Molecular Trail For Duck Hepatitis A Virus 3 Attenuated

Duck hepatitis A virus 3(DHAV-3)is one of the important pathogens that cause viral hepatitis in ducklings and can cause acute death of infected ducklings.Focusing on the scientific problem of analyzing the molecular basis of DHAV-3 attenuation,this article carried out a series of studies,and the results are as follows:1.Isolation and identification of a DHAV-3 virulent strainWe isolated one strain of DHAV-3 from the suspected cases of duck viral hepatitis,and the VP1 gene of this strain has high amino acid similarity to many Chinese strains of DHAV-3(95.7%?100%);rabbit anti-DHAV-3 serum can completely neutralize this strain(serum titer?1:160),and the rabbit antiserum of this isolate has a high neutralizing ability to other reference DHAV-3 strains(serum titer?1:200);the 1-day-old ducklings inoculated with this isolate showed typical symptoms such as ataxia and kyphosis,with an morbidity rate of 100%(10/10)and a mortality rate of 80%(8/10).The liver of the dead duckling was obviously bleeding and swollen,indicating that the isolated virus was a virulent DHAV-3 strain with good antigenicity.2.Passage attenuation of DHAV-3 and its immune protection effectThe isolated DHAV-3 virulent strain was serially passaged to the 120th generation on9-day-old duck embryos,and 5 different passages strains(CH-P10,CH-P30,CH-P60,CH-P90,and CH-P120 strains)were selected for detection.The results show:(1)During the passaging process,the proliferative ability of DHAV-3 is enhanced.The virus titers of these 5 different passages strains in duck allantoic fluid are 10^4.33ELD₅₀/0.2m L,10^6.55ELD₅₀/0.2m L,10^7.91ELD₅₀/0.2m L,10^8.37ELD₅₀/0.2m L,and 10^8.37ELD₅₀/0.2m L;(2)The results of the duckling pathogenicity test and challenge protection test show that the CH-P10 strain can cause the disease of all ducklings,with a mortality rate of 80%(8/10);CH-P30 strain can cause 80%(8/10)ducklings to get sick and 60%(6/10)ducklings to die;The pathogenicity of CH-P60 strain,CH-P90 strain and CH-P120 strain on ducklings has been significantly reduced.The ducklings have no clinical symptoms,and the ducklings can obtain good immune protection after vaccination.3.Whole genome comparative analysis of different passages of DHAV-3Whole-genome sequencing analysis of different passages of DHAV-3,the results show that:CH-P10 strain has no deletions or insertions in the genome during continuous passage to CH-P120 strain,but there are 7 nucleotide sites with stable mutations(C62T,T453C,T1142A,C4334A,G5215A,G6268A,and A7181C).Of these 7 nucleotide mutations,2 nucleotide mutations are located in the 5'UTR region(C62T and T453C),2nucleotide mutations are nonsense mutations(G5215A and G6268A),and 3 nucleotide mutations(T1142A,C4334A,and A7181C)caused mutations in the corresponding amino acid positions.Nucleotide mutation T1142A causes mutation of 164th tyrosine(Tyr)of the VP0 protein to asparagine(Asn);nucleotide mutation C4334A causes mutation of the 71st leucine(Leu)of the 2C protein to isoleucine(Ile);nucleotide mutation A7181C causes mutation of isleucine(Ile)to leucine(Leu)at position 378 of the 3D protein.Between CH-P10 strain(virulent)and CH-P60 strain(attenuated),only Y164N mutation of VP0protein and L71I mutation of 2C protein occurred,so we speculated that these two site mutations are related to the DHAV-3 virulence phenotype.4.Construction of DHAV-3 infectious cloneUsing PCR and fusion PCR,the CH-P10 parent strain was divided into three fragments of similar size for PCR amplification.The first fragment and the third fragment overlapped with the second fragment by 74 and 83 base pairs region,respectively.Introduced a synonymous base mutation in the 2A gene of the second fragment by fusion PCR,making the G mutation of the 3403th nucleotide of the reverse genetic strain virus to T(as a genetic molecular marker for infectious clones).Add p CMV at the 5'end of the first fragment and HDVR-SV40p A at the 3'end of the third fragment,together with the second fragment containing artificially marked genetic loci,they form the DHAV-3 infectious subgenomic replicons;then they were transfected with DEF cells,the lysis of DEF cell culture were collected and inoculated 9-day-old duck embryos,and the genetic markers of the allantoic fluid of dead duck embryos were identified.The results showed that the DHAV-3 infectious clone(ISA-P10 strain)was successfully constructed.The test results showed that the morbidity and mortality of 1-day-old ducklings infected with CH-P10strain and ISA-P10 strain were 100%(10/10)and 80%(8/10),respectively(indicating similar virulence),the ELD₅₀inoculated with 9-day-old duck embryos were 10^-4.33/0.2m L and 10^-4.50/0.2m L,respectively(indicating similar virus titers),they can be neutralized by DHAV-3 antiserum(indicating that the antigen is stable).The ISA-P10 strain was passaged10 times in 9-day-old duck embryos and the whole genome sequence determination and analysis showed that it had good genetic stability.These results indicate that we have successfully established a DHAV-3 reverse genetics operation platform.5.Molecular basis of the mechanism of the virus attenuationUsing the established DHAV-3 reverse genetics operation platform to carry out site-directed mutation on the DHAV-3 CH-P10 strain,we successfully rescued the strain containing the VP0 gene T1142A single point mutation(ISA-T1142A strain),the 2C gene C4334A single point mutant strain(ISA-C4334A strain),and double point mutant strain(ISA-T1142A-C4334A strain).The test results showed that the morbidity and mortality rate of ISA-T1142A strain and ISA-C4334A strain infected with 1-day-old ducklings were50%(5/10)and 30%(3/10),respectively.Their pathogenicity is lower than the parental CH-P10 strain;the ISA-T1142A strain and ISA-C4334A strain were inoculated with9-day-old duck embryos with ELD₅₀of 10^-7.55/0.2m L and 10^-6.55/0.2m L,respectively(indicating that the virus titer was higher than that of CH-P10 strain).Double point mutation ISA-T1142A-C4334A strain infection is not pathogenic to 1-day-old ducklings,similar to CH-P60 strain.In summary,infectious subgenomic replicons technology was used to construct DHAV-3 infectious clones,and a reverse DHAV-3 genetics operation platform was successfully established.Mutations in the VP0 gene T1142A(Y164N)and 2C gene C4334A(L71I)of the DHAV-3 genome can affect the pathogenicity of the virus to ducklings,and the combined effect of these two mutations can lead to a significant reduction in the pathogenicity of this strain to ducklings,which is the molecular basis of the attenuation of DHAV-3.