Reovirus infections in broilers are mainly related to two clinical syndromes: malabsorption and arthritis/tenosynovitis. However, they are also involved together with other viruses in cases of immunosuppression, enteric disease, hepatitis, myocarditis, malabsorption, and runting-stunting syndrome. These viruses represent a significant economic burden on the broiler industry worldwide due to their widespread prevalence and potential pathogenicity.
Classification and virus epidemiology
The avian orthoreoviruses (ARV) belong to the Reoviridae family, subfamily Spinareoviridae, order Reovirales, and are double-stranded RNA viruses with multiple tropisms in tendons, heart, and liver.
- The phylogenetic analysis revealed the existence of at least six major genotypes of ARV based on the σC gene coding (Figure 1).
- Genotypes I and IV appear to have the greatest distribution worldwide.
- Genotype I is frequently isolated from birds with respiratory disease, and genotype IV is frequently isolated from arthritis/tenosynovitis lesions, while genotype VI has been identified in a wide range of lesions (arthritis/tenosynovitis to malabsorption).
Strains
Reovirus have a heterogeneous geographical distribution and genetic diversity that complicates control measures with vaccination. The four strains frequently isolated from intensively raised broilers and against which vaccination is carried out are S1133, 1733, 2408, and 2177. However, due to the antigenic disparity between the commercially licensed vaccines and contemporary field strains, these vaccines offer limited protection against current ARV challenges
Reovirus infections are a relevant issue worldwide, but publications about challenges and isolations are more frequent in China, Brazil, and the USA. In all these countries, recent reports indicate that most prevalent ARV strains have undergone significant genetic mutations compared to traditional vaccine strains (Figure 2). These viruses are extremely resistant to the environment and disinfectants. The ARV can remain viable even after exposure to 50 oC. They can be inactivated by alcohol (70%) and iodinated compounds.
Infection
Infections increase in the spring and autumn and reduce in winter and summer conditions. The horizontal transmission route is the most important through fecal/oral contact, respiratory transmission or contact of viral particles with skin lesions. Vertical transmission is also possible. Wild birds are a natural reservoir, and vector of transmission. Chickens under two weeks of age are the most susceptible to ARV infection.
Lesions
The ARV infections can cause lesions in the gastrocnemius and digital flexor tendons, tendon rupture (Figure 3), causing swollen hock joints, hemorrhages, and leg discolorations called green legs at processing age.
The subclinical nature of ARV infections can lead to reduced weight gain, flock desuniformity, suboptimal feed conversion rates, increased lameness-related condemnations in processing plants, and compromised animal welfare.
Reovirus causes viremia in the first 24 hours post infection and distributes in all tissues in less than four days.
The σC gene is an attachment or binding protein and responsible for initiating the ARV replication cycle (Figure 4). Chickens become lethargic, sit and move using their hocks, and crowd near the feeding areas.
The inflammation of the synovial sheaths causes aplomb deformities, swelling of the limb joints, chronic inflammation, and lameness. There is hyperplasia of the synovial membrane and in the periarticular subcutaneous tissue with infiltration of macrophages, lymphocytes, plasma cells, and numerous lymphoid aggregates. Inflammation can become exudative or fibrinous in the synovial cavity. Muscles and tendons can have necrosis and resorption of fibers accompanied by cellular infiltrate rich in macrophages, plasma cells, lymphocytes and rare heterophils.
In case of enteric infections, chickens show persistent diarrhea, poor feed conversion and slow growth rate. The abnormal feathering of primary feathers due to ARV infection is referred to as helicopter wing feathers due to their ruffled and discolored patterns.
Diagnosis
Almost 80% of ARV isolates are considered non-pathogenic and can be isolated from clinically healthy birds. Most of the time, ARV infections are mixed with other viruses. Then, their presence is not indicative of infection or the cause of a disease. Over 94% of commercial broiler flocks are seropositive for ARV based on ELISA testing. However, the presence of antibodies is not always associated with clinical signs or specific lesions of the disease.
The presumptive diagnosis is based on epidemiological conditions, clinical signs, and lesional aspects. Confirmation requires virus isolation and identification using cultivation on cell lines, detection of viral RNA and amplification of the σC gene via PCR and RT-PCR, genomic sequencing, ELISA for determining serum antibodies, and immunohistochemical examination to identify viral antigens in the tissues. Virus isolation is conducted in embryonated eggs inoculated into the yolk sac of SPF embryos. The virus is later inoculated to liver of embryos to record cytopathic effects.
Next-generation sequencing (NGS) allows for comparative metagenomic analysis of gut contents from healthy/clinical birds, without the need for virus cultivation or prior knowledge of the target sequence, making it ideal for metagenomic studies and the identification of new and emerging pathogens. The main steps of the NGS technique involve sample collection and processing, viral DNA/RNA extraction, genome amplification, genomic library construction, actual sequencing, and bioinformatic analysis.
Prevention, control, and vaccination
The ARV are ubiquitous. Rigorous biosecurity with cleaning and disinfection of affected facilities reduce infection prevalence. Reovirus control starts with breeder vaccination to induce high levels of neutralizing anti-reovirus antibodies. These antibodies are passively transferred from the breeder hen to the chick through the yolk sac to provide temporary protection to the progeny. The level of protection afforded by these antibodies is influenced by various factors, including serotype similarity, virus virulence, host age, and antibody titer.
Broiler breeders are generally vaccinated with 1 to 3 live attenuated vaccines up to 12 weeks of age, followed by 1 to 3 inactivated vaccines. The S1133 strain is frequently used for breeders. In contrast, the S1133 strain disrupts the functionality of the gastrointestinal tract in young chicks, leading to poor feed conversion and reduced weight gain. Furthermore, maternally derived antibodies transmitted to chicks from breeder hens vaccinated with the commercial S1133 strain have failed to prevent infection with the live-modified S1133 strain in broilers.
Vaccination
Vaccination with live classical commercial attenuated strains, such as S1133 and 2177 (Reovirus genotype 1) has become ineffective in multiple countries. The inactivated vaccines contain combinations of S1133, 1733, 2408, and Miss B strains. A recent vaccine includes the antigenic variant reovirus serotypes 1/4455, 2/4455, and 3.
- It has been proposed that vaccines must contain all six genotypes of ARV to ensure total immunization.
- However, these have not been developed yet.
- Due to the multitude of ARV strain variants, autogenous vaccines have become the most effective control method.
- These autogenous vaccines require regular updates to maintain efficacy against evolving viral genotypes.
The efficacy of vaccines must be evaluated by continuous virological and serological monitoring. The efficacy of reovirus vaccines is commonly assessed through a challenge model involving footpad inoculation of day-old chicks with a virulent autogenous virus. While this method can be informative, interpretation of results can sometimes be challenging.
Recombinant poultry vaccines on viral vectors, such as fowlpox virus, and turkey herpesvirus have been developed and commercialized. Other vaccine technologies under development include lipid nanoparticle (LNP)-encapsulated mRNA vaccines, chimeric vaccines created from a known virus with antigens from a pathogen, baculovirus-based vaccines, and recombinant vaccines expressed in plants or bacterial vectors. The baculovirus are a double-stranded DNA viruses that specifically infect insects and arthropods. These experimental vaccines have demonstrated protective efficacy in broilers, but there are not available in the market yet.
Immunity and immunoevasion mechanisms
Chickens can generate humoral, cell-mediated, and mucosal immune responses against ARV. However, the ARV also have several immunoevasion mechanisms. The mucosal responses are mediated mainly by the production of IgA in the respiratory and digestive tracts of chickens. The ARV with higher multiplication rates generates elevated levels of pro- and anti-inflammatory cytokines (IL-6, IL-10 and IFN-γ). These viruses can also cause generation of anti-nuclear and anti-collagen antibodies which can be linked to their autoimmune responses.
The cell immunity initially is related to macrophage activation, followed by lymphocyte proliferation. The CD8+ T cells during acuate infection. In the subacuate response CD4+ and IgM+ B cells tend to play a role, and in the chronic infections mainly CD4+ T cells are present with limited B-cell activity. This immunological profile is indicative of an autoimmune disease. The ARV can suppress lymphocyte proliferation, which is the main cause of clinical immunosuppression. The ARV encode the protein σA to bind the double-stranded RNA and avoid triggering the antiviral response.
There is still a lot to learn to minimize the evasion of the virus to the immune response and stimulate specific immune responses with long-lasting protection. In the same way, it is necessary to develop advanced diagnostic tools to monitor virus evolution to adapt the vaccines timely.
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