Separation And Identification And Whole Genome Sequencing Of Feline Herpesvirus 1 In Guangzhou From 2016 To 2017

Feline herpesvirus 1?FHV-1?,family Herpesviridae,subfamily Alphaherpesvirinae,genus Varicellovirus,which is one of the Pathogens of upper respiratory tract disease.It can cause mixed infection with Feline calicivirus and prone to infect the kittens.Similarly,the adult cats could be the susceptible animals and the spread source in highly crowded environment.Its mainly clinical symptom is that mentally depressed,slurry flow and the conjunctivitis.No effective prevention and treatment measures have been found for the infection of FHV-1 and its secondary diseases.Since the scholar separated the first strain of FHV-1 from a kittern in 1957 at North America,scholars from several countries in Europe and Asia have successively reported the isolation and the epidemiology of the FHV-1.However,it was a difficult way led to the epidemiology because of the absence of specific epidemic features of FHV-1.Researchers in our country had made some progress of studying the FHV-1,though the separation was fall behind the other countries.Similarly,focusing more energy on the molecular means than before,but not that just on the clinical dignasis.In this study,by the molecular biological means that collecting samples,isolation,identification,purification and the second-generation sequencing to research the conservation and diversity of FHV-1 genome.The study was conducted in four aspect as follow:Firstly,112 swab samples were collected at pet hospitals in Guangzhou.At the time of collecting,recorded the age,gender,sterility,castration,and clinical symptoms of the collected objects.The samples were inoculated after pretreated on the Crandel feline kidney cells?CRFK?for isolation and culture.Then the PCR product was subjected to electrophoresis analysis and one-generation sequencing.As a result,there were 7 positive samples according to the isolation and the identification.The information of samples was counted and the correlation analysis among the samples,age and gender showed that age and gender couldn't be the epidemic feature of FHV-1 infection.Secondly,with the nucleotide homologous analysis,the homologous of Thymidine kinase?TK?gene reached to 100% among the positive samples in this study.With the further identification and electrophoresis,one of the positive samples could had been polluted or mixed infection.However,the one-generation sequencing results refuted the possibility.Then the positive sample that was named as the FHV-1 isolated strains was chosed to be purified on the CRFK.As its purification load to the fifth generation,the purity of this strain was determined by PCR.The result showed that the FHV-1 isolates was purified well.Thirdly,the isolates was then subjected to second-generation sequencing,which was relative to the complete genome of FHV-1 as the reference sequence?GenBank login ID:NC₀13590.2?on NCBI.,A data analysis,assembly overview,analysis of Single nucleotide polymorphism?SNP?and the alignment with the reference sequence were performed after the second-generation sequencing.From the perspective of available data and assembly,the results of this second generation sequencing were reliable.There were 20 SNPs relative to the reference sequence.One of the non-synonymous coding regions SNPs was located at the gG gene,and its function and effect on the gG gene was unknown.Other SNPs were located in unidentified proteins.In this study,the quantity of SNPs in FHV-1 isolates was less than 0.1% of the total quantity of bases in the whole genome.From the perspective of alignment,there was an unknown sequence in the whole gene sequence of the FHV-1 isolate that sequenced by the means of second-generation sequencing.Then this unknown sequence was sequenced by one generation of sequencing.It showed that none of the deletion was in the whole genome of the FHV-1 isolate.Forthly,the major virulence genes of FHV-1 isolates were determined by one-generation sequencing.The phylogenetic analysis of the whole gene sequence and the major virulence genes of the FHV-1 isolate showed that the evolutionary relationship between the FHV-1 isolate and the FHV-1 historical isolate was close,the homology of their nucleotide as well as the virulence gene in the conserved region were high,and the whole genome was highly conserved;instead that,the antigenic proteins and transmembrane proteins of FHV-1 isolates was low homology compared to the FHV-1 historical isolates.The above results confirmed the significance to combine the whole gene sequence and the major virulence genes for phylogenetic analysis.As the research above,there were three conclusions were drawn as follow: The genome conservation of a FHV-1 isolate was high in Guangzhou from 2016 to 2017;molecular epidemiological investigation of FHV-1 is more feasible,instead of the tracditionl epidemiological investigation methods;a more in-depth study of the major virulence genes of FHV-1 may improve the prevention and control of FHV-1.Through the isolation,identification and purification of FHV-1 isolates,the use of gene sequencing techniques analyzing the whole genome of FHV-1 isolates in Guangzhou,China at the first time.So that the study enriching the domestic research of FHV-1,and deepening the clinicians' understanding of FHV-1.