Coronavirus: proteomics analysis of chicken kidney tissue infected with variant 2 (IS-1494)-like avian infectious bronchitis virus.

Avian infectious bronchitis virus is one of the most important gammacoronaviruses, which causes a highly contagious disease. In this study, we investigated changes in the proteome of kidney tissue of specific-pathogen-free (SPF) chickens that were infected with an isolate of the nephrotropic variant 2 genotype (IS/1494/06) of avian coronavirus. Twenty 1-day-old SPF White Leghorn chickens were randomly divided into two groups, each comprising 10 chickens, which were kept in separate positive-pressure isolators. Chickens in group A served as a virus-free control group up to the end of the experiment, whereas chickens in group B were inoculated with 0.1 ml of 104.5 EID50 of the IBV/chicken/Iran/UTIVO-C/2014 isolate of IBV, and kidney tissue samples were collected at 2 and 7 days post-inoculation (dpi) from both groups. Sequencing of five protein spots at 2 dpi and 22 spots at 7 dpi that showed differential expression by two-dimensional electrophoresis (2DE) along with fold change greater than 2 was done by MS-MALDI/TOF/TOF. Furthermore, the corresponding protein-protein interaction (PPI) networks at 2 and 7 dpi were identified to develop a detailed understanding of the mechanism of molecular pathogenesis. Topological graph analysis of this undirected PPI network revealed the effect of 10 genes in the 2 dpi PPI network and nine genes in the 7 dpi PPI network during virus pathogenesis. Proteins that were found by 2DE analysis and MS/TOF-TOF mass spectrometry to be down- or upregulated were subjected to PPI network analysis to identify interactions with other cellular components. The results show that cellular metabolism was altered due to viral infection. Additionally, multifunctional heat shock proteins with a significant role in host cell survival may be employed circuitously by the virus to reach its target. The data from this study suggest that the process of pathogenesis that occurs during avian coronavirus infection involves the regulation of vital cellular processes and the gradual disruption of critical cellular functions.

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 analysis of H9N2 avian influenza viruses in Afghanistan (2016-2017).

Avian influenza A virus (AIV) subtype H9N2 is the most prevalent subtype found in terrestrial poultry throughout Eurasia and has been isolated from poultry outbreaks worldwide. Tracheal tissue specimens from 100 commercial broiler flocks in Afghanistan were collected between 2016 and 2017. After real-time RT-PCR, AI-positive samples were further characterized. A part of the HA gene was amplified using RT-PCR and sequenced. The results of real-time RT-PCR showed that 40 percent of the flocks were AI positive. Phylogenetic studies showed that these H9N2 AIVs grouped within the Eurasian-lineage G1 AIVs and had a correlation with H9N2 AIV circulating in the poultry population of the neighboring countries over the past decade. Analysis of the amino acid sequence of HA revealed that the detected H9N2 viruses possessed molecular profiles suggestive of low pathogenicity and specificity for the avian-like SAα2,3 receptor, demonstrating their specificity for and adaptation to domestic poultry. The results of the current study provide great insights into H9N2 viruses circulating in Afghanistan's poultry industry and demonstrate the necessity of planning an applied policy aimed at controlling and managing H9N2 infection in Afghan poultry.

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.

Clade 2.3.4.4 avian influenza A (H5N8) outbreak in commercial poultry, Iran, 2016: the first report and update data.

In 2010, H5N8 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage dramatically affected poultry and wild birds in Asia, Europe, and North America. In November 2016, HPAI H5N8 was detected in a commercial layer farm in Tehran province. The diagnosis was based on real-time reverse transcriptase PCR (RRT-PCR) and sequencing of haemaglutinin (HA) and neuraminidase (NA) genes from suspected samples. Genetic and phylogenetic analysis of the HA gene demonstrated that the Iranian HPAI H5N8 viruses belong to the HPAI H5 virus clade 2.3.4.4 and cluster within group B (Gochang-like). In particular, the highest similarity was found with the sequences of the HPAI H5N8 identified in Russia in 2016. To our knowledge, this clade has not been previously detected in Iran. Previous HPAI A (H5) epidemic in Iran occurred in 2015 and involved exclusively viruses of clade 2.3.2.1c. These findings indicate that Iran is at high risk of introduction of HPAI H5 of the A/Goose/Guangdong/1/1996 lineage from East Asia and highlight the need to maintain adequate monitoring activities in target wild and domestic bird species for HPAI early detection. This study is useful for better understanding the genetic and antigenic evolution of H5 HPAI viruses in the region and the world.

Spatial spread and emergence of reassortant H5 highly pathogenic avian influenza viruses in Iran.

Since 2005, H5Nx highly pathogenic avian influenza (HPAI) viruses of the Goose/Guangdong (Gs/GD) lineage have spread worldwide, affecting poultry and wild birds in Asia, Europe, Africa and North America. So far, the role of Western Asia and the Middle East in the diffusion dynamics of this virus has been poorly explored. In order to investigate the genetic diversity and the role of Iran in the transmission dynamics of the Gs/GD lineage, we sequenced the complete genome of twenty-eight H5Nx viruses which were circulating in the country between 2016 and 2018. We reported the first characterization of the HPAI H5N6 subtype of clade 2.3.4.4B in Iran and gave evidence of the high propensity of the Gs/GD H5 AIVs to reassort, describing six novel H5N8 genotypes of clade 2.3.4.4B, some of them likely generated in this area, and one H5N1 reassortant virus of clade 2.3.2.1c. Our spatial analyses demonstrated that the viruses resulted from different viral introductions from Asia and Europe and provided evidence of virus spread from Iran to the Middle East. Therefore, Iran may represent a hot-spot for virus introduction, dissemination and for the generation of new genetic variability. Increasing surveillance efforts in this high-risk area is of utmost importance for the early detection of novel emerging strains with zoonotic potential.

Efficacy of H120 and Ma5 avian infectious bronchitis vaccines in early challenge against QX strain.

Infectious bronchitis (IB) is a highly infectious avian pathogen, which affects the respiratory tract, gut, reproductive system, and kidney of chicks of all ages. Many different serotypes of IB virus (IBV) are recognized which cause different clinical manifestations. According to the antigenic differences, different serotypes of the virus do not cross-protect. Massachusetts serotype induces the best cross-protection against other serotypes. Recently, the IBV QX strain has been detected in Iran. QX strain causes permanent damage to the oviduct if it occurs in the early life cycle and is a significant factor in layer and breeder chicken flocks. In this study, we compare the H120 and Ma5 vaccines' protection against early challenge with the QX strain in commercial chicks. one-day-old commercial chicks were divided into six groups. Groups 1 and 2 were unvaccinated groups. Groups 3 and 5 were vaccinated with the H120 vaccine (eye drop) and groups 4 and 6 were vaccinated with Ma5 (eye drop) on the 6th day (5 days after vaccination). Groups 2, 3 and 4 challenged (oculonasal) with QX strain (10^4 EID50). Ciliostasis test, histopathology, and quantitative real-time RT-PCR were done at 11 days-old of age. Results showed that neither H120 nor Ma5 could induce proper cross-protection against QX early challenge, but the viral load and adverse pathological records in vaccinated chicks were less than that in the non-vaccinated groups. It can be concluded that vaccination on the first day of the life of a chick offers not full protection against the IBV QX strain but reduced the viral load and pathological damages in vaccinated chickens. Applying other forms of vaccination and using different genotypes on one-day-old chicks are suggested.

Correction to: Efficacy of H120 and Ma5 avian infectious bronchitis vaccines in early challenge against QX strain.

[This corrects the article DOI: 10.1007/s13337-017-0414-4.].

Prevalence of avian metapneumovirus subtype B in live bird market in Gilan province, Iran.

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and swollen head syndrome in chickens. Four aMPV subgroups (A-D) have been reported previously based on their genetic and antigenic differences. Evidence suggests that the live bird markets (LBMs) play an important role in the epidemiology of the avian viral diseases. A total number of 450 oropharyngeal samples from eight different species of birds (migratory and local) were collected from LBMs of Gilan province, Iran, from October to December 2016. The presence of aMPV was determined by reverse transcription polymerase chain reaction (RT-PCR) based on nucleoprotein gene. The aMPV was detected in 30.60% of the examined birds including chickens (37.00%), turkey (33.00%), Eurasian teal (25.00%), common blackbird (33.00%), and Eurasian woodcock (25.00%). Bioinformatics analysis and a phylogenetic tree based on partial nucleotide sequences of the N gene showed that the detected aMPVs were belonged to subtype B. This is the first report of aMPV in non-commercial birds in Iran. Knowledge of the frequency and types of infected birds with pneumoviruses allow a better understanding of the epidemiology of aMPV in Iran.

Pathogenicity study of Iranian genotype of avian infectious bronchitis virus (IR-1).

Avian infectious bronchitis (IB) is a major cause of economic losses in poultry industry. The IB virus primarily affects respiratory tract, but various strains differ in their tropism for other target organs such as kidney and alimentary tract. The objective of this study was to estimate the pathogenicity of Iranian IBV variant (IR-1), which is limited exclusively to Iran. Specific pathogen free chicks were inoculated intranasally. Sera, fecal swabs and different tissue samples were collected on different days post infection (DPI). Clinical signs, gross pathology and histological changes were recorded. The viral load was quantified in the RNA extractions from different tissue samples using real-time PCR. Anti-IBV antibodies were detected in serum samples. The IgG antibody were found on 21 and 28 DPI. Severe histological lesions were observed in the trachea and lung while the lesions in kidney were appeared to be milder. Viral RNA was detected in all tested tissues from 1 DPI to the last day of the experiment. The highest viral load was measured in the trachea and feces on 1st and 5th DPI, respectively. It can be concluded the IR-1 had broad tropism for respiratory tract, digestive system, and renal tissue, reflecting its epitheliotropic nature, but it caused the most severe lesions in the respiratory tract. This was the first pathogenicity study of Iranian IR-1 IBV. Further knowledge of IBV pathogenesis provides the groundwork to inform more effective prevention practices.

Molecular identification and phylogenetic analysis of chronic bee paralysis virus in Iran.

Chronic bee paralysis virus (CBPV) is an unclassified polymorphic single-stranded RNA virus. Among the viruses infecting honeybees, CBPV is known to induce significant losses in honeybee colonies. In this study, a total number of eighty-nine suspected apiaries from four regions of Iran (including Mazandaran, Khorasan Razavi, Hormozgan, and Kurdistan) were sampled and submitted for molecular identification. Three positive samples were detected by RT-PCR. All positive samples were confirmed by sequencing. The phylogenetic tree which displays the molecular relationship between the viruses of different Iranian geographic regions and references isolates was constructed. The Iranian isolates formed two distinct phylogenetic groups (Group 1 and Group 2). The IR-CPV-GMG-1, IR-CPV-GMG-2, IR-CPV-GMG-4, and IR-CPV-GMG-6 formed Group 1 and IR-CPV-GMG-3, IR-CPV-GMG-5, and IR-CPV-GMG-7 were in Group 2 as a distinct group. Iranian isolates in group 1 were similar to European and East Asian CBPVs. This research was the first phylogenetic analysis of CBPV in Iran. Further researches are needed to study the other aspects of this virus-like genetic characteristics and pathogenesis in Iran.