Understanding how IB viruses are tested, typed is first step to control in poultry
By Mark W. Jackwood, PhD
Poultry Diagnostic and Research Center
Department of Population Health
College of Veterinary Medicine
University of Georgia
Athens, GA 30602
Email: [email protected]
Infectious bronchitis remains one of the most economically significant diseases of commercial poultry. It causes not only respiratory disease but also decreased egg production, poor quality eggs and, with some strains, nephritis and high mortality.
New variants of infectious bronchitis virus (IBV) continually emerge, which underscores the need for active surveillance and timely identification of the IBVs circulating in chicken flocks. It’s the only way that producers and their veterinarians have the information they need to initiate an appropriate vaccine-management plan since different types of IBV do not cross-protect. The more you understand how we screen samples for IBV, the better you can understand the complexity of the disease and the need for strategic vaccination programs.
IBV identification is accomplished using molecular detection techniques. Some IBV samples are 100% similar to a known IBV, but when the similarity is not as clear-cut, the test results leave uncertainty about how to plan vaccine protocols.
Almost all molecular diagnostics are based on the polymerase chain reaction (PCR) test — and typing of avian IBV is no exception.
PCR synthesizes large quantities of DNA by copying the genetic sequence of an organism over and over again. This chain reaction generates lots of DNA quickly, which can then easily be detected and examined with common laboratory techniques.
Because each organism has a unique genetic code, PCR-generated DNA can be sequenced to identify the disease agent and, sometimes, even the strain or specific isolate in a sample.
To explain how this works, I’m afraid I’m going to serve up a large bowl of “alphabet soup,” but please stick with me over the next few paragraphs. I’ll then explain how all this relates to getting an accurate diagnosis of infectious bronchitis.
As noted earlier, PCR tests use DNA as a template to synthesize more DNA. However, because IBV is an RNA virus, we first have to convert the viral RNA to DNA. We do this with an enzyme called reverse transcriptase (RT), which uses RNA as a template to synthesize a DNA copy. The copied DNA can then be used in the PCR reaction to generate a lot of DNA for analysis. This procedure is called RT-PCR.
Still with me?
Once we obtain the copied DNA from a clinical sample, it needs to be detected and analyzed. We do this using a process known as electrophoresis, which separates DNA fragments by size. The process involves the use of ethidium bromide, a chemical that incorporates into the DNA and fluoresces when exposed to UV light, enabling us to see the individual fragments.
Real-time PCR — a technique that detects newly synthesized DNA in the PCR reaction as it’s being made — is essentially a conventional PCR test with a fluorescent marker incorporated into the reaction. Using a specialized thermocycler that includes a spectrophotometer, we can detect the fluorescent signal of the amplified DNA in real time as the reaction is taking place.
Real-time PCR is extremely fast — it takes only about 20 to 30 minutes — and can be conducted on a 96-well format. This makes it a good choice for screening hundreds of samples quickly.
Real-time PCR is also quantitative and is sometimes called qPCR. This means it can be used to determine how much of the pathogen’s genome was actually present in the original sample.
Still with me, right?
Identifies IBV presence
Now let’s look at how all this applies to diagnosing IBV and its many variants.
For IBV, we need to conduct two important tests:
First, we have to determine if the clinical sample is positive for IBV or not. This is typically done with a real-time RT-PCR test that targets a highly conserved region of the genome (Figure 1). It detects all IBV types and, thus, does not provide data on the type of IBV in the sample. Instead, it tells us which samples are positive for IBV and shows which samples contain the most virus.
Since different serotypes of IBV do not cross-protect, we need to use a second RT-PCR amplification test to determine the virus type. This is done by amplifying a portion of the viral-spike gene. The spike protein encoded by the spike gene is located on the outside of the virus (Figure 2). It mediates attachment to the host cell and induces neutralizing antibodies in the infected bird.
Because different serotypes of IBV have different spike glycoproteins, we can use the gene for the spike glycoprotein to identify the virus type. The spike is divided into two fragments — S1 and S2. It is the S1 fragment — specifically, the hypervariable region within the S1 fragment of spike — that contains unique sequences for each IBV type.
Typically, we use RT-PCR to amplify the hypervariable region or the entire S1 half of the spike (Figure 1). Using the nucleotide sequence of the PCR product, we can then identify the type of IBV in the sample.
Once we obtain the sequence of the spike-gene PCR product, we analyze it with “bioinformatics” computer programs. First we look at the spike-sequence data to determine the amino acid of the spike protein. Basically, the computer program identifies the sequence of nucleotides that code the different amino acids — and converts the nucleotide data into an amino-acid sequence.
Next, we take the spike protein amino-acid sequence and look for similar sequences. This involves searching the National Center for Biotechnology Information database.
We use a program called Basic Local Alignment Search Tool (BLAST) to find similarities between the query sequence (the unknown sequence) and sequences in the database. BLAST then provides a description of the matched sequences — specifically, the highest alignment similarity score, the percentage of the sequence covered by the alignment and the identification (accession number) of the matched sequences in the database. This helps identify the unknown sequence.
After BLAST identifies a number of similar sequences in the database, we use a sequence-alignment program to determine the percent similarity and differences among the sequences. Sequence alignments are very difficult to read, so the data is usually reported in a “percent similarity/distance” table (Table 1) or graphically in a phylogenetic tree (Figure 3).
For IBV, the million-dollar question is what amino-acid sequence known to be present correlates with cross-protection? Unfortunately, there is no hard rule because exceptions always exist. But we can make reliable, educated predictions based on previous studies. A case in point follows:
We used real time RT-PCR to identify an IBV-positive clinical sample from a 4-week-old broiler. We then amplified and sequenced the spike-gene hypervariable region. Figure 4 shows the BLAST analysis for the unknown sequence.
The first virus identified from the BLAST analysis was AL/0803/01, an IBV sequence from a virus isolated in Alabama in 2001.
After examining the next three sequences identified in the BLAST analysis, we saw that the most closely related viruses were Arkansas vaccine-type viruses. All of these viruses (including the AL/0803/01) were 100% similar to our unknown virus.
There were viruses in the database that were either not identified or were misidentified when they were submitted. When the AL/0803/01 sequence was entered into the database, it was not identified as an Arkansas-type virus. Often the second or third or even fourth virus identified by the BLAST analysis would reflect the true nature of the unknown virus. This is not unusual and we needed to consider that possibility when examining the data. The similarity/distance table (Table 1) and the phylogenetic tree (Figure 3) showed the relationship of our unknown virus with Arkansas-type viruses as well as other IBV genetic types. Clearly, this unknown virus was Arkansas vaccine-type virus.
In summary, molecular diagnostic methods are most always based on PCR. Our goals are to detect IBV in a clinical sample and analyze the spike-sequence data to determine the type of the virus.
Molecular diagnostic tests for IBV are fast and take only 1 or 2 days to complete. They provide data on the amount of virus in the clinical sample, the type of virus and the similarity of that virus to other known strains of IBV.
However, analysis of the data is not always straightforward, and some knowledge of the viruses in the database is necessary to interpret the results in a meaningful way.
Molecular diagnostic tests (RT-PCR and sequencing) now provide a high throughput, rapid and cost-effective surveillance tool that will enable poultry producers to be more responsive to emerging variant IBVs capable of causing disease.
This, in turn, will improve animal welfare and help reduce losses from a very serious and costly disease.