Co-circulation of multiple genotypes of ARV in poultry in Anhui, China

ABSTRACT
 Poultry production in China has been experiencing a high incidence of broiler arthritis /tenosynovitis caused by avian orthoreovirus (ARV) since 2013. In the spring of 2020 severe arthritis cases from broiler flocks were identified in a large-scale commercial poultry company in Anhui Province, China. Diseased organs from dead birds were sent for diagnosis to our laboratory. ARVs, including seven broiler-isolates and two breeder-isolates, were successfully harvested and sequenced. Interestingly, the genotypes of ARVs isolated from infected chickens were inconsistent between different flocks, or even between different houses on the same flocks. Pathogenicity testing in chicks confirmed that the seven broiler-isolates were pathogenic strains, which could cause arthritis in infected chickens. Subsequently, a total of 89.66% serum samples collected from apparently healthy adult broiler flocks not vaccinated against ARV tested positive for ARV antibodies, suggesting that low and high virulence reovirus strains may be co-circulating in the farm. To this end, we collected dead embryos of unhatched chicken eggs for pathogen tracing, and the two ARV breeder-isolates isolated indicated that vertical transmission from breeders to progeny should not be underestimated for the prevalence of ARV within broiler flocks. The findings have implications for the evidenced-based formulation of prevention and control strategies.


Introduction
Avian orthoreovirus (ARV) is an important cause of diseases in poultry.In particular, reovirus-induced arthritis/tenosynovitis, chronic respiratory diseases, and malabsorption syndrome result in considerable economic losses (Hieronymus et al. 1983;Shih et al. 2004).Reoviral arthritis/tenosynovitis causes extremely serious harm in chicken flocks, especially in broiler breeds.Gross lesions are characterized by unior bilateral lameness due to swelling of the hocks, periarticular fibrosis, tenosynovitis, occasional erosion of the articular cartilage on the distal tibiotarsus and rupture of the gastrocnemius or digital flexor tendon (Sharafeldin et al. 2014).ARVs are ubiquitous among poultry flocks and all commercial poultry flocks probably become infected at some time during the life of the flock (Jones, 2000;Benavente & Martínez-Costas, 2006), resulting in substantial economic losses to chicken farmers in the form of increased culling, increased condemnation rates, poor feed efficiency and low rates of weight gain (Lu et al., 2015).
ARV affects mostly young birds and is disseminated through the oral-faecal route, and vertically from breeders to progeny (Al-Muffarej et al., 1996;Mansour et al., 2019).The virus can be spread through inhalation or by the contact of viral particles with injured skin (al Afaleq & Jones, 1990;de la Torre et al., 2021).In China, the virus was identified and reported for the first time in 1985 from cases of arthritis in chicks, and at present, strains S1133 and 1733 are used as live and inactivated vaccines for active immunization of breeders and laying hens (de la Torre et al., 2021;Kumar et al., 2022).In the last decade, a progressive increment in severity of ARV-induced viral arthritis has been observed in China and other countries and regions.In addition, an increase in ARV infection has been observed even in vaccinated birds (Jiang et al., 2021), indicating that the vaccine currently used does not provide sufficient protection due to the variation in novel circulating strains.
In this study, we describe the widespread co-circulation of multiple genotypes of ARV in a large-scale commercial poultry company in Anhui province, China, and report molecular and serological evidence of ARV infection.The findings have implications for the evidenced-based formulation of prevention and control strategies.

Source and sample collection
In the spring of 2020, an outbreak of suspected ARV infection occurred in a large commercial poultry company in Anhui Province, China, which is a vertically integrated breeder breeding, hatching, broiler breeding, and poultry meat processing company.Clinical cases presenting with swollen and bleeding hocks and broken tendons were submitted to us for disease diagnosis.Tissue samples from seven houses of four broiler farms, including tendons and synovial fluid from broilers with arthritis symptoms (n = 130, online supplementary Table S1), were homogenized in phosphate buffered saline (PBS) to obtain a 10% tissue suspension.The suspensions were subjected to a freezethaw cycle before centrifugation at 11,300× g for 10 min and filtration sterilization passed through a 0.22 μm filter for virus detection, isolation, and identification.In an effort to resolve some of the confusion surrounding viral arthritis occurrence and prevalence, a series of tissue and serum samples were collected to determine some of the possible factors that cause infection and transmission of ARV.Tissue samples of liver of dead embryos in unhatched chicken eggs (n = 69, online supplementary Table S2) were collected for detection of ARV infection in embryos.Fifty-eight serum samples (online supplementary Table S3) were collected from four healthy broiler flocks (awaiting slaughter), ID denoted B5, B6, B7, and B8, for seroprevalence studies, of which 10 samples were collected from group B5, 24 samples from B6, 14 samples from B7, and 10 samples from B8.The ages of broilers in B5, B6, B7 and B8 groups were 39d, 38d, 37d and 36d, respectively.None of the flocks used for the seroprevalence study had a history of vaccination against ARV.The birds in the flocks investigated were apparently healthy and did not show any clinical signs of ARV infection.The ARV ELISA antibody titres in serum samples were determined by IDEXX Avian Reovirus (REO) Antibody Test Kit (IDEXX, Westbrook, ME, USA), following the manufacturer's protocols.The mean titres, above the the cut-off point of 396 (S/P ratio greater than 0.20) were considered positive as specified in the IDEXX guide (Sawale et al. 2019).The greater the concentration of the specific antibody in the serum sample, the higher the titre.

Virus isolation
Those tissue samples identified as ARV-positive by RT-PCR were used to isolate ARV on Leghorn Male Hepatoma (LMH) cells.Briefly, LMH cell monolayers were cultured in 6-well plates, and maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with penicillin-streptomycin (Biochannel, Nanjing, Jiangsu, China) supplemented with 10% foetal bovine serum (FBS) (Biochannel, Nanjing, Jiangsu, China), and incubated at 37°C with 5% CO 2 (Lu et al. 2015).Sterile treated ARV positive supernatant was overlayed onto LMH cells.After 1 h absorption, the supernatant was discarded and replaced with fresh DMEM/F12 supplemented with 2% FBS.Stock virus was harvested when approximately 75% of the cell monolayer of virus-infected plates manifested a marked giant or bloom-like cytopathic effect (CPE).After a freeze-thaw cycle, harvested virus supernatant was used for the next round of virus propagation, RT-PCR, and nucleotide sequencing.

Cloning and nucleotide sequencing
Positive RT-PCR products were tested by electrophoresis in 1% agarose gels and purified by using E.Z.N.A.-Gel Extraction Kit (Omega, Guangzhou, China).All gel-purified PCR products were cloned into a pMD18-T vector (TaKaRa), and transformed into E. coli DH5α competent cells (TaKaRa).At least two positive clones of each PCR fragment were selected for DNA sequencing by Tsingke Biotechnology Company (Beijing, China).

Sequence comparison and phylogenetic analysis
The viral open reading frames (ORFs) prediction and amino acids (aa) translation were performed by using the EditSeq of DNASTAR Lasergene.The other nucleotide reference sequences for ARV were obtained from GenBank by searching sequence numbers (https://www.ncbi.nlm.nih.gov/).Sequence alignment, pairwise sequence comparisons and sequence distance were done by MegAlign.Phylogenetic analysis of the σC gene was carried out using MEGA7 program by using the neighbor-joining (NJ) method and the bootstrap validation method with 1000 replications (Yang et al., 2018;Jiang et al., 2021).Multiple alignment and protein disorder analysis were accomplished by using Jalview desktop version 2.11.2.0.

Pathogenicity in chicks
Tissue culture 50% infectious dose (TCID 50 ) of seven broilerisolates was determined according to Reed and Muench (1938).Virus-containing liquid collected from cell cultures was inoculated into 1-day-old Ross broiler chicks using 0.1 ml per chick by footpad puncture.Each strain was administered to 10 chickens fed in separate isolators, and PBS (n = 10) was administered as a control.Clinical manifestations were observed and recorded.

Anti-serum (ARV) preparation and serum neutralization test (SNT)
AH02 strain was isolated from arthritic broilers with sample code B2H2F2 and was identified in this study.The ARV -AH02 strain used as vaccine preparation was purified and propagated in LMH cells.The TCID 50 was determined according to the Reed and Muench method (Reed & Muench, 1938).Water-inoil emulsion (OE) vaccines were prepared with virus antigen concentration the equivalent of 10 8.67 TCID 50 per 0.1 ml of virus before formaldehyde inactivation.Vaccine was prepared as described by Stone et al. (1978).Day-old White Leghorn SPF chickens were inoculated subcutaneously in the dorsal anterior neck region with 0.5 ml of monovalent emulsion vaccines and were housed in isolation.Immunized chickens were re-inoculated every 10 days by the same dose and inoculation manner and anti-ARV sera from chickens were collected 7 days after the fourth immunization.
The sera obtained from immunized chickens were also tested for serum neutralization antibodies against virus strains AH01, 02, AH03 and AH07 to characterize the homologous and heterologous virus-neutralizing capability.Briefly, the heat inactivated serum samples were 2-fold serially diluted in a 96-well plate (20-fold dilution in the first well), and 25 µl of ARV preparations (100 TCID 50 ) was added to all wells except negative control wells.Subsequently, the virus-serum mixture was incubated at 37°C for 1 h.LMH cells were added in a concentration of 2 × 10 5 cells with 10% foetal calf serum per well and incubated for 4 days at 37°C in an incubator with 5% CO 2 .Virusinfected and uninfected cells were used as positive and negative controls, respectively.Virus controls, cell controls, and serum controls were included on each plate.The plates were observed daily for the appearance of reovirus specific CPE.Finally, neutralization titres were recorded as the reciprocal of the highest dilution of serum that inhibited virus-induced CPE in at least 50% of the cell monolayer (Loeffen et al., 2012).

Ethical statement
This study was approved by the Animal Care and Use Committee of Shandong Agricultural University (permit number: 20190322) and performed in accordance with the "Guidelines for Experimental Animals" of the Ministry of Science and Technology (Beijing, China).All broilers in this study were bred and cared for in accordance with humane procedures.

Statistical analysis
All data were analyzed using the GraphPad Prism software 8.0 (GraphPad Software Inc.) and results were expressed as the means ± SD.

Morbidity profile in broiler flocks
A large number of arthritis and tenosynovitis cases first occurred in commercial broiler farms, which showed a significant increase in feed/meat ratio and higher elimination rate.Arthritis cases occurred in meat-type broiler chickens that were 13-33 days old (average 24.14 days).The morbidity rate of broiler chickens in different farms varied from 20% to 60%.One hundred and thirty hock joints (classification shown in Table 1) from commercial broiler flocks suspected of ARV infection, due to gross lesions in hocks characterized by swollen and bleeding, were examined.Of the 130 submitted hock joint samples, 106 (81.54%) tested positive for ARV, and the epidemiological details of the ARV-positive cases are described in Table 1 and visualized in Figure 1B.Three positive sample supernatants from each group (21 in total) were selected to overlayed onto the LMH cells for ARV isolation.The LMH cells showed specific CPE including cell swelling, syncytia formation, detachment from monolayer at 24 h post-infection (hpi) (Figure 1C).A total of 21 ARV isolates were harvested with 100% isolation rate.The gene sequences of σC of all 21 ARV strains were determined and the three strains isolated from the same group were genetically identical.The RT-PCR results of the isolated strains showed that the 1081-bp fragments were successfully amplified (Figure 1D).

Pathogenicity in chicks
We selected seven broilerisolates to study the pathogenicity of the viruses in chickens.Significant abnormal gaits in infected chickens were observed (Figure 2A).Gross lesions included swollen hocks and purple-cyanic blood stasis in ventral tarsal joints thereafter (Figure 2 B, C).The pathological changes showed hock joint capsules with swollen hocks were thickened and had serosanguineous exudate in the joint space (Figure 2 D).The results showed that the seven broiler-derived ARVs were pathogenic strains.

Serological test
Subsequently, 58 serum samples were collected from four apparently healthy broiler flocks awaiting slaughter (derived from four broiler farms) for seroprevalence study.A total of 89.66% of serum samples (i.e.52 of 58 serum samples) tested positive for ARV antibodies with the lowest Titre of 505 and highest Titer of 6681 (Figure 3).The overall mean Titer and Titer range of positive antibodies in broiler flocks were 1738, and 505 to 6681, respectively.Table 2 presented the seroprevalence of ARV infection in seven different apparently healthy broiler flocks, including mean Titer, Titer range and standard deviation (SD).The age of broilers sampled in group B5 was 39 d.The positive rate of ARV antibody in group B5-1 was 100% (5/5) with a Titer range of 505-2141, and the positive rate in group B5-2 was 80% (4/5) with a Titer range of 661-1336.The broilers in group B6-1 with an ARV antibody-positive rate of 89.47% (17/ 19) and group B6-2 with 80% (4/5) were 38 days old, and the titres ranged from 527-668, 866-3177, respectively.ARV antibody positivity in broilers (37 days old) of B7 group was 92.86% (13/14) with titres of 527-3383.The 36-day-old broilers of group B8 were from two groups B8-1, and B8-2, and their ARV antibody positivity was 80% (4/5) and 100% (5/ 5) with titres ranging from 505-1649 and 1265-3771, respectively.The flocks considered under this investigation were not vaccinated against ARV.Severe arthritis cases tested positive for ARV, and high seroprevalence of ARV was detected in apparently healthy flocks.

Investigation of ARV infection in dead embryos of unhatched chicken eggs
Of 69 dead embryos of unhatched chicken eggs collected from breeder farms, two (2.9%) samples tested positive for ARV, the epidemiological details of which are described in Table 1.Isolation and identification of ARVs in breeder flocks suggested that the factor of vertical transmission from breeders to progeny should not be underestimated for the prevalence of ARV within broiler flocks.

Sequence comparison and phylogenetic analysis of the σC gene
The nucleotide (nt) and amino acid (aa) sequences of σC gene from seven broiler-isolates and two breederisolates showed different sequence identity values with reference chicken-origin reovirus strains, including historical and pathogenic strains (online supplementary Table S4), and the results of pairwise comparisons of nt and aa are summarized in Table 3.
Phylogenetic analysis of σC sequences of the seven broilerisolates and two breederisolates was carried out, and their correlations are shown in Figure 4.The phylogenetic tree based on the nucleotide sequences of σC was distinctly divided into six major branches, genotypes 1 -6 (Lu et al. 2015).Seven ARV broiler-strains in the study were clustered into three groups: genotype 1, genotype 2 and genotype 5 and two breeder-isolates were clustered into genotype 1.Seven strains, namely breeder 1, breeder 2, AH01, AH02, AH04, AH05 and AH06, were grouped into the genotype 1 represented by S1133.However, five broiler-isolates differed from S1133 in that they showed low similarity with S1133 (74.6-76.2%nt, 74.0-76.1% aa) and could be further classified into variant subgroups of genotype I. Further observations revealed that the strain AH01 was distinct from AH02/ AH04/AH05/H06 with similarities of 83.7-84.1% (nt) and 84.4-85% (aa).One strain, AH03, was classified in genotype 2 and was closely genetically related to K1800134-01.0027strain.Another strain, AH07, closely related to the LY383 strain, presented a low similarity (81.2% nt, 84.1% aa) with GEI10 97M and genotyped closely with PA15511(94.7% nt, 95.7% aa).Analysis of the σC protein, including the nine obtained sequences and two vaccine strain sequences, showed no deletion of aa, while position 31-164 was a high-variable region of aa (Figure 5).
For further analysis, ARVs (AH02/03/04) isolated from samples coded B2H2F2, B2H3F2 and B2H4F2, collected from different houses of the same broiler farm (B2) and originated from the same batch of hatching eggs in breeding hens farm F2, showed inconsistent genotypes (1, 2), indicating that natural infection of field strains in the rearing environment may be another main obstacle to ARV prevalence within broiler flocks.

Serum neutralization
To test whether ARV antibodies can neutralize virus strains with multiple-variant σC, we obtained chicken hyperimmune serum against ARV-AH02.Anti-ARV-AH02 sera were used against the AH01/02/03/07 strains to characterize the homologous and heterologous virus-neutralizing capability.Table 4 shows the neutralizing antibody titres, including mean titre, titre range, standard deviation (SD), of anti-ARV-AH02 sera against different strains.The highest neutralization titre was 275 with a mean titre of 187.75 from AH02 group.The lowest neutralization titre was 25 with a mean titer of 56.58 from AH07 group.The results further indicated that the neutralization and protection ability of ARV antibodies against homologous strains was superior to that of heterologous strains.

Discussion
ARV is associated with viral arthritis and tenosynovitis in breeding hens and broilers, causing economic losses in the poultry industry due to increased culling, increased condemnation rates, poor feed efficiency and low rates of weight gain.It affects mostly young birds and is disseminated through the oral-faecal route and vertically from breeders to progeny (Mansour et al., 2019).Molecular surveillance is crucial to   strategize control and prevention of endemic diseases (Egaña-Labrin et al., 2019).Understanding the evidence of the high prevalence of ARV in broiler flocks can help in the prevention and control of the disease.In this study, a large number of arthritis and tenosynovitis cases first occurred in a large commercial poultry company in Anhui Province, China, which is vertically integrated breeder breeding, hatching, broiler breeding, and poultry meat processing company.Both the ARV detected by RT-PCR and virus isolated in LMH cell culture were present in all of the seven broiler flocks submitted, indicating infection by pathogenic ARV.In addition, most of the apparently healthy adult birds unvaccinated against ARV showed a higher mean titre with a higher percentage of positive birds for ARV antibodies indicating that the birds may have been exposed to ARV of low virulence during their lifetime as suggested by Herdt et al. (1999) who studied reovirus serology in broiler parents and their progeny in Belgium and its correlation with performance (Herdt et al., 1999;Sawale et al., 2019).However, the ELISA (IDEXX Laboratories) kit plates are coated with a whole virus lysate and the kit is not strain-specific and detect a wide range of pathogenic and nonpathogenic strains, therefore antibody level and disease are not directly associated (Sawale et al., 2019).Or it could be argued that broilers with a higher percentage of positive birds for ARV antibodies are simply exposed to natural ARV infection.But, with the isolation and identification of multiple genotypes of ARV and the high seroprevalence of ARV detected in apparently healthy flocks in the study, we prefer the possibility of a co-circulation of low virulence and pathogenic ARVs in this company.Detailed analysis of seven ARV broiler-isolaters, AH02/03/04 isolated from samples code B2H2F2, B2H3F2, and B2H4F2, collected from different houses of the same broiler farm (B2) and originated from the same batch of hatching eggs in breeding hens farm F2, showed inconsistent genotypes (1, 2), indicating that natural infection of field strains in the rearing environment may be a hurdle in the prevalence of ARV within broiler flocks.Identification of two breeder-isolates suggested that the factor of vertical transmission from breeders to progeny should not be underestimated for the prevalence of ARV within broiler flocks.Although the virus isolation rate from breeder embryos was a low value of 2.9%, the rate of horizontal transmission by the oral-faecal route occurring in chicks hatched from infected breeders is incalculable (Mansour et al., 2019).Some efforts have been made to prevent infections caused by ARV, which include biosecurity controls and immunization with both live and attenuated vaccines (de la Torre et al., 2021).The σC protein, as a minor component of the outer capsid, has been identified that is responsible for host cell attachment and which elicits neutralizing antibodies (Benavente & Martínez-Costas, 2007).Protein σC is involved in the main roles of avian reovirus infection and pathogenesis.As a result, it has become the important protein in vaccine development.In the present study, a pairwise comparison of σC genes between seven broiler-isolates, two breeder-isolates and reference ARV strains showed that they shared very low identity values.The recombination analysis of σC showed that none of the seven broiler-isolates and two breeder-isolates were recombinant, suggesting that most of the variation in σC originates from mutation in nucleotides rather than recombination.The results of the serum neutralization test showed that the neutralization titres of homologous sera were higher than those of heterologous sera, and sera with high ARV σC homology were higher than those with low homology, further indicating that the protective effect of ARV vaccine was variable because of the differences in infected field strains.However, the emergence of variant avian reoviruses is not just a case of the high divergence of the outer capsid protein σC.ARVs, as segmented RNA viruses, have the capacity to exchange genome segments in toto during co-infection through a process called reassortment (McDonald et al., 2016).Specifically, when two or more viruses infect a single host cell, they can package each other's genome segments in toto in a nascent virion, thereby producing hybrid progeny, so their variability possibilities are numerous (McDonald et al., 2016).For example, reassortment can produce an antigenically novel variant that is not recognized by the host immune system.The presence of this more suitable recombination in the host is responsible for the continued viral infection and inadequate  protection provided by commercial vaccines in the vaccinated flock and their progeny (Kumar et al., 2022).Research has also shown that the S3 segment encoding the σB protein is the target for group-specific antibodies and the λC protein is another target for group-specific antibodies (Yang et al., 2010).But the factors or proteins related to ARV antigenicity need to be further studied.From this perspective, in order to further develop vaccines with high protective efficacy and protection against multiple infecting strains, it is urgent to further study the recombination mechanism or protein function of ARV.

Figure 2 .
Figure 2. Clinical signs and gross lesions in broilers after challenge with ARV isolates.(A) Significant abnormal gaits in infected chickens; (B/C) Swollen hocks and purple-cyanic blood stasis in ventral tarsal joints; (D) Serosanguineous exudate in the joint space.

Figure 4 .
Figure 4. Phylogenetic tree constructed for avian orthoreovirus (ARV) based on nucleotide sequences of the σC homologous genome segments or genes.Note: The seven broiler-strains are marked with solid circles and the two breeder-strains are marked with solid triangles.

Figure 5 .
Figure 5. Analysis of the σC protein disorder including the nine obtained sequences and two vaccine strain sequences.

Table 1 .
Details of ARV strains isolated from broiler and breeder flocks.

Table 2 .
Seroprevalence of ARV in apparently healthy broiler flocks awaiting slaughter.

Table 4 .
Neutralization titres of anti-ARV-AH02 chicken hyperimmune serum (100 TCID 50 per well and 4 days of incubation of the SNT).