Characterisation, whole-genome sequencing and phylogenetic analysis of three H3N2 avian influenza viruses isolated from domestic ducks at live poultry markets of Iran, 2017: First report.

BACKGROUND: Avian influenza type A viruses (AIV) can infect a broad range of hosts including human and birds, making them an important viral pathogen with zoonotic potential. Ducks are a known reservoir for many avian viruses including the AIV. OBJECTIVES: To sequence the entire genome of duck-derived H3N2 and ran comprehensive phylogenetic analysis on them to study their origin. METHODS: In this study, 962 cloacal swabs were collected from domestic ducks at several live poultry markets (LPMs) of Gilan, Mazandaran and Golestan provinces of Iran in the year 2017. RESULTS: Preliminary assays such as haemagglutination inhibition assay (HI), Neuraminidase Inhibition assay(NI) and RT-qPCR suggested that 0.5% of the birds were infected by H3 low pathogenic influenza viruses (LPAI). Three isolates were selected for whole genome sequencing. The cleavage site of the HA genes showed a PEKQTR/GLF motif, an indicator of LPAI. Furthermore, BLAST and phylogenetic analyses of the HA gene showed high homology to the Eurasian lineage of H3N8 AIV (95.5%-97.1% to several European and East Asian isolates). However, the NA genes showed high homology (at most 96.5-96.9%) to those belonging to AIV N2 subtype. Furthermore, internal genes showed high homology (96%-98%) to a variety of duck-origin subtypes and glycoprotein combinations, which were different for each segment. This showed a complex reassortment between different subtypes. DISCUSSION: This report is the first whole genome sequencing and complete characterisation of H3N2 AIV from Iran. CONCLUSION: Such surveillance should continue to study the evolution and possible emergence of viruses with pandemic potential.

Predominance of Fourth Panzootic Newcastle Disease Virus Subgenotype VII.1.1 in Iran and Its Relation to the Genotypes Circulating in the Region.

Following recent Newcastle disease virus (NDV) outbreaks in Iranian poultry farms which were mostly associated with lesions of the avian gastrointestinal tract, it was speculated that the scale of the outbreaks could be attributed in part to co-circulating infectious agents or a new NDV genotype/subgenotype. This speculation was due to the isolation of a few 5th panzootic subgenotype VII.2 viruses from Iranian poultry farms in 2017. Samples from different species of commercial and domestic birds were collected from different provinces of Iran, 19 of which were selected for the current study. Phylogenetic analyses showed that the recent outbreaks have been caused by only one agent, i.e. the distinctive NDV subgenotype VII.1.1 (previously known VIIl) viruses that seem to be circulating predominantly in Iran, but have also been sporadically reported from Iraq among neighbouring countries. At most, 96.3-96.7% BLAST identity to non-Iranian VII.1.1 isolates was observed. Genetic distance values of <1% were indicative of high similarity between the isolates, but the values were approaximately 2% when the current isolates were compared to the earliest recorded Iranian VII.1.1 viruses isolated in 2010. Using Bayesian analysis, annual mutation rates of 1.7156E-3 (strict) and 1.9902E-3 (relaxed) over 11 years were obtained. In addition, we report that our laboratories have not detected any genotype XIII strains since 2011. Following up on previous reports, we concluded that currently, and except in Columbiforms, subgenotype VII.1.1 may likely be the predominant subgenotype in many bird species in Iran despite the subgenotype VII.2 being predominant in neighbouring countries.

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.

High frequency of Mycoplasma pneumoniae among patients with atypical pneumonia in Tehran, Iran.

INTRODUCTION: Mycoplasma pneumoniae is a major cause of atypical community-acquired pneumonia (CAP) with a prevalence range of 15-20% and up to 40% in adults and children, respectively. In Iran, the recorded frequency ranges between 1-6.15%. We aimed to investigate the frequency of M. pneumoniae among patients with atypical pneumonia acquired from the community. METHODS: Over a period of 5 months between January and June 2017, 520 patients with suspected CAP, who had been to the hospital outpatient clinics of Tehran University, were enrolled in this study. Throat swab specimens were obtained from 110 outpatients who presented with symptoms of atypical pneumonia. M. pneumoniae was identified via culture and biochemical tests, such as fermentation of glucose and arginine, hemolysis, and hemadsorption. For confirmation, PCR was performed to amplify the gene fragment coding for p1 adhesin. RESULTS: The major and minor clinical signs of the patients were dyspnea (67.3%) and nausea (15.5%), respectively. Out of 110 specimens, 25 (22.7%) and 29 (26.4%) isolates were identified to be M. pneumoniae via culture and molecular assay, respectively. Comparing the results of the two methods, the PCR showed better sensitivity and rapidity for the detection of M. pneumoniae. There was a high congruence between culture and the PCR assay; kappa level was 'almost perfect' (κ=0.90). CONCLUSION: This is the first report of high frequency of M. pneumoniae in our region. This finding can serve as baseline information for further investigation and confirmation of the potential epidemics of M. pneumoniae pneumonia in our community.

Phylogenetic characterization of virulent Newcastle disease viruses isolated during outbreaks in northwestern Iran in 2010.

The northwest of Iran shares long borders with three neighboring countries; therefore, it is considered one of the main entry portals of Newcastle disease virus (NDV) into the country. Ten virulent NDVs were recovered from 19 poultry farms of various prefectures in northwestern Iran during Newcastle disease outbreaks in 2010. The isolates were genotypically analyzed using an F-gene-specific reverse transcription polymerase chain reaction (RT-PCR) assay. The amplified F gene (nucleotides 189-1666) sequences of the NDV isolates were compared phylogenetically with those of previously published strains in GenBank. All of the NDV isolates belonged to genotype VIIb and were closely related to some isolates from Iran, Russia, and Sweden. Therefore, it can be postulated that these isolates evolved from previously reported strains. The velogenic viruses carried the motif (112)R-R-Q-K-R/F(117) at the F0 cleavage site and a unique substitution of (190)L→F which had never been reported in any NDV genotype VIIb isolate. They shared high sequence similarity with each other but were distinct from current NDV vaccines and NDV strains reported from other countries. This information is fundamental for improving the efficacy of controlling strategies and vaccine development for NDV.

Molecular characterization of H9N2 avian influenza viruses isolated from vaccinated broiler chickens in northeast Iran.

Avian influenza is a highly contagious respiratory disease of poultry caused by influenza A viruses, family Orthomyxoviridae. H9N2 avian influenza outbreaks are a major problem of the poultry industry in Iran. To determine the genetic differences between field viruses and the vaccine strain, the genomes of four strains isolated in 2011 from vaccinated broiler flocks with a history of respiratory illness were sequenced. Genetic and serological comparisons were made. Sequence analysis of the hemagglutinin (HA) and neuraminidase (NA) genes indicated that the isolated strains shared nucleotide homologies of 91.6-93.9 and 90.2-91.7% with the vaccine strain, respectively. Phylogenetic analyses of HA and NA genes showed that all strains isolated in this study fell into the same group and belonged to the influenza A virus (A)/quail/Hong Kong/G1/97 H9N2 sublineage. Several amino acids have changed at the antigenic sites in HA in the field viruses. Extra potential glycosylation sites were observed in the HA and NA proteins expressed by the current isolates relative to those in the vaccine strain. The deduced amino acid sequence at the cleavage site of HA in recent isolates is the KSSR/GLF motif, whereas it is RSSR/GLF in the vaccine strain. A serological analysis revealed that the currently circulating strains are antigenically distinct from the vaccine strain. These results suggest that the commercial vaccine is insufficiently genetically and antigenically similar to the viruses currently circulating in the region. These findings confirm that it is important to monitor the genetic and antigenic variations in H9N2 influenza viruses when selecting a vaccine strain.

Molecular typing of Iranian field isolates Mycoplasma synoviae and their differentiation from the live commercial vaccine strain MS-H using vlhA gene.

1. The single-copy domain of the N-terminal region of the vlhA gene of Mycoplasma synoviae was sequenced, analysed and verified and used to type Iranian field isolates of M. synoviae and the MS-H live vaccine strain. In addition, a restriction fragment length polymorphism (RFLP) method was developed to differentiate between field isolates of Iranian and MS-H vaccine strains. 2. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and dendrograms were constructed. Based on single nucleotide polymorphism (SNP) that existed in all field isolates in Iran, the PCR-RFLP method allowed the differentiation of all M. synoviae field isolates from the vaccine strain. 3. Using phylogenetic analysis, the isolates were assigned to 8 unique genotypes and, within each group, DNA had a high level of similarity. 4. DNA sequence analysis and PCR-RFLP of the amplicon based on percent similarity and evolutionary relationship appeared to be useful tools for strain differentiation whether M. synoviae clinical isolates from infected chickens were derived from the vaccine strain or wild-type strains. 5. This study confirms the potential value of strain typing for epidemiological purposes and suggests that phylogenetic studies are essential to understand the true relationships between strains.