome is a rare disease in our times that occurs after an oropharyngeal infection and is manifested by septic thrombophlebitis of the internal jugular vein and metastatic infections. It is produced by anaerobic germs, Fusobacterium necrophorum being the most frequent. Besides the microbiological isolation of the germ, image tests such as Doppler ultrasound and computed-tomography (CT) scan are fundamental for its diagnosis. Treatment consists in early, prolonged antibiotic treatment at high doses and sometimes surgical excision of the jugular veins.We present the case of an adolescent who had serious septic shock that evolved to multiorganic failure after being diagnosed of this syndrome. As a special finding, we discovered the presence of thrombosis in the left external jugular vein.

In spite of hepatitis B virus (HBV) vaccination, HBV infection remains an important public health problem worldwide. Although the HBV genotype distribution has been determined in some parts of South Central Asia, no survey has been conducted to determine the HBV genotype in Afghanistan. Twelve Afghan patients infected with HBV living in Afghanistan were enrolled in this study. Partial HBsAg and basic core promoter, precore, and core (BCP/preC/C) regions were amplified and subjected for direct sequencing. In parallel, precore G1896A mutation was also determined by an amplification-created restriction site method. Results revealed HBV genotype D (95% bootstrap value), sub-genotype D1 (98% bootstrap value), and subtype ayw2 in all Afghan isolates. Afghan isolates clustered in a separate branch in the D1 sub-genotype called D1', while supported by 82% bootstrap value. The percentage of intra-genotypic distance among Afghan isolates was 1.05% and inter-genotypic distance with the other genotype D was 2.87% and with other genotypes was 7.50%-11.1%. The wildtype, mixed infection, and precore mutant were found in six, two, and four HBV isolates, respectively. The A1762T/G1764A BCP dual mutation was found in one isolate. Three isolates presented single mutation in the BCP dual mutation region, whereas two showed a novel G1764T mutation. In conclusion, this preliminary study revealed HBV genotype D, sub-genotype D1, and subtype ayw2 of HBV among hepatitis B infected patients from Afghanistan. Further investigation should be carried out.

Objective Dot Blot (DB) assay provides highly specific results, but usually is not reliable for quantification of antibody production. The need for a more objective DB assay to provide a better definition of the immune status, against HIV antigens, promoted this study to develop a quantitative DB assay. Materials and Methods Dot blot (DB) strips for antibodies, directed to human immunodeficiency virus (HIV) type 1 and 2, were analyzed by a video densitometer. This method was used to quantify the antibody response to different HIV proteins in infected patients. In order to increase reproducibility, reagents and protocols were accurately standardized and internal controls were added. In the first format, an internal control band consisting of Human IgG was added to each dot to minimize the effects of band intensity variation. In the second format, antibody concentrations were calculated from the ratio of the densities produced by test sera and by positive and negative standard sera. Results The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the obtained results were compared with those of the corresponding DB. A statistically significant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titers were compared to corrected DB values (p = 0.001). Conclusion Densitometric analysis of DB assays led to quantify the antibodies against HIV-1 and 2 Gag and Env proteins and might be useful to investigate possible humoral immune correlates of production in HIV vaccine studies and antibody production in the early phase of infection.

Background: Macrophages have important role in defense against Herpes Simplex Virus type-1 (HSV-1). The present study was performed to determine the viability and nitric oxide (NO) production by HSV-1 infected mouse peritoneal macrophages (HIM).Method: The viability of macrophages was evaluated using MTT reduction assay and the production of nitrite using Griess method.Results: The ability of infected macrophages to reduce Tetrazolium (MTT) was diminished at virus to cell ratios of multiplicity of infection (MOI) of one, three and 10; but not at 0.01and 0.1. Induction and inhibition of NO production by HIM were MOI dependent. The basal NO production by these cells was inhibited at MOI of three and ten. In contrast virus to cell ratios of 0.01 and 0.1 induced low but significant enhancement in NO production. The inability of HIM to reduce MTT at MOI of three was significant after 12-hrs and inhibition of NO production was initiated between 12-20 hours after infection.Conclusion: High doses of HSV-1 seem to decrease the normal activity of macrophages by inhibiting the production of nitric oxide.

Hepatitis C is a major cause of liver related morbidity and mortality worldwide and represents a major public health problem. Depending on genomic organization, the virus is divided into six genotypes and a number of subtypes. Different genotypes are seen in different parts of the world. Genotype one is difficult to treat, while genotypes 2 and 3 are easy to treat. Therefore, identification of HCV genotype in patients is necessary to begin and follow up the treatment. In this study, viral genomic materials of 214 patients’ sera were detected by nested-RT PCR. Based on genomic differences among different genotypes, the PCR products were digested with proper enzymes and studied by RFLP. Except for one, sequencing of 14 samples, representative of all genotypes, confirmed the results of PCR-RFLP. The results of PCR-RFLP were as follows: 1a (52.88%), 1b (14.01%), 3a (27.57%), 2a (2.1%), 4 (3.44%). This indicates that a high percentage of HCV infected patients in Iran are infected with 1a or 3a genotypes. These findings reveal that the pattern of HCV genotypes in Iran differs from those of other middle-eastern countries.

OBJECTIVES:High-yield isolation and purification of human leukocyte subpopulations from whole blood is fundamental to many biological and medical applications including qualitative and quantitative PCR-based techniques of determining human cytomegalovirus infection. Several procedures have been reported to purify morphologically and functionally intact human leukocyte subpopulations for diagnostic proposes. Here, we report and evaluate a technique for high-yield purification of intact and viable human leukocyte subpopulations based on modification of a previous methodology.MATERIALS AND METHODS:One hundred peripheral blood samples were collected from bone marrow transplant recipients (n = 60), bone marrow donors (n = 20), and healthy blood donors (n = 20). The samples were tested in parallel using 4 different leukocyte separation methods. The methods were evaluated based on the concentration, purity, and viability of the isolated leukocyte subpopulations.RESULTS:When compared with standard methods, our methods produced 99% purity for both polymorphonuclear or mononuclear leukocytes. The corresponding viability for the methods was determined to be 98%. No erythrocyte contamination was demonstrated. However, the maximum concentration for polymorphonuclear or mononuclear leukocytes obtained by standard methods was 70%. The corresponding viability for all the methods was determined to be 98%.CONCLUSIONS:Our results indicate that in patients with decreased whole blood leukocyte numbers, using either a modified Ficoll NH(4)Cl or a modified dextran method would be valuable for simultaneous separation of polymorphonuclear and mononuclear leukocytes with high purity, viability, and concentration.

The aim of the study was to determine the prognostic value of a double primer PCR assay to detect human cytomegalovirus (HCMV) infection or disease in bone marrow transplant (BMT) recipients. A total of 209 blood samples including peripheral blood mononuclear cells (PBMN), polymorphonuclear (PMN) leukocytes and plasma from 26 BMT recipients were tested by PCR assay. To discriminate between latent and active HCMV infection, 177 blood samples were also tested by a quantitative antigenemia assay. HCMV serology status of donors and recipients was determined before transplantation by an enzyme immunosorbent assay method. Using the double primer PCR assay, the number of positive samples increased by an average of 11.6%. Symptomatic active HCMV infection was diagnosed in 14 (53.8%) out of 26 BMT patients. There was a good association between double primer PCR assay of PMN leukocytes and antigenemia assays for detection of active HCMV infection in all patients. Detection of HCMV DNA in PMN leukocytes of BMT patients by double primer PCR assay can be an alternative method for antigenemia assay. However, quantitative PCR methods will be necessary for monitoring antiviral treatment.

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.