Whole-genome characterization of avian picornaviruses from diarrheic broiler chickens co-infected with multiple picornaviruses in Iran.

Gastrointestinal symptoms in poultry are caused by several factors, such as infecting viruses. Several avian picornaviruses can cause diarrhea in these valuable animals. Poultry flocks in Iran suffer from gastrointestinal diseases, and information on picornaviruses is limited. In this study, two genera of avian picornaviruses were isolated from poultry and identified by the viral metagenomics. Fecal samples were collected from broiler chicken flocks affected with diarrhea from Gilan province Iran. The results showed that Eastern chicken flocks carried two genera of picornaviridae belonging to Sicinivirus A (SiV A) and Megrivirus C (MeV C). The Western chicken flocks carried SiV A based on whole-genome sequencing data. SiV A had type II IRES and MeV C contained a type IVB IRES 5'UTR. Phylogenetic results showed that all these three picornaviruses were similar to the Hungarian isolates. Interestingly, two different picornavirus genera were simultaneously co-infected with Eastern flocks. This phenomenon could increase and facilitate the recombination and evolution rate of picornaviruses and consequently cause this diversity of gastrointestinal diseases in poultry. This is the first report and complete genome sequencing of Sicinivirus and Megrivirus in Iran. Further studies are needed to evaluate the pathogenic potential of these picornaviruses.

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.

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.

Evaluation of the Newcastle disease virus genotype VII-mismatched vaccines in SPF chickens: A challenge efficacy study.

Newcastle disease virus (NDV) strains, while falling under a single serotype, are classified into distinct genotypes. Genotype VII virulent NDVs pose a significant threat to poultry due to their association with high mortality rates and economic losses. This study aimed to evaluate the efficacy of three commercial live vaccines based on genotype II against genotype VII virulent NDV (vNDV) in specific pathogen-free (SPF) chickens. Forty one-day-old chickens were randomly divided into four groups (n = 10) and inoculated with one dose of each ND pneumotropic vaccine-B1, Clone.12IR, and La Sota-or received phosphate-buffered saline (PBS) as a control at eight days of age via eye drop. At 28 days of age (20th post-vaccination days), chickens were intramuscularly challenged with genotype VII virulent NDV (≥ 105 LD50). Serum samples were collected at 28 days of age (challenge day), 7 and 14 post-challenge days to measure NDV antibodies via the hemagglutination inhibition (HI) test. Cloacal and oropharyngeal swabs were taken on the 3rd, 5th, 7th, and 10th post-challenge days to evaluate virus shedding. Vaccinated groups exhibited significantly higher antibody titers and greater protection levels compared to the control group (P≤ 0.001). While HI antibody titer was not different at 28 and 35 days of age between vaccinated chickens, the Clone.12IR groups showed higher HI antibody titer compared to B1 at day 42 of age (9.43 vs. 7.42; P≤ 0.002). La Sota and Clone.12IR vaccines demonstrated superior protection against mortality compared to the B1 vaccine (90 %, 80% vs. 60 %, respectively) with 6.0 and 2.67 odds ratio of survivability. All three mismatched vaccines effectively curbed the shedding of virulent genotype VII NDV, with 0 % to 11 % positive cloacal samples up to the 3rd post-challenge day. These findings demonstrate that in the experimental setting, the administration of mismatched ND vaccines, particularly La Sota and Clone.12IR, confer protection against genotype VII virulent NDV and control viral shedding, which can help to develop effective vaccination strategies to mitigate the impact of vNDV outbreaks in the poultry farms.

Thermoresistant Newcastle disease vaccine effectively protects SPF, native, and commercial chickens in challenge with virulent virus.

BACKGROUND: Due to the more stability and a better homogenecity in immune response, the use of thermoresistant vaccines in different chicken types has been increased. OBJECTIVE: This study aimed to evaluate the efficacy of a newly developed Newcastle disease vaccine (ND.TR.IR) originating from I-2 strain in specific pathogen-free (SPF) and native and broiler chickens. METHODS: Following determination of pathogenicity indices on the candidate seed, three efficacy examinations were conducted. In the first experiment, 120 1-day-old SPF chickens were randomly allocated to six groups and either vaccinated with ND.TR.IR via eye drop at 1, 7, and 21 days of age (V1 , V7 , and V21 ), or considered as non-vaccinated control groups (C1 , C7 , and C21 ). At 20th post-vaccination day, sera hemagglutination inhibition (HI) antibody titres against ND virus (NDV) were measured and then the chickens were challenged by virulent NDV (vNDV). In the second and third experiments, the efficacy of ND.TR.IR vaccine was compared to routine vaccination program (B1 and LaSota) in native and broiler chickens that were vaccinated at 10 and 20 days of age, respectively. The HI antibody titres were measured on 10, 20, 30, and 40 days of age, and also challenge efficacy test with vNDV was conducted on 30 days of age. RESULTS: The studied virus, as a vaccinal seed, complied with the pathogenicity indices of avirulent NDV and molecular identity of I-2 strain. In the efficacy evaluation trials, the vaccinated chickens had higher HI antibody titres against NDV compared with their corresponding control chickens (p < 0.05). Results of the challenge tests indicated 95% and 100% protection against vNDV in native, SPF, and broiler-vaccinated chickens, respectively. CONCLUSIONS: The present findings indicated that administration of ND.TR.IR induced appropriate HI antibody titres against NDV in SPF, native, and broiler chickens associated with good protection in efficacy test.

Phylogenetic analyses on Marek's disease virus circulating in Iranian backyard and commercial poultry indicate viruses of different origin.

As neoplastic viruses have been affecting Iranian chicken farms more frequently in recent years, the first step in prevention may therefore be to genetically characterize and systematically identify their source and origin. Recently, we published a phylogenetic analysis based on the meq gene of Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1), that circulated in Iranian backyard and commercial chickens. In the current study, we are reporting for the first time the identification of a 298 aa meq protein containing only two PPPP motifs from an MDV-1-infected unvaccinated backyard turkey. This protein length has never been reported from any turkey species before. According to phylogenetic analysis, a close genetic relationship (0.68%) to several chicken-origin isolates such as the American vv + 648A strain was found. In addition, we identified a standard meq protein from a MDV-1-infected commercial chicken farm. In corroboration with our previous finding from other Iranian provinces, it is likely that the highly identical MDV-1 viruses currently circulating in Iranian chicken farms, which may be indicative of human role in the spread of the virus, have similar Eurasian origin. Our data suggest that regardless of the meq size, MDV-1 circulating in Iran are from different origins. On the other hand, meq sequences from bird species other than chicken have been reported but are very few. Our investigation suggests MDV-1 circulating in turkey do not have species-specific sequences.

Comparison of the Impact of Different Administration Routes on the Efficacy of a Thermoresistant Newcastle Disease Vaccine in Chickens.

Route of vaccine administration has a great impact on immunization and protection outcomes in chickens. This study was conducted to compare the effect of different administration routes on the efficacy of a thermoresistant Newcastle disease (ND) vaccine (ND.TR.IR) in chickens. A total of 100 one-day-old specific pathogen-free chicks were divided into five groups (n = 20 chicks per group) and vaccinated through different routes at 10 and 20 days of age. Treatments included no vaccination (control [C]), 1 dose inoculation through eye drop (ED), 1 dose inoculation through drinking water (DW), 1 dose inoculation through feed (FV1), and 10 doses inoculation through feed (FV10). At 20 and 34 days of age, antibody titers were measured against ND virus (NDV) in all the chickens by hemagglutination inhibition (HI) test. Chicks immunized with ND.TR.IR vaccine through different routes of administration also were intramuscularly challenged with a local virulent NDV (vNDV) (Ck/ir/Beh/2011) 14 days after booster vaccination (at 34 days of age). Our results showed that in comparison with the FVs groups, the immunized chicks through ED induced a higher HI antibody titers at 20 days of age (p < 0.05). Meanwhile, vaccination through ED induced higher HI antibody titers at day 34 of age compared with all other groups (p > 0.05). The percentages of the protective HI antibody titers (≥log23) detected in ED and DW groups at 20 days of age were higher than those detected in the FV1 group (p < 0.05). However, routes of vaccination had no significant effect on the rate of protective titers at day 34 of age (100%, 90%, 75%, and 85% for ED, DW, FV1, and FV10, respectively). The percentage of post-NDV challenge survived chickens was not affected by the route of vaccination (p > 0.05), but immunization of chicks with ND.TR.IR in FV1 group provided relatively poorer protection when compared with the other groups (90% vs. 100%, respectively). Altogether, immunization of chicks with ND.TR.IR vaccine through different routes of administration induced protective NDV antibody HI titers, and provided protection against vNDV. However, when the vaccine was administrated through feed, a higher dose of vaccine is recommended.