Analysis of Genomic Genetic Diversity of Cucurbit Aphidborne Yellows Virus

[Objectives]This study was conducted to investigate the molecular characteristics,genetic structure and evolutionary mechanism of cucurbit aphid-borne yellows virus(CABYV),which is of great significance for clarifying the epidemic laws of the disease in the field and formulating long-term sustainable control strategies.[Methods]120 melon leaves suspected to be infected with CABYV were randomly collected from Aksu City,Xinjiang.RT-PCR was used to detect the virus.MEGA 7.0 was used to construct a phylogenetic tree.DnaSP v5.10 was used to analyze the genetic diversity among different sequences,and RDP v.4.31 software was used to analyze possible recombination events of CABYV sequence.[Results]The detection rate of CABYV was 31.67%.A new CABYV-2 was isolated,sequenced and cloned from these positive samples.The length of the genome is 5497 bp,encoding six open reading frames.Compared with 23 CABYV strains isolated from different countries in NCBI database,CABYV-2 shared the highest homology with KR231942.1 and the lowest homology with JF939812.1.The results of evolutionary tree analysis showed that the 24 isolates were divided into two branches due to different geographical factors,including CABYV-2 and KR2319421,which were clustered together and belonged to branch 1.The results of genetic diversity and neutrality tests showed that CABYV was highly variable and the population was expanding.The results of recombination analysis showed that there were two recombinants,EU636992.1 and KR231949.1,which exacerbated the variation of CABYV.[Conclusions]The CABYV isolate from Aksu City,Xinjiang had the closest relationship with the Korean isolate KR231942.1,and the farthest relationship with the isolates from European countries.There were large genetic differences between the isolates of branch 1 and branch 2,and the recombination of CABYV in different hosts aggravated the variation of CABYV.Recombination and negative selection may be important reasons for the genetic variation of CABYV.

Pathogenic Effects of Cloned Genomic DNA of Porcine Circovirus-like Virus P1 on Neonatal Mice via Different Inoculation Routes

[Objective] The paper was to explore the pathogenicity of cloned genomic DNA of porcine circovirus-like virus Pl to neonatal mice via different inoculation routes （brain, liver and muscle）. [Method] Cloned genomic DNA of P1 was inoculated to neonatal mice via different routes of brain, liver and muscle. Tissues of heart, liver, spleen, lung, kidney and brain were taken from neonatal mice at 7, 14 and 21 d post inoculation, re- spectively. Pl in various tissues were qualitatively and quantitatively detected by using ordinary PCR and quantitative real-time PCR. Meanwhile, histopathological changes were analyzed. [Result] Pl was detected in neonatal mice inoculated through three different routes. The viral load of tis- sues at 7 d post inoculation was significantly higher than those at 14 and 21 d post inoculation. Moreover, muscle inoculation led to the highest viral load in all tissues of neonatal mice. [Conclusion] Pl infection caused different degrees of pathological damage to heart, liver, lung, kidney and brain in neonatal mice.

Modification of the full-length cDNA clone of Newcastle disease virus isolated from an outbreak in the goose

A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length cDNA clone of the Newcastle disease virus(NDV)strain ZJI of goose origin,and thereafter it was self-ligated to form a high quality plasmid for mutagenesis.Site-directed mutagenesis was used for inserting three additional G nucleotides(nts)into the region between the T7 promoter and the leader sequence of the NDV genome.RT-PCR was employed to amplify the F/HN gene fragments,and then they were ligated by the shared restriction enzyme BsmBI.Finally,the corresponding fragment in the mutant full-length cDNA was substituted with the new one.The sequencing results showed that the three additional G nts were successfully inserted and the mutant nts in the full-length cDNA were corrected.This study lays a good foundation for research on the reverse genetics of NDV strain ZJI.