Joseph J. Giambrone and Hung Jen Liu

Avian reoviruses cause a number of economically important, debilitating diseases of commercial chicken and turkey flocks worldwide. Reovirus is an abbreviation for respiratory, enteric orphan virus. These viruses got their
names because they were originally isolated from the enteric and respiratory tract of clinically normal children some 40 years ago.

In susceptible chickens, reoviruses can cause lameness and suboptimal weight gain and feed conversion, as well as immunosuppression. Immunosuppression results in poor vaccine responses and renders the birds more susceptible to other infectious agents.

Reoviruses can also result in increased mortality and high processing plant condemnation, which will increase the cost of production. Other economic loses come from the cost of medications to treat the disease as well as for diagnostic procedures and vaccines to prevent infection.

Despite new vaccines and treatments, losses due to reovirus infections still occur. The virus has the ability to mutate and evade the chicken's immune response. Diagnosis of reovirus-induced diseases is difficult, because they are clinically indistinguishable from a number of other common problems. Therefore, a rapid laboratory diagnostic test for reovirus infections in poultry is needed. Researchers in the AAES have developed a rapid sensitive laboratory test for the diagnosis of avian reoviruses. The test uses the very latest molecular biological procedures for the detection of the presence of reovirus nucleic acid in the tissues of infected chickens. In situ (in the tissue) hybridization (the union of two strands of nucleic acid) was used to detect viral genes in infected tissue sections, that had been fixed in formalin and paraffin-embedded. This histologic procedure of formalin fixing and paraffin embedding is common in the poultry diagnostic laboratory for the microscopic observation of lesions (abnormalities) in the tissues of sick chickens.

The in situ hybridization (ISH) procedure can assess infection in a small percentage of the total number of chicken cells, without loss of sensitivity due to large numbers of cellular nucleic acids. This allows for early detection (24 hours after infection) with only a small number of viral particles. The ISH test has been widely used for the detection of viral infections. A clinically unapparent viral state in which a particular viral marker is synthesized at levels below the threshold of detection by serology (antibody detection) or immunohistochemistry can be detected using ISH.
The AAES ISH test used a nonradioactive, cloned cDNA (copy of deoxyribonucleic acid) probe amplified in E. coli bacteria for the safe and rapid (one day of processing) detection of viral nucleic acid.

The probe was labeled with the nonradioactive chemical digoxigenin (DIG). The DIG labeled probe is safe, sensitive, and stable for more than one year in the refrigerator. DIG labeled probes accurately define viral nucleic particles and show less nonspecific (background) staining than with other nonradioactive probes. With this test the viral RNA (ribonucleic acid) can be seen in close proximity to the microscopic lesions indicating that the reovirus caused the lesions. The figure shows an arrow, which indicates a positive ISH signal (presence of reovirus RNA). This is a heart tissue showing microscopic lesions from a reovirus infected chicken. With this test, viral containing particles could be localized in the liver, pancreas, and synovial membrane of the tendon of infected chickens. These tissues also had macroscopic and microscopic lesions indicative of reovirus infection. The microscopic lesions were in close proximity with the positive ISH particles indicating that the reovirus caused the lesions. Tissues showing the most amount of microscopic lesions had the most amount of positively stained particles, which further showed that reoviral replication was responsible for the microscopic lesions. In addition, tissues from noninfected chickens or chickens infected with nonrelated poultry viruses did not show positive stained particles. This indicated the specificity and reliability of the test for routine diagnosis of reovirus infections.

The ISH test can be adapted to many other poultry viruses by simply making specific probes for each piece of viral nucleic acid. Therefore, this test represents a major breakthrough for the diagnosis of all viral diseases of poultry where the disease cannot be diagnosed based solely on the clinical picture.