Whole genome characterization and evolutionary analysis of bovine ephemeral fever virus isolated in Iran.

Bovine ephemeral fever virus (BEFV) is an economically important arthropod-borne virus of cattle and water buffaloes which is enzootic in Africa, Australia, and Asia. We characterized the entire length of BEFV BA/RZ/IR strain genome isolated in Iran and compared to the all BEFV full genomes available in the GenBank. The BEFV genomes were phylogenetically classified as 4 lineages including the Middle Eastern, East Asian, Australian, and South African lineages. The Iranian BA/RZ/IR strain, which displayed maximum sequence identity (96.72%) to the Chinese JT02L strain was clustered as a separate branch in the East Asian lineage of the virus. Using Shannon entropy analysis, amino acid variations were detected in the all proteins encoded by BEFV genomes. Particularly, the polymerase L and the accessory proteins Gns, α2 and ß exhibited the highest amino acid variations suggesting their significance in the viral replication efficiency. Our bioinformatics analyses also predict the occurrence of recombination event within the East Asian lineage of BEFV genomes. Our data show that the Chinese Henan 1 may be a hybrid strain constructed of the Chinese JT02L and Iranian BA/RZ/IR BEFV strains as the major and minor parents, respectively. These computational analyses suggest that the homologous recombination may be an evolutionary mechanism for BEFV as a member of the Rhabdoviridae family.

Sperm and oocyte as carriers for bovine viral diarrhoea virus biotypes during in vitro fertilization.

Bovine viral diarrhoea virus (BVDV) is an important viral agent causing the reproductive failure in cattle. The objectives of the study were to assess the role of male and female gametes, as carriers of cytopathic (CP) and non-cytopathic (NCP) BVDV to embryonic cells during in vitro fertilization. In this respect, sperm and oocytes were separately exposed to concentrations of 104.5 or 105.5 TCID50 /mL CP and NCP BVDV, for 2 h before fertilization. After washing, the intact gametes with the infected gametes were inseminated. Seven days post-fertilization, the virus-exposed embryos were examined for presence of the viral genome by RT-PCR. One-way anova with post-hoc Tukey's HSD test and an independent samples t-test were used to compare within and between groups, respectively. The results presented a significant decrease in the blastocyst rates for CP-infected groups than NCP-infected groups (p ≤ .01). Compared to the controls and the infected oocyte groups, the cleavage rates of the infected sperm groups (NCP and CP BVDV) were significantly reduced both in low (104.5 TCID50 /mL) and high (105.5 TCID50 /mL) titres of the virus (p ≤ .01). The proportion of embryos which was developed to blastocyst stages was significantly lower for CP and NCP-infected groups than the control groups (p ≤ .001). According to the molecular results, all samples of the retarded/degenerated embryos (at least one blastocyst within each one) in CP and NCP groups, one sample (at least one blastocyst in that) within a CP-infected group, and six samples (at least one blastocyst in each one of those) of NCP-infected groups contained the viral nucleic acid. Likewise, the results of viral enrichment showed all reactions in which RT-PCR were positive induced CPEs in MDBK monolayers. In conclusion, it is clear that CP and NCP BVDV were able to traverse zona pellucida during fertilization, and they had also negative effects on embryo development.

Prevalence of bovine herpesvirus 1 antibodies and risk factors in dairy cattle of Iran's central desert.

Bovine herpesvirus type 1 (BoHV 1) is a major bovine pathogen spreading worldwide and causing extensive damage to the livestock industry. BoHV causes respiratory, genital, and neurological disorders. A cross-sectional study was performed for the first time to estimate the seroreactivity to BoHV 1 and related risk factors among Iran's central desert dairy cattle. A total of 800 blood samples was randomly collected from 76 unvaccinated herds. Samples were tested with an indirect enzyme-linked immunosorbent assay (ELISA) commercial kit to detect BoHV 1 antibodies. The logistic regression model was used to analyze the data. BoHV 1 seroreactivity at animal and herd levels was 50% and 65%, respectively. Herd size was recognized as a risk factor (OR = 2.65, CI = 1.61-4.37) for seroreactivity to BoHV using GLM (p < 0.05). The high prevalence of BoHV 1 antibodies in the study area indicates the need to implement educational programs on the importance of the disease and design methods to control and prevent virus distribution.

Seroprevalence of bovine viral diarrhea virus antibodies and risk factors in dairy cattle from the central desert of Iran.

Bovine viral diarrhea virus (BVDV) infects cattle worldwide and causes one of the most important economic diseases of the dairy industry. BVDV infection reduces reproductive efficiency, suppresses the immune system, and causes gastrointestinal and respiratory diseases. A first cross-sectional study was conducted in the central desert of Iran (Yazd and South Khorasan provinces) to estimate the seroprevalence and identify BVDV-related risk factors in dairy cattle. A total of 800 cows were randomly selected of 76 herds, and their serum samples were tested by the indirect enzyme-linked immunosorbent assay (ELISA) method for BVDV antibody detection. Data were analyzed using the logistic regression model. The serum prevalence of BVDV at animal and herd levels was 66.83% and 91.6%, respectively. Traditional housing system (OR = 3.22; 95% CI = 1.20-9.09) and cattle introduction to the herd (OR = 2.12; 95% CI = 1.21-3.70) were the important risk factors for BVDV seropositivity (p < 0.05). Increasing of age per year caused adding in 0.33 log (odds) of BVDV seropositivity (p < 0.05). It is necessary to implement control and eradication programs because of the high seroprevalence at the individual level and at the herd in the central desert of Iran.

Antigenic variation of bovine ephemeral fever viruses isolated in Iran, 2012-2013.

Bovine ephemeral fever virus (BEFV) is an economic arthropod-borne virus distributed in Africa, Asia, and Australia. Based on the sequence of the gene encoding the surface glycoprotein G, the viral antigenic determinant, BEFV has been phylogenetically classified into three clusters, including Australia, East Asia, and the Middle East. Here, we provide evidence for antigenic variations among the BEFV isolates in Iran during the period of 2012 to 2013 and also the exotic YHL strain, which are all classified into the East Asian cluster of the virus. For this propose, the entire length of the G gene of the viruses were sequenced and phylogenetically compared. The corresponding antigenic sites (G1-G4) were analyzed and antigenic relatedness among these viruses was measured. The two Iranian viruses, which displayed substitutions at residues E503K in the site G1 and E461K in the predicted site G4, were partially neutralized by each other's antisera (R value = 63.23%); however, these two viruses exhibited much lower cross-neutralization that measured by R value as 28.28% and 22.82%, respectively. The crucial substitution at amino acid R218K in the site G3a is believed to be the foremost cause of these declines. The data emphasize the frequent evolution of BEFV in different time periods and geographic regions, in which the new variants can emerge and likely escape from the pre-existing immunities. Thus, continuous monitoring of the circulating viruses is necessary for understanding the viral evolution and evaluation of protective immunity induced by the heterologous viruses.

Seroprevalence and risk factors associated with bovine viral diarrhea virus and bovine herpes virus-1 in Zanjan Province, Iran.

Bovine viral diarrhea virus (BVDV) and bovine herpes virus-1 (BHV-1) remain as the major pathogens with heavy economic consequences in Iran. The prevalence of antibodies against BVDV and BHV-1, the rate of BVDV persistently infected (PI) animals, and associated risk factors were evaluated in a cross-sectional study carried out in Zanjan Province, Northwest Iran, in December 2011. A total number of 562 cattle in 10 herds and five cities were randomly selected, and their serum samples were tested to detect antibodies to these viruses and also BVDV antigen-positive (PI) animals. The data were analyzed with Pearson's correlation coefficient, chi-square, and logistic regression test. In total, nine and eight of the selected herds were seropositive to BVDV and BHV-1, respectively. The overall seroprevalence of these infections were estimated at 28.6 and 10.7% for BVDV and BHV-1, respectively, and 0.53% of the samples were detected as persistently infected. Statistical analysis revealed that sex, age, and farming system are risk factors for both infections (P < 0.05), while breed was determined as a strong risk factor only for BVDV (P < 0.001). In addition, the present study certainly identifies that infection with BVDV is associated with infection to BHV-1 (OR = 4.52, 95% CI: 2.60-7.80; P Ë‚ 0.001). The results add our knowledge about the prevalence and associated risk factors of BVDV and BHV-1 in Iran and imply that the prophylactic and surveillance strategies need to be implemented to reduce the risk of spread of these viruses.

Effect of bovine viral diarrhea virus biotypes exposure on bovine gametes in early embryonic development in vitro.

Bovine viral diarrhea virus (BVDV) is an important viral agent causing reproductive failure in cattle. The objectives of the current study were to investigate the interaction between two BVDV biotypes, cytopathic (CP) and Non-cytopathic (NCP) and bovine gametes during in vitro fertilization (IVF) processing, the existence of the virus within embryonic cells and early embryonic development rates. Sperm and ova were exposed separately to CP and NCP BVDV at two concentrations of 104.5 and 105.5 tissue culture infectious dose 50.00% (TCID50) mL-1 prior to IVF, respectively. After five days post-IVF, early embryonic development rates of infected groups were assessed. Several embryos of each group, normal and degenerated, were selected for a viral assay using reverse transcription polymerase chain reaction technique. The result showed that the early embryonic development rates were decreased in treatment groups. The rates in the CP groups were lower than the NCP groups. In the CP groups, the proportions were, respectively, 10.00, 6.00 and 11.00, and 6.00% in the infected sperm and oocyte groups (104.5 and 105.5 TCID50 mL-1) that were higher than 50.00% in the control group. In NCP groups, the rates were, respectively, 25.00, 18.00 and 24.00, and 21.00% in the infected groups compared to 48.00% in the control group. In the CP groups, no BVDV was detected in normal embryos, whereas, all degenerated embryos were completely virus-positive. In the NCP groups, the virus was detected in both normal and degenerated embryos. In conclusion, this study supported detrimental impacts of CP and NCP BVDV on early embryonic development and the role of sperm and the zona pellucida layer as carriers of the virus.

Structural features of the Seneca Valley virus internal ribosome entry site (IRES) element: a picornavirus with a pestivirus-like IRES.

The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId(2) domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.

Identification of Cytotoxic T lymphocyte (CTL) Epitope and design of an immunogenic multi-epitope of Bovine Ephemeral Fever Virus (BEFV) Glycoprotein G for Vaccine Development.

Bovine ephemeral fever (BEF), a vector-borne disease of cattle and water buffalo, is enzootic in tropical and subtropical zones of Asia, Australia, and Africa. Since cytotoxic T lymphocytes (CTL) responses may play a key role in the control of bovine ephemeral fever virus (BEFV) infection, it is important to identify and characterize the CTL target epitopes of BEFV antigens. The current study has been designed to identify and characterize the potential CTL epitopes using the Immuno-informatics tools, and it helped find the potent vaccine candidates against BEF. Antigenicity, toxicity, allergenicity, and immunogenicity testing of predicted CTL epitopes was done. Total four CTL epitopes for BEFV G protein, have been identified as potential epitopes. Prediction of the 3D structure of multi-epitope (final structure) was performed using I-TASSER server. Model 1 was selected as the best model with C-Score: -3.71. The modeled G protein structure and multi-epitope structure were validated by the Ramachandran plots Prosa and Verify 3D server. Epitopic regions of 3D protein structure were identified by Chimera UCSF software. Physicochemical properties of the Multi epitope were evaluated using ProtParam server. This is the first report of CTL epitope in the G protein of BEFV. In this manner, they would play an important role in evoking the immune response as well as vaccine development. However, in vitro and in vivo experimental studies are required for suggested epitopes verification. The multi-epitope was designed from regions of the G protein sequence that lacked mutation and genomic diversity. Therefore, it can be introduced as a protein vaccine from all strains of BEFV as a vaccine candidate for design.

Large-scale seroprevalence and risk factors associated with Bluetongue virus in Iran.

Bluetongue virus (BTV) remains as an economically major concern in the world. Seroprevalence and potential risk factors of BTV were assessed in a cross-sectional study at both the herd and animal levels in Iran. A total of 73 Epidemiologic Units (E.Unit), defined as a herd, flock or village including animals with equal chance of exposure to infectious agents, were randomly selected. Serum samples from all animals (n = 34,575) within the E.Units were collected and tested for BTV sero-group antibodies by using commercially competitive ELISA test. Using cluster analysis, 90.41 % (95 %, CI: 80.85 %-95.47 %) of the E.Units and 56.13 % (95 % CI: 55.61 %-56.66 %) of the tested animals were detected seropositive against BTV. A seroprevalence rate of 57.59 % (95 % CI: 48.01 %-66.63 %), 65.65 % (95 % CI: 59.10 %-73.74 %) and 27.63 % (95 % CI: 14.40 %-46.43 %) was estimated for sheep, goats and cattle, respectively. At E.Unit (herd) level, density was identified as a great risk factor for the infection (r2 = 0.891; P = 0.000), and particularly density of cattle significantly correlated with BTV infection within the E.Units (r2 = 0.247; P = 0.019). Using multilevel logistic regression, adjusted odds ratios (ORs) were estimated at individual level. A significantly less risk of BTV infection was evaluated for cattle than for sheep (OR = 0.42, 95 % CI: 0.38-0.47, P < 0.001), while no significant difference was observed between sheep and goat (OR = 1.03, 95 % CI: 0.97-1.10, P = 0.345). Animals over 2 years and between 6 months and 2 years expressed 2.22 (OR = 2.22, 95 % CI: 1.96-2.52, P < 0.001) and 2.18 (OR = 2.18, 95 % CI: 1.92-2.49, P < 0.001) times higher chance for the infection than animals under 6 months. Males were at significantly less risk of the infection than females (OR=0.68, 95 % CI: 0.63-0.74, P < 0.001). Animals kept in industrial farming systems displayed 0.46 (OR=0.46, 95 % CI: 032-0.66, P < 0.001) times less chance than animals kept in traditional farming system for BTV, while animals lived in semi-industrial farming system were found to be at 2.97 (OR=2.97, 95 % CI: 2.41-3.66, P < 0.001) times higher chance for BTV than animals lived in traditional farming system. Furthermore, seropositive animals exhibited a high amount of antibodies against BTV (s) suggesting that viral exposure may have frequently occurred during their lifetimes. This large - scale study yielded information on epidemiology of BTV in Iran that is prerequisite for further research, and also for evaluation of any cost-benefit control measure to be established in an enzootic zone of the virus.

The picornavirus avian encephalomyelitis virus possesses a hepatitis C virus-like internal ribosome entry site element.

Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5' untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3' end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.

Expression of G1- epitope of bovine ephemeral fever virus in E. coli : A novel candidate to develop ELISA kit.

Bovine ephemeral fever is an acute and arthropod-borne viral disease of cattle and water buffalo which occurs seasonally in most of the world tropical and subtropical regions. The epizootic feature of the disease has been reported in Iran with serious economic consequences. The surface glycoprotein G of bovine ephemeral fever virus (BEFV) is composed of 4 antigenic sites (G1-G4) and plays the main role for eliciting neutralizing antibodies and protective immunity. The G1 - epitope is a linear antigenic site and conserved among BEFV strains. In order to develop an ELISA test based on G1-epitope as coating antigen, this study was carried out to express the recombinant G1-epitope of BEFV in prokaryotic system. Using PCR and specific primers, a length of 88 amino acid of the G glycoprotein of BEFV including G1- epitope was amplified and cloned into the expression vector pGEX-4T-1, with the GST moiety. The recombinant plasmid (pGEX-4T-1-G1) was then transformed into Escherichia coli BL21 and expression of fusion protein was induced by 0.10 mM IPTG. The maximum expression of the fusion protein was obtained at 16 hr post induction as verified by SDS-PAGE electrophoresis, and it was also confirmed that this protein bearing G1- epitope is sufficiently biologically active to bind to anti-BEFV serum in western blot experiment.

Lack of Evidence for a Relationship between High Risk Human Papillomaviruses and Breast Cancer in Iranian Patients.

BACKGROUND: Whether there is any relationship between human papilloma virus (HPV) and breast carcinoma is not clear. Some previous studies have indicated a possible role in oncogenesis in the breast. In this study, we therefore analyzed the presence of HPV infection in breast tissues of Iranian women from Yazd city. MATERIALS AND METHODS: In a cross-sectional study, formalin-fixed paraffin-embedded tissues from 87 patients with breast cancer and 84 cases with breast fibrocystic lesions (control group) were selected from a tissue archive. Grade of tumors and fibrocystic tissues were determined by two pathologists. The nested-PCR method was performed for detection of HPVs in samples. HPV genotypes were determined by sequencing and the phylogenetic tree depicted by MEGA software. RESULTS: Of the 87 women with breast cancer, 22.9% (20 isolates) had positive results for HPV DNA. In the control group no HPV was detected. The HPV genotypes in positive samples were HPV-16 (35%) HPV-18 (15%), HPV-6 (45%) and HPV-11 (5%). The data did not approved a significant correlation between tissue pathology of breast cancer and the HPV genotype frequency. CONCLUSIONS: The data did not provide any evidence for a role of high risk HPV types in oncogenesis in the breast.

Bovine Ephemeral Fever in Iran: Diagnosis, Isolation and Molecular Characterization.

BACKGROUND: Bovine ephemeral fever (BEFV) is an arthropod-borne disease of cattle and water buffaloes. BEFV occurs seasonally in tropical, subtropical and temperate regions of Africa, Asia and Australia. It has been known for the past decades in Iran based on clinical signs but lack of an accurate diagnosis has made the real feature of disease obscured. This is the first scientific report on isolation and identification of the agent in which molecular diagnosis of BEFV was also set up with high sensitivity and specificity. METHODS: The viral agent was successfully isolated through serial passages in brain of suckling mice and cell culture. In addition, the circulating virus during the autumn 2012 in Iran was molecularly characterized based on partial G gene. RESULTS: Alignment of 3 virus sequences from different parts of Iran revealed that they are identical suggesting that the circulating viruses were most likely the same in this period. Phylogenetic analysis of the Iranian sequences with 17 sequences in the GenBank from the world showed that it is identical to the virus circulated in Turkey during the same period suggesting that the virus was circulated in a large geographic region. CONCLUSION: These results offer primary information about BEFV in Iran. To better understanding the epidemiology of the virus, further studies based on seroepidemiology, molecular epidemiology, entomology and meteorology together with finding the model of animal transportation in the region are necessary.