In Ovo Competition between Distinct Virus Populations in an Avian Influenza Isolate

Abstract

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.

A viral race for primacy: co-infection of a natural pair of low and highly pathogenic H7N7 avian influenza viruses in chickens and embryonated chicken eggs

“…Although inoculation was targeted to the allantoic sac, accidental lesion of the amnion cannot be excluded 50 , 51 . We confirmed previous results suggesting an increased resistance of 14-day old versus 10-day old ECEs to AIV infection 29 , 52 , 53 .…”

Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico

“…Primary chicken embryo fibroblast (CEF) cell cultures were prepared from 10-day-old embryos as previously described (Perdue et al, 1989) and inoculated with infected allantoic fluid. Virus was radiolabelled with [35S]methionine for 16 h during infection of fibroblast cultures with no trypsin added, as previously described (Perdue, 1992).…”

Genetic changes that accompanied shifts of low pathogenic avian influenza viruses toward higher pathogenicity in poultry

“…The virulent strain induced rapid embryo lethality of SPF chicken embryonated eggs and mortality in four-week-old chicks while the avirulent pathotype induced slowly embryo lethality (more than 72 h) and was nonlethal to chicks. Both virus populations were stable through 12 serial passages in SPF eggs 144 . Two amino acid differences in the NS gene were found 145 : valine and phenylalanine (in the nonlethal phenotype) or leucine and serine (in the virulent phenotype) at position 119 in NS1 and 48 in NS2, respectively.…”