Effects Of F Gene Start Codon Mutation On Biological Characteristics Of Class ? Newcastle Disease Virus

Newcastle disease(ND)is a highly contact infectious disease in poultry caused by Newcastle disease virus(NDV).The epidemiological investigation of NDV in recent years showed that class ? NDV has been widely distributed in China.The transmission and epidemic of class ? NDV in poultry may lead to the mutation and the increased virulence of virus.The whole genome of multiple class ? NDV strains isolated from 19 provinces and cities in China in 2017 were sequenced and several variants with F gene start codon mutated into ATA and CTA were identified.The FJ28 strain was isolated from ducks in Fujian province.Genetic evolution analysis showed that FJ28 belongs to genotype 3 in class ?.Compared with most class ? NDVs,the start codon of F gene of FJ28 was mutated from ATG to ATA.Further analysis found that the start codon of the F gene of FJ28 shifted forward by 33 nucleotides,resulting in the F gene coding sequence consisted of 1695 nucleotides,and the encoded F protein consisted of 564 amino acids.Compared with most of other class ? NDVs,the length of other genes of the FJ28 did not changed.The results of virulence index showed that the mean death time(MDT)in chicken embryo of FJ28 strain was 72 h,and the intracerebral pathogenicity index(ICPI)in 1-day-old chicks was 1.413,indicating that the FJ28 was a mesogenic strain.Since most class ? NDVs are lentogenic,we speculated that the increased virulence of FJ28 may be related to the elongated coding sequence of F gene caused by the mutation of start codon.To verify the above inference,the F gene of class ?I avirulent La Sota strain was replaced by the F gene of FJ28 strain via the reverse genetics and recombinant virus La Sota-FJ28 F was obtained.The results of virulence index showed that the ICPI of La Sota-FJ28 F was 0.7321,which was higher than 0.19 of the parent La Sota strain,indicating that La Sota-FJ28 F is a mesogenic strain and the replacement of F gene in La Sota led to the increased virulence.Then both the F and HN genes of La Sota strain were replaced with the F and HN genes of FJ28 strain simultaneously and the recombinant virus La SotaFJ28 FHN was rescued.The results of virulence index showed that the ICPI of La Sota-FJ28 FHN was 0.736,indicating that the La Sota-FJ28 FHN was mesogenic,and there was no significant difference between La Sota-FJ28 FHN and La Sota-FJ28 F.Besides,though the MDT of La Sota-FJ28 FHN was 126 h,but the survival chicken embryo incubated with La Sota-FJ28 FHN showed obviously bleeding and lesion.These results indicated that the replacement of HN genes did not result in virulence increase,and the replacement of F genes was the key factor.In order to prove above conclusion,the F gene start codon of La Sota-FJ28 FHN was mutated from ATA to ATG and the recombinant virus La Sota-FJ28FHN-FATG was rescued.The result of virulence index showed that the ICPI of La Sota-FJ28FHN-F-ATG was 0.238,indicating La Sota-FJ28FHN-F-ATG was lentogenic.These results indicated that F gene start codon mutated to ATG was the key factor of the decreased virulence of recombinant virus La SotaFJ28FHN-F-ATG.To further verify that the mutation of F gene start codon was the key factor contributed to the increased virulence of FJ28,we developed the reverse genetic system of class ? FJ28 strain,and the parental FJ28 strain was rescued.Then the F gene start codon was mutated to the conventional start codon(ATG)and the mutant virus FJ28FL-ATG was obtained.The results of NDV epidemiological investigation showed that the F gene start codon of some NDVs have been mutated from ATG to CTA when compared with other class ? NDV.In order to evaluate the effect of different mutations in the F gene start codon on the biological characteristics of NDV,the F gene start codon was mutated to CTA and the mutant virus FJ28FL-CTA was rescued.The results of virulence index showed that the ICPI of FJ28,FJ28FL-ATG and FJ28FL-CTA were 0.875,0.266 and 0.406,respectively,indicating that FJ28 was a mesogenic strain and FJ28FL-ATG and FJ28FL-CTA were lentogenic strains.Besides,the MDT of FJ28,FJ28FL-ATG and FJ28FL-CTA were 136 h,134 h and 121 h,respectively.And the survival chicken embryo incubated with FJ28 showed obviously bleeding and lesion,while no bleeding and lesion were observed in survival chicken embryo incubated with FJ28FL-ATG and FJ28FL-CTA.The above results indicated that the amino acid encoded by the F gene start codon ATA was a determinant of the increased virulence of FJ28.Moreover,in this study,the growth curves of parental FJ28,recombinant FJ28FL-ATG and FJ28FL-CTA were measured in chicken eggs.The results showed that all three viruses had similar growth kinetics,while the replication rate of parental FJ28 was fastest.The virus titer of FJ28 at each time point was higher than those of FJ28FL-ATG and FJ28F-CTA.These results suggested that the change of encoded F protein size caused by the mutation of F gene start codon is the key factor contribute to the difference of virulence and growth kinetics between FJ28 and other class ? lentogenic strains.In this study,we reported for the first time that the special mutation of the F gene start codon is the key factor leading to the increased virulence of class ? NDV.The results obtained here could contribute to the better understanding of the genetic evolution and pathogenic mechanism of class ? NDV.