In ovo competition between distinct virus populations in an avian influenza isolate

Embryo lethality patterns induced by an avian influenza virus isolate (A/Tk/Ws/68/H5N9) suggested that it contained more than one genetic form. Two different virus populations were recovered from the isolate by plaque isolation and limit-dilution cloning and were characterized with respect to their biological and molecular properties. They were very closely related but exhibited strikingly different mean death times (MDT) in 10-day-old chick embryos. One was rapidly embryo lethal (REL), while the other was slowly embryo lethal (SEL). The REL isolate demonstrated a small but measurable mortality rate in 4-week-old chicks, as did the parental isolate. The SEL isolate, however, was nonlethal to 4-week-old chicks. The embryo MDT induced by the parental isolate revealed a biphasic death pattern reflecting expression of both REL and SEL populations. Mixing experiments, using different amounts of the two cloned populations, demonstrated that expression of their unique phenotypic property (either REL or SEL) was competitive. The number of early or late embryo deaths was directly related to the input levels of each respective virus. The only molecular difference thus far detected between the two populations is in the nonstructural (NS) gene, with the REL clone possessing a faster migrating electrophoretic form of that RNA than the SEL clone. Both forms of the NS gene were present in the original parental isolate. This study thus demonstrates the competitive coexistence of two closely related virus populations within a single natural isolate.