Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

Evaluation of the different methods to detect Salmonella in poultry feces samples.

Salmonella is one of the most common causes of foodborne outbreaks and infection worldwide. The gold-standard detection method of Salmonella is cultivation. There is a need to investigate rapid and accurate processes with time-consuming cultivation. The study evaluated different approaches to detect Salmonella in poultry feces samples. Poultry farm feces samples from 21 cities in Iran were collected from January 2016 to December 2019. Microbiological cultures, serological assays, and multiplex PCR (m-PCR) were used to detect and characterize Salmonella spp. isolates. Serological assays and m-PCR were used to determine the serogroups A, B, C1, C2, D1, E, H, and FliC. The m-PCR was used to detect seven Salmonella serovars, and a Chi-square test was performed to compare the discriminatory power of the methods. Of 2300 poultry feces samples, 173 (7.5%) and 166 (7.2%) samples were detected as Salmonella spp. by cultivation and m-PCR, respectively. The sensitivity of the molecular method was equal to cultivation at 0.96 (CI = 95%). Assessment of H antigenic subgroups showed the same for both m-PCR and serological tests. Therefore, the matching rate of the two methods for detecting all H antigenic subgroups was 100%. Thus, the relationship between the results obtained from both methods was significant in the contingency table test (P < 0.01). The PCR-based approach confirmed the detection of Salmonella in a shorter period (24-36 h) compared to the conventional microbiological approach (3-8 days).

Circulation of at Least Six Distinct Groups of Pigeon-Derived Newcastle Disease Virus in Iran Between 1996 and 2019.

According to the latest Newcastle disease virus (NDV) classification system, Iranian PPMV-1 isolates were classified as either XXI.1.1 or XXI.2 subgenotypes only. However, a few recent studies have suggested the possible existence of other Iranian PPMV-1 genotypes/subgenotypes. Recently, we isolated a PPMV-1 closely related to the African origin subgenotype VI.2.1.2 from an ill captive pigeon in a park aviary in central Tehran (Pg/IR/AMMM160/2019). This subgenotype had never been reported from Iran or neighboring countries. We also isolated a subgenotype VII.1.1 NDV (Pg/IR/AMMM117/2018), usually reported from non-pigeon birds in Iran. The nucleotide distance of AMMM117 was 1.0-2.5% compared to other Iranian subgenotypes VII.1.1 isolates. However, usually the same year VII.1.1 viruses that we isolate from Iranian poultry farms show negligible distances (0.0-0.5%). More isolates are required to study if this difference is due to subgenotype VII.1.1 being circulated and mutated in pigeons. Here, we also characterized two other isolates, namely Pg/IR/AMMM168/2019 and Pg/IR/MAM39/2017. The latter is the first Iranian subgenotype XXI.1.1 to be featured in the NDV datasets of the international NDV consortium. We also investigated the phylogenetic relation of all the published Iranian pigeon-derived NDV to date and updated the grouping according to the latest classification system. We have concluded that at least six different groups of pigeon-derived NDV have been circulating in Iran since 1996, four of which have been reported from just one city over the last seven years. This study suggests that the Iranian pigeon-origin NDV have been more diverse than the Iranian poultry-derived NDV in recent years.

The fowl adenovirus (Fadv-11) outbreak in Iranian broiler chicken farms: The first full genome characterization and phylogenetic analysis.

Fowl adenoviruses D and E (FAdV-D and E) can cause inclusion body hepatitis (IBH) in commercial chicken flocks. Recently, IBH outbreaks have been increasingly reported in different regions of Iran, particularly in broiler farms. The present study was conducted to perform, for the first time, a complete genome characterization of a FAdV isolate from an IBH outbreak in Iran. Briefly, liver samples were collected from affected broiler flocks and following viral DNA extraction and confirming by PCR technique; one positive sample was selected from an affected flock to conduct a complete genome sequencing. The current FAdV, named "Fowl_Adenovirus_D_isolate_iran/UT-Kiaee_2018", was placed into FAdV-11 serotype (D species). According to the complete genome sequence analysis, UT-Kiaee had high homology with Chinese and Canadian FAdV. The partial sequence of the hexon gene revealed that UT-Kiaee shared 100% identity with previous Iranian FAdVs. The present study was the first to report full genome FAdV in Iran and complete the puzzle of molecular epidemiology of FAdV in Iran through determining the possible origin of Iranian FAdvs, which are the causative agents of recent IBH outbreaks in Iran.

Seroprevalence and genotyping of avian infectious bronchitis virus detected from Iranian unvaccinated backyard chickens.

BACKGROUND AND OBJECTIVES: Different epidemiological studies have found that backyard chickens are a reservoir for poultry diseases. Most backyard chicken flocks have a poor level of biosecurity, which increases the risk of spread of diseases. In recent years, the number of backyard chickens has been on the rise in Iran. However, the health status of backyard flocks is still poorly documented. Thus, this study aimed at examining the seroprevalence of antibodies against infectious bronchitis virus (IBV) and molecular surveillance and genotyping of IBV among backyard chickens (without vaccination history) in Mazandaran province, North of Iran, 2014. MATERIALS AND METHODS: A total of 460 blood samples of unvaccinated backyard chickens in the mentioned area were tested for antibodies against IBV using commercial ELISA. Also, cecal tonsils were collected from 75 chickens in the same area. Real time RT-PCR (for detection) and RT-PCR and sequencing spike gene were performed. RESULTS: The seropositivity rate was 54.5%. In addition, we detected 793/B, Variant 2, and QX in the backyard flocks and performed phylogenetic studies on them. The phylogenetic study revealed that the detected genotypes had high homology with IBV strains that were infected broilers, pullets, and layers in Iran. CONCLUSION: There is a need for continuous monitoring of IBV among avian species to complete the epidemiological map and work on the pathogenesis of Iranian IBV strains in Iranian backyard chickens.

Fowl Adenoviruses D and E Cause Inclusion Body Hepatitis Outbreaks in Broiler and Broiler Breeder Pullet Flocks.

Twenty-four fowl adenoviruses (FAdVs) were isolated from broiler and broiler breeder pullet flocks in Iran during 2013-2016 and were identified and characterized. All FAdVs were from inclusion body hepatitis (IBH) cases, showing an enlarged and pale yellow liver with multiple petechial hemorrhages. Phylogenetic analyses of partial hexon gene sequences are an adequate and quick method for differentiation and genotyping. The isolates were subjected to PCR to amplify a 590-bp fragment from the hexon gene. Sequence analysis revealed the presence of two species D and E. Eighty FAdV isolates were genetically related to the strain EU979378 of FAdV-11 (96.5% to 97.6% identity), and six isolates were related to the strain EU979375 of FAdV-8b (97% identity). The results indicated that two FAdV serotypes (11 and 8b) are high prevalence serotypes of FAdVs in Iran and are pathogenic enough to cause IBH in young chicks. Therefore, preventive measures against FAdV infection on poultry farms should be implemented.

The first comprehensive molecular detection of six honey bee viruses in Iran in 2015-2016.

At least 18 viruses have been reported in the honey bee (Apis mellifera L.). However, severe diseases in honey bees are mainly caused by six viruses, and these are the most important in beekeeping. These viruses include: deformed wing virus (DWV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), sacbrood virus (SBV), kashmir bee virus (KBV), and black queen cell virus (BQCV). In this study, we evaluated 89 Iranian honey bee apiaries (during the period 2015-2016) suffering from symptoms of depopulation, sudden collapse, paralysis, or dark coloring, by employing reverse transcription-PCR. Samples were collected from four regions (Mazandaran, Hormozgan, Kurdistan, and Khorasan Razavi) of Iran. Of the 89 apiaries examined, 16 (17.97%), three (3.37%), and three (3.37%) were infected by DWV, ABPV, and CBPV, respectively. The study results for the other viruses (SBV, KBV, and BQCV) were negative. The present study evaluated the presence of the six most important honey bee viruses in bee colonies with suspected infections, and identified remarkable differences in the distribution patterns of the viruses in different geographic regions of Iran.

Phylogenetic study-based hemagglutinin (HA) gene of highly pathogenic avian influenza virus (H5N1) detected from backyard chickens in Iran, 2015.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have been diversified into multiple phylogenetic clades over the past decade and are highly genetically variable. In June 2015, one outbreak of HPAI H5N1 in backyard chickens was reported in the Nogardan village of the Mazandaran Province. Tracheal tissues were taken from the dead domestic chickens (n = 10) and processed for RT-PCR. The positive samples (n = 10) were characterized as HPAI H5N1 by sequencing analysis for the hemagglutinin and neuraminidase genes. Phylogenetic analysis of the samples revealed that the viruses belonged to clade 2.3.2.1c, and cluster with the HPAI H5N1 viruses isolated from different avian species in Bulgaria, Romania, and Nigeria in 2015. They were not closely related to other H5N1 isolates detected in previous years in Iran. Our study provides new insights into the evolution and genesis of H5N1 influenza in Iran and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Iran.

Phylogenetic analysis of the glycoprotein gene of viral hemorrhagic septicemia virus from Iranian trout farms points towards a common European origin.

Viral haemorrhagic septicaemia virus (VHSV), a member of family Rhabdoviridae and genus Novirhabdoviridae, causes mortality in numerous marine and freshwater hosts located in northern hemisphere. To evaluate the genetic diversity of VHSV from the North and South West of Iran, the sequences of a 1483bp nt region of the glycoprotein gene were determined for four Iranian isolates. These sequences were analysed to evaluate their genetic relatedness with 86 worldwide isolates representing the four known genogroups of VHSV. Phylogenetic analysis by nucleotide sequences showed that all the VHSV isolates studied were closest related to the 19 fresh water strains from Germany grouped within the European genogroup Ia-2. This finding indicates that Iranian VHSV most likely was introduced to Iran by the movement of contaminated fish fry from a source in Europe.

Molecular characterization of infectious bronchitis viruses isolated from broiler chicken farms in Iran, 2014-2015.

Infectious bronchitis (IB) is a viral avian disease with economic importance in the world, including Iran. S1 gene sequencing has been used for molecular epidemiological studies and genotypic characterization of infectious bronchitis virus (IBV). A total of 118 IBV isolates were obtained from tissue samples from chickens with clinically suspected IB from Iranian broiler farms (eight provinces, 200 samples). The isolates were confirmed by real-time polymerase chain reaction (PCR) and characterized by sequencing the spike glycoprotein gene. The isolates formed six distinct phylogenetic groups (IS/1494/06 [Var2] like, 4/91-like, IS/720-like, QX-like, IR-1 and Mass-like) that were related to variants isolated in the region. The most frequently detected viruses were of the Var2-like (IS/1494/06-like) genotype, with an overall prevalence of 34 %. Twenty-one percent of the isolates formed a cluster together with the 4/91 IBV type, 10 % were of the QX genotype, and 8 % were of the IS/720 genotype. In addition, 4 % and 3 % of the isolates belonged to the Massachusetts and IR-1 genotype, respectively. For the first time, we have isolated and characterized IBV variants from broiler farms in different provinces of Iran. This study demonstrates a constant evolution of IBV in Iran, demonstrating the need for continuous monitoring and development of new vaccines based on indigenous viruses.

Molecular characterization and phylogenetic study of Newcastle disease viruses isolated in Iran, 2010-2012.

Newcastle disease (ND) is a highly contagious viral disease and has been a constant threat to the poultry industry worldwide. In this study, partial sequences of ND virus (NDV) fusion genome collected from some provinces in Iran during 2010-2012 in vaccinated commercial farms were characterized and compared with other NDV sequences. All viruses showed the amino acid sequence 112 RRQKRF117 at the C-terminus of the F2 protein and phenylalanine at the N-terminus of the F1 protein, residue 117. These amino acid sequences were identical to a known virulent motif. The phylogenetic analysis showed that the Iranian ND isolates in this study are closely related to the genotype VIId of class II NDV strains. The emergence and identification of new sublineages provide an insight into the high rate of genetic drift occurring in NDV strains in Iran, and raise many concerns about the efficacy of current ND control measures in the country.

An active serological survey of antibodies to newcastle disease and avian influenza (H9N2) viruses in the unvaccinated backyard poultry in Bushehr province,Iran, 2012-2013.

OBJECTIVE: To test the antibodies against newcastle disease virus (NDV) and avian influenza virus (AIV, H9N2) in the unvaccinated backyard poultry in Bushehr province, Iran from 2012 to 2013. METHODS: A total of 1 530 blood samples from unvaccinated backyard chickens in Bushehr province, south of Iran, were tested for antibodies against NDV and AIV (H9N2) by hemagglutination inhibition test according to International Epizootic Office (OIE) recommendation. RESULTS: Of these, 614 (40.13%) and 595 (39.00%) were positive for NDV and AIV (H9N2) respectively. CONCLUSIONS: The findings of the present study indicated that NDV and AIV (H9N2) were endemic and widely distributed in backyard areas of Bushehr province which should be incorporated in the control strategies. Further studies are needed to identify the circulating virus genotypes, model their transmission risk, provide adapted control measures and design proper and applicable vaccination program.

Elevated carbon monoxide to carbon dioxide ratio in the exhaled breath of mice treated with a single dose of lipopolysaccharide.

BACKGROUND: Analysis of volatile organic chemicals in breath holds promise for noninvasive diagnosis and monitoring of patients, but investigation of this in experimental mouse models has been limited. Of particular interest is endogenous production of carbon monoxide as a biomarker of inflammation and, more particularly, during sepsis. METHODS: Using a nose-only collection procedure for unanesthetized individual adult mice and sensitive gas chromatography of carbon monoxide (CO) and carbon dioxide (CO2) of sampled breath, we investigated the responses of mice to one-time injections with different doses of purified Escherichia coli lipopolysaccharide. Two strains of mice were examined: BALB/c and C3H, including an endotoxin-resistant mutant (HeJ) as well as the wild type (HOuJ). RESULTS: The CO to CO2 ratio increased in a dose-responsive manner within hours in treated BALC/c mice but not control mice. The CO/CO2 values declined to the range of control mice within 48-72 h after the injection of lipopolysaccharide. Breath CO/CO2 values correlated with systemic inflammation biomarkers in serum and heme oxygenase-1 gene expression in blood. C3H/HOuJ mice, but not the HeJ mice, had similar increases of the CO/CO2 ratio in response to the endotoxin. CONCLUSIONS: Carbon monoxide concentrations in exhaled breath of at least 2 strains of mice increase in response to single injections of endotoxin. The magnitude of increase was similar to what was observed with a bacteremia model. These findings with an experimental model provide a rationale for further studies of normalized CO concentrations in human breath as an informative biomarker for staging and monitoring of sepsis.