Iranian Journal of Veterinary Research, Shiraz University 105 Molecular identification of reovirus in broiler type flocks in Golestan province, Iran Mayahi, M.1*; Boroomand, Z.1; Jafari, R. A.1; Khademian, S.2 and Hoseini, H.3 1Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz; Ahvaz, Iran; 2Ph.D. Student in Poultry Health and Diseases, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran; 3Departmet of Clinical Sciences, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran *Correspondence: M. Mayahi, Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran. E-mail: mansoormayahi@scu.ac.ir (Received 9 Jun 2018; revised version 21 Oct 2018; accepted 12 Nov 2018) Summary Background: Avian reovirus (ARV) has a global distribution in nature and most clinical signs are found in broiler type chickens. Aims: This study was conducted to detect and identify reovirus infections from vaccinated breeder chickens and their progenies. Methods: A total of 20 tissue and blood samples were collected from vaccinated broiler breeders and their progenies with gastrointestinal or performance problems during peak production. Antibody titers were measured by indirect enzyme-linked immunosorbent assay (ELISA) tests. RNA extraction from tissue samples was performed and cDNA was prepared and directly used in the polymerase chain reaction (PCR). Nucleotide sequences were bilaterally determined using internal primers. The analysis of the nucleotide sequences and their related amino acids was performed by the specialized Molecular Evolutionary Genetics Analysis software (6th version). Results: The virus variant was detected in two vaccinated broiler breeders and five broiler flocks. The vaccine strains in breeder flocks included S1133, SS412, 1733, 2408 belonging to genotype 1 from the reovirus phylogenetic tree. Sequence 7 from the isolated reovirus based on the σC revealed that they were different from the reovirus vaccine, and that 6 isolates belonged to genotype 1 of the phylogenetic tree while 1 isolate belonged to branch 4 of the phylogenetic tree. Conclusion: The study showed that the new reovirus strain isolated from vaccinated birds differs from common strains used in the vaccines. It is therefore essential to prevent the effects of the field reovirus on the performance of industrial poultry, by updating and inventing new commercial vaccines, live and killed, against the reovirus. Key words: Broiler, Broiler breeder, Phylogenetic tree, Reovirus, σC protein Introduction Avian reovirus (ARV) has a global release in nature and creates a wide range of diseases in different bird species like poultry, pheasants, turkeys, ducks, geese, pigeons, quails, hunting birds, and parrots (Jones and Swane, 2013; Lu, 2015). Avian reoviruses belong to Orthoreovirus genus of the Reoviridae family with 10 segments of double-stranded RNA (dsRNA) without any covers, all of which being classified according to their electrophoretic stimulation into three large (LI-L3), medium (M1-M3) or small (S1-S4) groups (Mcnulty et al., 2008; Jones, 2009; Jones and Swane, 2013). The virus size ranges from 4/0 to 5/2 kbp (Tang and Huaguang, 2015) and the segmented genome includes eight structural proteins (λA, λB, λC, µA, µB, σA, σB, σC) and four non-structural proteins (µNS, P10, P17, σNS) (Tang and Huaguang, 2015). The virus has 6 branches in the phylogenetic tree (Huang et al., 2015). Most clinical signs are observed in broiler poultry and breeder chickens (Lu, 2015). Reovirus infections in domestic birds have numerous noticeable destructive economic effects. Clinical signs in young chickens include tenosynovitis, malabsorption syndrome, runtingstunting syndrome (RSS), digestive problems, immune system suppression, and secondary infections caused by a bacterium or a virus (Jones and Swane, 2013; Lu, 2015). Simultaneous infection with other pathogens such as mycoplasma sinwellia leads to severe immune deficiency, faintness, weight loss, egg production reduction, and more particularly, the creation of slaughterhouse waste (Senties-Cue et al., 2005; Reck, 2013). The findings indicate that the σC protein encoded by genomic S1 is a cell binding agent and the main determinant of the antigenicity of ARVs (Hoseini et al., 2015; Lu, 2015). Genomic S1 segment in the current ARVs strain has been well identified and evaluated in poultry viruses (Spackman et al., 2005; Mcnulty et al., 2008). Avian reoviruses are usually based on molecular functions especially the nucleotide and the amino acid sequence determination of the σ protein (Jones and Swane, 2013; Hoseini et al., 2015). Recent studies based on the phylogenetic analysis of the σC immunogenic protein indicate many varieties that are different from the current cluster of ARVs (Kant et al., 2003). New isolates seem to be distinct from conventional vaccine strains (Kant et al., 2003; Hoseini et al., 2015). Recent studies in Australia showed that there is a potential for genetic reassortment when the cell is infected with more than one reovirus, hence the emergence of new variants. Reoviruses are found everywhere and a large number of the isolates (over 80%) are non-pathogens (Rosenberger IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111 Iranian Journal of Veterinary Research, Shiraz University 106 et al., 1989). Horizontal transmission of the virus is one of the main ways of infecting birds (Robertson et al., 1989; Mcnulty et al., 2008; Jones and Swane, 2013). Horizontal transmissions are often carried out through faecal contamination and can also be transmitted through the respiratory tract. Reoviruses can also be transmitted vertically (Mcnulty et al., 2008; Jones and Swane, 2013). Vaccination of breeder flocks is essential to transfer breeder antibodies to the progenies and protect the birds from infection (Kant et al., 2003). However, breeder vaccination will be effective in protecting the progeny when there is a homologue between the strains of the vaccine and the virus circulating in the fields. There have been recent reports of reovirus infections despite the vaccination of breeder flocks and their progeny (Jones, 2009; Troxler, 2013; Hoseini et al., 2015; Mayahi et al., 2015). The present study was conducted to identify possible reoviruses in vaccinated breeder chicken flocks and their progenies. Materials and Methods Sampling Initially, a history of target broiler breeder flocks from the Golestan province was gathered including information about location, capacity, age, flock performance, reovirus infection signs and vaccination programs against reovirus. This information was then documented in a work sheet (Hoseini et al., 2015). During peak production season, 5 to 10 cm sections of duodenum, jejunum, ileum, and rectum of 10 birds of each of the 20 reovirus-vaccinated broiler breeder flocks with digestive disorders or performance problems were collected, and placed in a container consisting of saline solution. These samples were then slowly mixed and numbered as one sample. After a 30 min centrifuge cycle, the supernatant was separated, transferred to a test tube and immediately sent to the laboratory on dry ice. In flocks with lameness symptoms, samples was taken from the tibiotarsal joint and gastrocnemius and finger retraction muscle tendons, all pooled and numbered as one sample (Table 1). In addition, blood samples were collected from the wing vein of the 20 birds of each breeder broiler flock. Twenty broiler farms that raised chicks from the same broiler breeder flocks were investigated at the age of about three weeks, for signs of gastrointestinal, enteritis problems, growth retardation or limb problems. Blood samples from the wing vein and Table 1: Breeder flock features and vaccination programs 1 Breeder, hybrid flock codes 1-Ross 308 2 3-Hubbardf15 Golestan 3 5-Ross 308 Golestan 4 6-Ross 308 Golestan 5 9-Arbor acres Golestan 6 11-Arbor acres Golestan 7 13-Ross 308 Golestan 8 15-Ross 308 Golestan 9 17-Hubbardf15 Golestan 10 19-Ross 308 Golestan 11 21-Ross 308 Mazandaran 12 23-Ross 308 Golestan 13 25-Ross 308 Golestan 14 26-Arbor acres Golestan 15 27-Ross 308 Golestan 16 29-Hubbardf15 Golestan 17 32-Ross 308 Mazandaran 18 33-Ross 308 Mazandaran 19 34-Ross 308 Mazandaran 20 39-Hubbardf15 Golestan Number Site of flocks Golestan Vaccination program A live vaccine (merial) at 12 days and two killed vaccines (ceva) at weeks 12 and 20 A live vaccine (MSD) at 11 days and a killed vaccine (MSD) at week 20 A live vaccine (MSD) at 11 days and a killed vaccine (MSD) at week 20 A live vaccine (MSD) at 12 days and a killed vaccine (ceva) at week 20 A live vaccine (merial) at 35 days and a killed vaccine (MSD) at week 20 A live vaccine (MSD) at 30 days and a killed vaccine (MSD) at week 20 Two live vaccines (merial) at week 1 and 10 and two killed vaccine (ceva) at weeks 7 and 18 Two live vaccines (merial) at 7 and 27 days and a killed vaccine (MSD) at week 18 Two live vaccines (merial) at10 and 35 days and a killed vaccine (ceva) at week 20 A live vaccine (merial) at 12 days and two killed vaccines (MSD&ceva) at weeks 12 and 20 A live vaccine (MSD) at 12 days and a killed vaccine (MSD) at week 20 Two live vaccines (merial) at 32 days and week 21 and a killed vaccine (ceva) at week 20 A live vaccine (merial) at 12 days and a killed vaccine (MSD) at week 20 Two live vaccines (merial) at week 4 and 8 and a killed vaccine (ceva) at week12 A live vaccine (MSD) at 41 days and a killed vaccine (ceva) at week 20 Two live vaccines (merial) at 19 days and week 10 and a killed vaccine (Biomune) at week 17 A live vaccine (MSD) at 12 days and two killed vaccines (MSD&ceva) at weeks 12 and 20 A live vaccine (merial) at 12 days and two killed vaccines (ceva) at weeks 7 and 18 A live vaccine (merial) at 12 days and two killed vaccines (ceva) at weeks 7 and 18 Two live vaccines (MSD) at 9 and 70 days and two killed vaccines (MSD) at week 5 and 20 IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111 Age of sampling (week) Breeder flocks Progeny 35 2 37 3 33 4 38 2 40 2 33 2 32 3 35 3 28 3 35 3 36 2 29 3 36 2 34 3 30 2 33 3 40 2 36 2 35 2 30 3 Iranian Journal of Veterinary Research, Shiraz University 10 intestine samples (pieces of duodenum, jejunum, ileum, and rectum) were collected along with samples from the tibiotarsal joint, gastrocnemius and finger retraction muscle tendons of 20 birds (Hoseini et al., 2015). Experimental method Serology The anti-reovirus antibody titer in the blood serum of broiler chickens and their progeny was measured using Biocheck Company’s enzyme-linked immunosorbent assay (ELISA) kit. Autopsy Birds with clinical signs were euthanized and autopsy was carried out to take tissue samples from joints, tendons, and the gastrointestinal tract (Troxler, 2013). RT-PCR Identification of the reovirus RNA extraction: Homogeneous tissue samples were centrifuged at 4°C and 3000 rpm for 10 min. The supernatant solution of the intestinal contents was transferred to a microtube for RNA extraction. The RNA extraction from samples was performed using the RNXplus solution (CinnaGen, Tehran, Iran) according to the manufacturer’s protocol. Isolated RNAs were either used immediately for the reverse transcription-polymerase chain reaction (RT-PCR) or stored at -80°C. Synthesis of cDNA (RT reaction) After extracting RNA for cDNA synthesis, the Random Hexamer Primer and Canadian Revert aid first stand cDNA Synthesis kit were used. These primers are attached to the RNA nonspecifically and as a result, the entire RNA was converted to cDNA (Troxler, 2013). The cDNA was prepared and used in the PCR reaction immediately. In order to run the PCR reaction, two pairs of primers were used as Nested-PCR. These primers are specific to the S1 gene of reovirus and allow the detection of reovirus with high sensitivity from clinical specimens (Hoseini et al., 2015). The primer’s sequence was as follows: S1C 5´ ATT GAA TTC TCT CTG TTA TCT AAC CTTG3´738 bp, S1D 5´AAG GAA TTC GTT GAG AAC AGA AGT AGG3´738 bp, S1E 5´TCT GAA TTC ATC CGC AGC GAA GAG AGG TG3´324 bp and S1F 5´AGT GAA TTC AGT ATC GCC GTG CGC AG3´ 342 bp (Tang and Huaguang, 2015). In all cases, positive and negative controls were used simultaneously. The master mix used contained 2 μL PCR buffer 10X, 1 μL MgCl2 (50 mM/μL), 0.25 μL dNTPs (10 mM/μL), 0.25 μL of each 10 mM/μL primer, 6 μL cDNA, 10 μL distilled water, and the final addition of 0.25 μL Taq DNA polymerase (5 IU/μL). The thermal programs were similar to those presented in Table 2. Finally, the PCR products were separated in 2% agarose gel containing safe stain (CinnaGen, Tehran, Iran), using an ultraviolet trans-illuminator. 107 Table 2: Reverse transcription-polymerase chain reaction thermal program of reovirus Phase Early denaturation Denaturation Annealing Elongation Final elongation Temperature (°C) Time 94 94 53 72 72 3 min 10 s 15 s 20 s 15 min Determination of nucleotide sequences and phylogenetic analysis In order to determine the sequence, PCR products of the identified positive samples were purified using a Roche commercial kit. The determination of nucleotide sequences using internal primers was bilaterally conducted by BioNeer Co., South Korea. Analysis of the nucleotide sequences and their related amino acids was conducted using the specialized Molecular Evolutionary Genetics Analysis Software (6th version). Nucleotide sequences of the S1 gene of these isolates were compared with each other and with the number of different reoviruses in the GeneBank. Multiple alignment was then performed using the CLUSTAL W method for all nucleotide sequences. The nucleotide sequences were then evaluated based on the codes equivalent to the corresponding amino acids. The amino acid sequences were then investigated along with those of the nucleotides. Phylogenetic analysis based on the nucleotide sequence and the amino acid was performed by the neighbor joining (NJ) method using the TamuraNEI model. The validity and accuracy of the phylogenetic tree were then evaluated using the bootstrapping method and 1000 replications. All analyses were performed with the Pro Molecular Evolutionary Genetics Analysis software (6th version). Results Despite being vaccinated, some clinical symptoms like lameness and gastrointestinal problems were observed in the broiler breeder flocks. Moreover, through a molecular study and PCR test, in two cases of broiler breeder flocks (codes 13 and 29), positive reovirus variants were reported. In 25% of the broiler flocks (codes 4, 7, 14, 30, and 40) originating from the broiler breeder flocks vaccinated against reovirus, the PCR test was positive for the virus variant (Table 3, Fig. 1). In reovirus-positive flocks, clinical symptoms like lameness and lack of growth were observed and in the autopsy, swelling and rupture of gastrocnemius tendons were found. The sequence of the isolates of code samples (4, 14, 29, 30, and 40) was determined and classified in the phylogenetic tree (Figs. 2 and 3). Samples 7 and 1 were related to the gastrointestinal tract and tendon, respectively. Table 4 shows the degree of similarity of the isolates of this study with the vaccine strain. In the serologic test, the highest antibodies titer were measured in the breeder flocks which received live and killed vaccines twice. In the progeny, the maternal IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111 Iranian Journal of Veterinary Research, Shiraz University 108 Table 3: ELISA test and RT-PCR results Breeder, hybrid flock codes Tissue sample Clinical features PCR result Mean Ab titre CV (%) Broiler flock codes Tissue sample PCR result MAS/VA Mean Ab titre CV (%) 1 1-Ross 308 MAS/VA -/- 8321 38 10 gut - MAS 2496 46 2 3-Hubbardf15 MAS/VA -/- 7739 27 2 MAS/VA 3224 51 5-Ross 308 MAS/VA -/- 7500 45 4 -/+ MAS/VA 6016 32 4 6-Ross 308 MAS/VA -/- 10700 23 20 Tendon & gut Tendon & gut gut -/- 3 - MAS 2996 41 5 9-Arbor acres MAS/VA -/- 8300 34 7 gut + MAS 16709 110 6 11-Arbor acres MAS/VA -/- 22352 32 12 MAS/VA 3751 36 13-Ross 308 MAS/VA + 9707 29 14 -/+ MAS/VA 2890 63 8 9 15-Ross 308 17-Hubbardf15 MAS/VA MAS/VA -/- 8420 9638 32 48 16 18 - MAS MAS 2216 2641 91 30 10 19-Ross 308 MAS/VA -/- 11643 33 22 MAS/VA 7034 47 21-Ross 308 MAS/VA -/- 14845 47 8 Tendon & gut gut -/- 11 - MAS 2924 31 12 23-Ross 308 MAS/VA -/- 11906 35 24 MAS/VA 5988 74 25-Ross 308 26-Arbor acres VA MAS/VA -/- 7120 10954 40 38 38 28 Tendon & gut gut gut -/- 13 14 - MAS MAS 270 1971 91 44 15 27-Ross 308 Tendon Tendon & gut Tendon & gut Tendon & gut Tendon & gut Tendon Tendon & gut gut Tendon & gut Tendon & gut gut gut -/- 7 Tendon & gut Tendon & gut Tendon & gut Tendon & gut Tendon & gut Tendon & gut gut MAS - 13255 33 31 -/- MAS/VA 3438 56 16 29-Hubbard f15 MAS/VA -/+ 6854 35 30 + MAS 4220 50 17 32-Ross 308 MAS/VA -/- 10086 44 35 gut - MAS 1561 59 18 33-Ross 308 MAS/VA -/- 9611 38 36 gut - MAS 2607 60 19 34-Ross 308 MAS/VA -/- 3065 45 37 gut - MAS 2240 56 20 39-Hubbard f15 Tendon & gut Tendon & gut Tendon & gut Tendon & gut Tendon & gut Tendon & gut gut MAS/VA -/- 6481 44 40 Tendon & gut +/+ MAS/VA 512 128 Number The sample related to the gastrointestinal tract and malabsorption (MAS: Mal absorption syndrome), the sample related to joint problems (VA: Viral arthritis), positive sample (+), negative sample (-), first or second positive or negative sample (+/-), Coefficient of variation: CV, and Ab: Antibody Fig. 1: The 324 base pair RT-PCR products. M: DNA marker (100 bp), Lines 1-2 and 4-6: Positive samples, Line 3: Negative sample, No. 7: Positive control, and Line 8: Negative controls IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111 Fig. 2: The phylogenetic tree based on the sequence of amino acids in Iranian poultry along with isolates of the poultry virus whose sequences existed in the GeneBank. The isolates related to the present study are marked with a bold circle Iranian Journal of Veterinary Research, Shiraz University 109 Table 4: Similarity of this study’s isolates with vaccine strains based on amino-acid sequences Number 1 2 3 4 5 6 7 8 Strain Accession number % Identity with vaccine strain S1133 Phylogeny phylum Farm codes IR/kH1996.71/17 IR/KH2003.50/17 IR/KH1996.70/17 IR/KH1996.45/17 IR/KH1996.11/17 IR/KH1996.74/17 IRK/H1996.76/17 IR/H1996.31/17 MG922798 MG922799 MG922800 MG922801 MG922802 MG922803 MG922804 MG922805 81 81 81 81 81 79.5 81 57.5 1 1 1 1 1 1 1 4 40 40 29 29 4 29 30 14 IR is related to the isolated country, code 1996 belongs to gastrointestinal samples, code 2003 belongs to tendon samples, numbers are related to the samples, and 17 is related to the year of isolation Fig. 3: The phylogenetic tree was based on the sequence of amino acids in Iranian poultry along with isolates of the poultry virus whose sequences existed in the GeneBank. Branch spacing represents the degree of sequence difference. The isolates related to the present study are marked by a bold circle antibody derived from the breeder flock was detectable. In the infected flocks, however, the level of antibody in the ELISA test increased (Table 3). Discussion Evaluation of lameness and growth retardation in broiler flocks showed a high prevalence of reovirus in industrial poultry farms (Mayahi et al., 2015). In the past, good protection was provided by vaccination in the breeder flocks due to the similarity of the vaccine virus and the circulating virus. In the broiler flocks, on the other hand, this protection was due to breeder antibody transmission. Recently, however, genetic changes in the virus have reduced vaccine effectiveness. The σC gene is the main cause of cellular connection and the induction of neutralizing antibodies and is considered to be the variable segment of the virus. The most important way to control the activity of the virus in poultry farms is to vaccinate the breeder flocks with an effective vaccine to protect the vertical transmission of the virus as well as the progeny through the breeder’s antibody against the circulating virus. The sequencing of the σC gene of S1 segment of reovirus helps differentiate the reovirus vaccine virus from the circulating reovirus virus. The present study was hence conducted to identify possible reoviruses in the vaccinated breeder flocks and their progenies. Bokaie et al. (2008) investigated the seroprevalence of reovirus infections in broiler chicken flocks of Tehran on 582 serum broiler chickens originating from non-vaccinated breeders using the ELISA test. Of the total samples, 572 serum samples were positive with a serum prevalence of 98.3% (Spackman et al., 2005). Hedayati and Shojadoust (2012) examined the reovirus of tenosinovitis in birds from Iranian breeder flocks using RT-PCR and restriction fragment length polymorphism (RFLP) methods. The analysis of the segments obtained from the enzymatic digestion of PCR products in all positive samples indicated that the enzymatic digestion patterns of the samples were consistent with the standard digestive pattern of each S1133 vaccine. Mayahi et al. (2015) reported the occurrence of reovirus in broiler chicks originating from vaccinated broiler breeders in Golestan province. According to their clinical study, signs of lameness were detected from the second week in a number of rooster chicks of broiler farms, and the presence of reovirus in the gastrointestinal tract and gastrocnemius tendons of the infected chicks was confirmed using RT-PCR. In the present study, the investigation of the phylogenetic tree based on the σC segment showed that most isolates belonged to branch one, and one isolate belonged to branch 4 of the tree. Vaccine strains such as S1133 and 1733 belonged to branch one of the phylogenetic tree, but the isolates studied in this research were different from the vaccine strains. Most closely related to the isolates were German GEL06 97M and GEL12 98M isolates which were identified in 1997 and 1998 with malabsorption symptoms from branch one of the phylogenetic tree. A single case of one isolate from broiler flocks and growth retardation syndrome belonged to branch 4 and showed the most similarity with the isolate of the American AVS B in branch four of the phylogenetic tree. In the study IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111 110 conducted by Hoseini et al. (2015), cases of tenosynovitis and lameness in broiler flocks were observed despite the vaccination of breeder flocks. In all cases, hock joint inflammation, flexor digital tendon, swelling of the foot and subsequently poor performance of the flocks were observed. In the serological study, all affected flocks were found to have high levels of antibodies in the ELISA test. S1 segment sequences that codify the σC gene were examined. After being separated from the first broiler flock in the Ardehal area in central Iran, this isolate was named Ardehal variant. Investigations showed that the Ardehal variant belonged to branch one of the phylogenetic tree but differed from the strains of the vaccine which also belonged to branch one of the phylogenetic tree. Broiler breeder flocks in Iran are protected against reovirus by live and killed reovirus vaccine. Thus, chicks of these breeders are also expected to be protected against reoviruses. However, recent cases of prevalence of the reovirus in broiler flocks indicate that vaccination of breeder flocks with current vaccines is not enough to provide immunity to chicks. Tang et al. (2015) reported severe clinical disease and economic loss in Pennsylvanian turkey breeder flocks since 2011. The study of molecular identification of the 114 cu gene of field isolates from 2011 to 2014 indicated that the isolated reovirus included only 21.93% of the 114 farm isolates of a genotypic branch (genotypic branch1) such as the vaccine strain (S1133, 1733, 2408). The remaining 78.7% of the strains belonged to a different genotypic branch of the vaccine (Branch 2, 3, 4, 5, and 6) and indicated the emergence of new strains of reovirus. In this report, the emerging genotype six was reported for the first time (Lu, 2015). Troxler (2013) reported a severe economic loss in French broiler breeder farms due to reovirus infection despite routine vaccinations in broiler farms. Clinical signs included lameness, growth retardation, and lack of uniformity in flocks. Observations of autopsy and bacteriological and serologic tests confirmed the presence of tenosynovitis caused by reovirus infections. Moreover, sequencing the cσ gene and the phylogenetic evaluation of the RT-PCR product determined the new genotype of the reovirus. The virus was not neutralized by monovalent serums of the vaccinated chickens in the neutralization test. It was concluded that these reovirus isolates were serologically and genetically distinct from conventional reoviruses used in commercial vaccines and could not, therefore, provide protection and prevention against disease. Kort et al. (2013) identified, classified, and determined the genotype of the Tunisian strains of bird reovirus using the RT-PCR test, which was performed on both σC and σB genes, and subsequent RFLP tests to better identify Tunisian isolates. The replicated segment in the RT-PCR test showed 15 Tunisian strains with lengths of 738 and 540 bp for σC and σB. Using Acil and Msel enzymes in the RFLP test showed that all isolates could be clearly distinct from the vaccine strain. Tang and Huaguang (2015) determined genotypes of the reovirus isolates in Pennsylvania. The reovirus strain (Reo/PA/Broiler/05682 /12) was isolated from broiler breeders suffering from IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111 Iranian Journal of Veterinary Research, Shiraz University tenosynovitis and severe joint infections, all reovirus genomes being sequenced by RT-PCR and rapid amplification of cDNA ends (RACE) methods. The size of the complete isolated reovirus was 23494 bp and contained C+G 50%. A phylogenetic study of the nucleotide sequence of 10 genome segments in Pennsylvania showed moderate to highly noticeable differences in the nucleotide sequence of the strain used in the reovirus S1133 vaccine and 138 isolates. This genomic data indicated that the new Pennsylvania field strain is a reovirus that produces tenosynovitis and differs from the common strain used in the vaccine. In the present study of broiler breeder flocks, despite their good performance, some clinical signs such as growth retardation, lameness, and gastrointestinal problems were observed. Vaccinating the broiler breeder flock helped prevent clinical symptoms; however, as shown in codes 13 and 29, both breeder and broiler flocks (codes 14 and 30) showed severe cases of growth retardation. In the molecular investigation of other breeder flocks, the virus was not detected despite clinical signs. In some broiler breeder flocks reovirus was not detected but the virus was detected in their progenies. Despite that the virus was not detected in some broiler breeder flocks, their progenies (codes 4, 7, and 40) showed clinical signs of growth retardation, gastrointestinal problems, and lameness. Environmental contamination seems to play a major role in developing infections in growing broiler flocks and due to the heterologous nature of the some reovirus and vaccine strains, the breeder’s antibody does not have the ability to protect and prevent infection. Comparison of the nucleotide sequence σC of 8 virus isolates with the standard virus suggests the emergence of a variant in the circulating virus that does not show enough vaccine immunity against the reovirus. Genetic rearrangement is a noticeable characteristic of the reovirus (Troxler, 2013), and in the σC gene, segment S1 appears more often than other gene segments. In addition to the horizontal transmission of the reovirus that is very common among the flocks, vertical transfer is observed in codes 13, 14 and 29, 30. Regarding the breeder flock (code 13) with the isolate belonging to branch one of reovirus and the broiler flock (code 14) with the isolate belonging to branch four of the reovirus, the possibility of vertical transmission of the virus is rejected. However, regarding the breeder flock (code 29) and the broiler flock (code 30), while both isolates belonged to the same branch, there is a high possibility of the vertical transmission of the reovirus. On the other hand, in cases such as code 7, in spite of the detection of severe lameness in the flock, a segment of the bird’s intestine was removed and examined, indicating the traceability of the reovirus in the tissues as well as the body’s capability to clear the virus. Moreover, it was shown that the gastrointestinal tract was the main reovirus replication location. This genomic data suggests that the new field strain of reovirus in Iran has been responsible for the development of tenosynovitis and growth retardation problems. Iranian Journal of Veterinary Research, Shiraz University The study showed that the new reovirus strain isolated from vaccinated birds was different from common strains used in the vaccines and that it is essential to prevent the effects of the field reovirus on the performance of industrial poultry by updating and making new commercial vaccines, live and killed, against the reovirus. It is necessary, therefore, to design further experiments to show this. Acknowledgement This manuscript was extracted from a dissertation project approved by vice-chancellery for research at Shahid Chamran University of Ahvaz. Conflict of interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. References Bokaie, S; Shojadoost, B; Pourbaksh, SA; Pourseyyed, SM and Sharifi, L (2008). Seroprevalence survey on Reovirus infection of broiler chickens in Tehran province. Iran. J. Vet. Res., 9: 181-183. Hedayati, M and Shojadoust, B (2012). Identification of tenosynovitis inducer reovirus in birds originating from Iranian breeder flocks by using RT-PCR & RFLP methods. Iranian J. Vet., 7: 135-142. Hoseini, H; Bozorgmehrifard, M and Charkhkar, S (2015). Aredhal variants involves in reovirus-associated Arthritis and Tenosynovitis outbreaks in broiler flocks. 5th International Veterinary Poultry Congress. P: 58. Huang, WR; Chiu, HC; Liao, TL; Chuang, KP; Shih, WL and Liu, HJ (2015). Avian reovirus protein P17. Functions as a nucleoporin Tpr suppressor leading to activation of p53, p21 and PTEN and inactivation of PI3K/AKT/mTOR and ERK signaling pathways. PLOS ONE. 10: e0138627. Jones, RC (2009). Avian reovirus infection. OIE. 19: 614-625. Jones, RC and Swane, DE (2013). Reovirus infection. In: Swane, DE; Glisson, JR; Mcdougald, LR; Nolan, LK; Suarez, DL and Nair, V (Eds.), Diseases of poultry. (13rd 111 Edn.), Ames, Iowa, Iowa State University Press. PP: 351363. Kant, A; Balk, F; Born, L; Van Roozelaar, D; Heijmans, J; Gielkens, A and Huurne, A (2003). Classification of Dutch and German avian reovirus by sequencing the Cσ protein. Vet. Res., 34: 203-212. Kort, YH; Bourogâa, H; Gribaa, L; Hassen, J and Ghram, A (2013). Genotyping and classification of Tunisian strains of avian reovirus using RT-PCR and RFLP analysis. Avian Dis., 59: 14-19. Lu, H (2015). Isolation and molecular characterization of newly emerging avian reovirus variants and novel strains in Pennsylvania, USA, 2011-2014. Sci. Rep., 5: Article No. 14727. Mayahi, M; Khademian, S and Hoseini, H (2015). Report of reovirus infection in broiler farms from vaccinated breeders. 5th International Veterinary Poultry Congress. P: 159. McNulty, MS; Jones, RC and Gough, RE (2008). Reoviridae. In: Pattison, M; McMulin, PF; Bradbury, JM and Alexander, DJ (Eds.), Poultry diseases. (6th Edn.), Canada, Toronto, Elsevier Publication Company. PP: 382391. Reck, C (2013). Rapid detection of Mycoplasma synovial and avian reovirus in clinical samples of poultry using multiplex PCR. Avian Dis., 57: 220-224. Robertson, MD and Wilcox, GE (1989). Avian reovirus. Vet. Bull., 56: 155-174. Rosenberger, K; Sterek, FF; Botts, K; Lee, KP and Margolin, A (1989). Invitro and Invivo characterization of avian reovirus. I. Pathogenicity and antigenic relatedness of several avian reovirus isolates. Avian Dis., 33: 535-544. Senties-Cue, G; Shivaprasad, HL and Chin, RP (2005). Systemic Mycoplasma synovial infection in broiler chickens. Avian Pathol., 34: 137-142. Spackman, E; Pantin-Jackwood, M; Day, JM and Sellers, H (2005). The pathogenesis of turkey origin reovirus in turkeys and chickens. Avian Pathol., 34: 291-296. Tang, Y and Huaguang, L (2015). Genomic characterization of a broiler reovirus field strain detected in Pennsylvania. Infect. Genet. Evol., 31: 177-182. Tang, Y; Lin, L; Knoll, EA; Dunn, PA; Wallner-Pendleton, EA and Lu, H (2015). The σC gene characterization of seven turkey arthritis reovirus field isolates in Pennsylvania during 2011-2014. J. Vet. Sci. Med., 3: 7. Troxler, S (2013). Identification of a new reovirus causing substantial losses in broiler production in France, despite routine vaccination of breeders. Vet. Rec., 172: 556. IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111