| Newcastle disease(ND)is a severe infectious disease that seriously endangering the poultry breeding industry,and its pathogen is Newcastle disease virus(NDV).The prevention and control of the disease is mainly dependent on vaccination,and the heat-resistant live attenuated vaccine is convenient for storage and transportation,and has important application value in tropical areas and remote rural areas.The heat-resistant V4,1-2 and other vaccine strains have been used in the vast rural areas of the tropical regions of Southeast Asia and Africa,and have produced good prevention and control effects.Although China had introduced the V4 and other heat-resistant strains,and also developed a number of heat resistant attenuated strains suitable for our country to use through further heat-resistant screening,it has not been popularized in our country due to limitation of intellectual property rights.A heat-resistant and high virulent HR09 strain was isolated by our laboratory previously.The biological characteristics of HR09 strain had been identified and a reverse genetics platform had been constructed.But the heat-resistant molecular basis of HR09 strain hasn't been fully discovered.In this study,the heat-resistant passaging test is carried out on the basis of previous researches.The genetic variation of virus under thermal compression is studied by measuring the heat-resistant related capsule membrane protein genes(F,HN gene)of different generations,and the bioinformatics analysis method is used to study the genetic variation of virus and heat-resistant genes at the nucleic acid and amino acid level,preliminarily exploring the heat-resistant molecular basis of the viral capsule protein.At the same time,the chimeric strain based on HR09 strain is constructed by replacing the fusion protein gene of the HR09 strain with the LaSota strain,for further researching the influence of F protein to HR09's heat-resistant property.And we look forward to obtaining a heat-resistant attenuated NDV strain,laying foundation to creating our independent heat-resistant attenuated NDV vaccine.1.Genetic variation patterns and sequence comparison of envelope protein of HR09 strainHeat resistant strain HR09,preserved in our laboratory,was transmitted to 30 generations in a hot bath at 56 C(The first 5 generations proved the heat tolerance limit of the strain.The last 25 generations were treated for 105min.).Then,the F and HN genes of the heat-resistant 5,20 and 30 generations of the HR09 strain were amplified,sequenced and analyzed,to research the genetic variation patterns of F and HN genes after heat treatment.The results showed that the heat tolerance limit of the strain is 56?-120min,and the F and HN genes of the 5th,20th and 30th generations produced some same sense and missense mutations.The 30th generation showed the most mutations and in F gene,there are 2 same sense mutations(A1071T,T1320C),3 missense mutations(G311A,A1295T,A1476T),and the according amino acid mutation sites of F protein are G104E,K432I,K492N;in HN gene,there are 3 same sense mutations(T748C,T1116C,G1122A),2 missense mutations(T755C,C1368G),and the according amino acid mutation sites of F protein are F252S,H456Q.Then,the sequences of F and HN gene of heat resistant HR09-30 and the original HR09 strain and the currently known heat resistant strains(V4,1-2,NDV4-C,TS09-C,AF2240)and the non-heat-resistant strain LaSota were analyzed and compared by SMS online sequence analysis tools(http://www.bio-soft.net/sms),Megalign and MEGA7.The results showed that,at the nucleotide level,the GC content of the heat-resistant strain F gene is higher than that of the non-heat-resistant strain(45.15%/44.46%);and the GC content of the HN gene in the heat-resistant strain is higher than that of the non-heat-resistant strain in addition to the genotype VIII strains,AF2240 and HR09.The GC content of the HN gene in the 30th generation of HR09 strain after heat treatment is higher than that of the original strain.In terms of homology,the homologies of the F and HN genes between the 30th and original HR09 strain are both 99.7%.Among all the known heat-resistant strains,HR09 strain has the biggest difference with non-heat-resistant LaSota strain.Gene phylogenetic analysis of the F and HN genes showed that the known heat-resistant strains are concentrated in NDV genotype I and VIII.At the amino acid level,the F protein of heat-resistant HR09 strain and non-heat-resistant strain have the greatest difference(10.90%).When the amino acid composition was analyzed,the content of proline in the F protein of non-heat-resistant strain is higher than that of the heat-resistant strain,whereas in the HN protein is opposite(5.23%/5.00%).The content of acidic and hydrophobic amino acid in HN protein of heat-resistant strain is generally higher(acidic amino acid:7.00%/6.70%;hydrophobic amino acid:40.89%/39.00%).2.Structure simulation and analysis of F and HN proteinThe secondary and tertiary structure simulation and comparison analysis were carried out by using PDB structure automatic simulation software(https://www.predictprotein.org/and https://swiss-model.expasy.org/)for the F and HN protein of HR09 original strain,the 30th generation of HR09 strain after heat treatment,the V4 strain and LaSota strain.Using VMD,Discovery Studio 2.5 for structural display and analysis.The results of structural simulation showed that the F and HN proteins are consistent with the homologous protein template,but there are some internal structural differences.From the results of the secondary structure prediction,it was found that the number of helical-structure and betastructure in F protein is not related to heat resistance,and the lower of the content of the irregular curl structure in the F protein,the stronger of the heat resistance of NDV;the number of helical-structure in HN protein is also not related to heat resistance,the lower of the content of the betastructure and the higher of the content of the irregular curl structure in HN protein,the stronger of the heat resistance of NDV.In the position,the helical-structure between 34-3 8aa,the betastructure between 408aa and 448-450aa,the irregular curl between 56-57aa and 244-245aa of F protein are related to heat resistance;the helical-structure with 6aa length between 605-611aa,the betastructure between 562aa and 565aa,the irregular curl between 421 aa and 423aa of HN protein are related to heat resistance.The ternary structure prediction showed that,the number of secondary structure between 94-130aa and the number of the secondary structure after the 466aa of the protein tail of F protein influence the heat resistance;in HN protein,the absence of bridge structure between 149-173aa,broader range of the irregular curl,turning corner to helix between 293aa and 533-545aa,and more a-helix around 473aa will improve the heat resistance.Combining the amino acid mutation of F and HN protein of the 30th generation of HR09 strain and the structure prediction of the 30th generation and the original HR09 strain,it showed that G104E in the second hydrophobic region(fusion zone)in the F protein is located in the random coil in HR09 strain,but in the helix of the heat resistant HR09 30 generation,and is located in the HB-B(seven peptide repetition).There was no change in the secondary structure of K432I near B and K492N located in the third hydrophobic region(protein transmembrane region)in HR09 strain and heat-resistant HR09 30 generation strain.The F252S and H456Q in HN protein were located in the extracellular domain,and were completely conserved near the cysteine residues,and the secondary structure did not change.3.Construction of full-length clone of rHR09-LaSota-F chimeric virus genomeUsing the HR09 strain reverse genetics platform constructed in our laboratory,the F gene of LaSota strain and F gene of HR09 strain in R2 plasmids(4157-6898nt)were replaced by overlap RCR,and the rR2-LaSota-F plasmid was constructed.Then,using BsmB I and Spe I double-digestion and Overlap RCR,rR2-LaSota-F plasmids were joint with R1 and R3 plasmids to construct rT123-LaSota-F plasmids(2249-9535nt).Secondly,using Spe I and FspA I double-digestion,rT123-LaSota-F plasmid and TL2 plasmid were joint to construct rTM-LaSota-F plasmid(2296nt-13048nt).Finally,using Apa I and Mlu I double-digestion,the plasmid rTM-LaSota-F and TVT-V were stitching together,after PCR identification,the full length of rHR09-LaSota-F chimeric virus genome.In this study,we only constructed the full-length plasmid of rHR09-LaSota-F chimeric virus and failed to rescue the virus.The virus rescue program needs to be optimized,and the virus's heat tolerance needs to be tested after saving the virus. |
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