Update on vvIBDV in the U.S. There have been several more outbreaks that have involved viruses with the vvIBDV genotype and phenotype. These viruses are indistinguishable from vvIBDV in other parts of the world.

We have now found a different IBDV in US chicken flocks that has the vvIBDV genome segment A sequence but a different segment B sequence. Previous reports by He et al., Hon et al., and Le Nouen et al. indicate these viruses may be less virulent because of this apparent recombination event. We still need to test the virulence of these viruses in SPF chickens. We also need to identify the source of the segment B in these viruses.

When diagnosing vvIBDV make sure genome segment B is identified as well as segment A.

Thank you Daral for the news. In my lab we routinely detect IBDV in clinical samples but we got a rapid method based on VP2 PCR-RFLP adapted from Zierenberg et al (Av. Path. 2001.30:55-62). This is a very convenient assay and give us results quite coherent with the clinical presentation. However, since these viruses change a lot with time, and taking into account your comments, I wonder whether we have to update our method. When you ask people to look for both viral segments....what do you mean exactly? ¿Sequence determination? ¿Probes or REA on both segments? ¿Which is the most appropiate method to perform IBDV detection and surveillance for this pathogens nowadays?

Jaime - We used RFLP for many years but have now moved on to a TaqMan assay that can differentiate the VP2 gene of vvIBDV from non-vvIBDV strains. Any positive samples can be sequenced directly from the TaqMan amplicon. That is usually not necessary but it is nice for some of the more interesting samples. I can send you the primers, probes and procedure if you want them. I am working on a TaqMan that will also differentiate the B genome segments. Currently, we use conventional RT-PCR to amplify segment B and then sequence it to determine if it is the vvIBDV or non-vvIBDV type.

We like the TaqMan assay because it is faster and cheaper than RFLP. Plus we have a 96-well machine that can do many more samples in a day than we could with RFLP.

Daral, I agree with your comments about RT-PCR advantages. The only thing that makes me wonder about using probes to catch an RNA target is the possibility of probe binding failure due to mutations in the binding site. I got several procedures already established in the lab based on RT-PCR, but most of them use SYBRgreen with no probes at all. Do you think that, for the particular issue of IBDV detection, using TaqMan probes is possible to detect "all" strains regardless the genetic variability?

I agree with you, detecting RNA viruses with probes can be dangerous. The SYBRgreen is a better way to do it if you are just looking for positive or negative. We have used melting curve data with SYBRgreen to differentiate IBDV and it works OK. I have been experimenting with probes because we wanted a quick vvIBDV assay. All the vvIBDV segment A sequences are very similar, so the vv probes work very well but we may be missing some of the non-vvIBDV using our non-vv probes. There are some very conserved sequences in the IBDV genome so it should be possible to setup a TaqMan assay that detects all viruses and then use a second probe to just detect the vvIBDV. Maybe I will try that.

Aprox 5% of the IBDV PCR+ samples in our DLab cannot be cut by the enzymes we use. 95% of them are either vv or classical. What I am really concern about is the 5% of viruses that are non-typable by the conventional method. For me the best assay is the one that combines 100% detectability (even detection of "variants" or mutants) with a second step of further characterization (in case of unexpected results). When we face a sample that is IBDV PCR+ but is non-typable by REA, then we perform seq+phylogenetics. I don't know whether other molecular approaches could fill this gap (High Resolution Melt (HRM) analysis for instance?)

What region are you amplifying and what enzymes are you using? We found that BstNI and MboI will always cut somewhere in the hypervariable region of VP2. We are amplifying the entire hypervariable region of VP2. I know some labs that only amplify a part of the hypervariable region and since their PCR product is short, it will not cut with some of the enzymes.