Reply to “May Newly Defined Subgenotypes Va and Vb of Newcastle Disease Virus in Poultry Be Considered Two Different Genotypes?”

We appreciate Dr. Desingu’s comments (1) on our paper entitled “Separate Evolution of Virulent Newcastle Disease Viruses from Mexico and Central America” (2). Dr. Desingu points out that the mean evolutionary distance between viruses in subgenotypes Va and Vb is higher than the threshold (10%) set for subgenotype assignment (3) and, therefore, that subgenotypes Va and Vb should be considered two distinct genotypes. In addition, he suggests that subgenotype Vc may be considered ancestral to both genotypes Va and Vb. In response to the first comment made by Dr. Desingu, we believe that subgenotypes Va, Vb, and Vc should remain subgenotypes within genotype V for the following reasons. (i) Although we reported an evolutionary distance between subgenotypes Va and Vb (10.9%) that is slightly higher than the threshold (2, 3), the supporting data for a new genotype are not sufficiently strong. In fact, the nucleotide distance criterion ( 10%) for designating a new genotype should be met for any group of viruses that is compared to all other groups. If subgenotype Va is to be considered a putative new genotype, it should have an evolutionary distance of 10% compared to all other genotypes. Using the same data set as in our previous paper (2), analysis comparing Va (putative new genotype) to merged Vb and Vc (as these will remain and constitute genotype V) shows that the distance between them is 9.8% (Table 1) and thus does not meet the 10% rule. Similarly, using a new data set with all currently available sequences, the evolutionary distances between these three subgenotypes within genotype V fall under 10% (Table 2), confirming our previous results. These observations suggest that above-the-threshold evolutionary distances between subgenotypes do not justify automatic assignment of a new genotype. Calculation of evolutionary distances between subgenotypes is influenced by how homogeneously such distances are distributed within the genotype and by the number of taxa available for analysis (groups with more strains weigh more heavily in the analysis). Adding or removing few sequences in an analyzed set may affect distances and topology. This is among the reasons explaining why our group has followed a very conservative approach in the creation of new genotypes in the past. (ii) There are currently no defined criteria for naming genotypes that originate by continued evolution of existing subgenotypes. Under the current classification, a new genotype should receive a different Roman numeral (e.g., genotype XIX) and this will inevitably result in additional confusion among researchers due to lack of consistency with existing literature. Regarding the second comment of Dr. Desingu stating that subgenotype Vc should be considered ancestral to both subgenotype Va and subgenotype Vb, we believe that there is no evidence supporting such a statement. As presented in Fig. 1 in our original paper (2), the phylogenetic tree demonstrates that subgenotypes Vb and Vc diverged from a common ancestor (bootstrap value, 65%) genetically closest to a Newcastle disease virus (NDV) strain