Construction Of The Genetic Deletion Strain Of Feline Herpesvirus Type Ⅰ And The Evaluation Of Vector Vaccine

As pets become an increasingly important part of people’s lives,viral infections pose a significant threat to the health of cats.Felid herpesvirus 1（FHV-1）,feline calicivirus（FCV）and feline parvovirus（FPV）are the infectious agents of common diseases in cats.FHV-1 has become an important source of upper respiratory tract infections in cats,with a prevalence rate of 50-75%.It has a 100%morbidity and mortality rate of approximately 50%and can remain latent for life after initial infection.Feline herpesviruses have good genomic stability and can insert a large number of exogenous genes into the genome.The inserted exogenous genes can be stably expressed in the host cells and cause strong cellular and humoral immune responses.Currently,the immune efficacy of the existing triple inactivated vaccines against FHV-1,FCV and FPV is not satisfactory and requires multiple immunizations with a short duration of immune protection.In this study,a new generation polyvalent vaccine against feline herpesvirus type I,feline culex virus and feline microvirus was developed using a live virus vector and a weak strain of feline herpesvirus type I gene deletion.The following studies were conducted in this thesis:1.In this study,a strain of FHV-1,named strain FHV WX19,was successfully isolated from clinical samples of suspected feline infectious rhinobronchitis infection.Animal pathogenicity tests demonstrated that infection of kittens with FHV WX19 strain caused typical clinical signs:sneezing,fever,increased ocular and nasal plasma discharge and dyspnoea and coughing,with a 100%incidence rate.The high-dose test group was characterised by acute infection,with a gradual increase in clinical scores at 2 dpi,a peak at 9 dpi,and severe organ damage with a 100%mortality rate.The low-dose group had a slower onset with an incubation period of about 3 d.The clinical score began to rise at 6 dpi and peaked at 9-13 dpi,causing relatively little organ damage and a mortality rate of 33.3%.The above results establish the basis for subsequent FHV-1 vaccine development.2.Using FHV WX19 as the standard strain,TK deletion gene,g I and g E double deletion gene and TK,g I and g E triple deletion gene strains（FHV△TK m Cherry,FHV△g I/g E e GFP and FHV△g Ig E/TK e GFP-m Cherry）were constructed using CRISPR/Cas9 technology.Firstly,these deletion strains were purified by 10 rounds of null spotting on CRFK cells,and then passed for 20 consecutive generations,and no base mutations were identified by sequencing,demonstrating that the constructed deletion strains all had good genetic stability.Then,the proliferation pattern of the deletion viruses was analysed.The results showed that the TK deletion strain and the g I/g E double deletion strain had the same growth curve as the parental strain,with no significant difference in virus titer(10^7.83TCID₅₀/0.1m L and 10^7.24 TCID₅₀/0.1m L,respectively);while the virus titer of the triple deletion（Δg Ig E/TK）strain was slightly lower(about 10^6.28 TCID₅₀/0.1m L).10^6.28 TCID₅₀/0.1m L).Finally,the three deletion strains were evaluated for safety and immunogenicity.The safety test results showed that the FHV?g I/g E e GFP and FHV?g Ig E/TK e GFP-m Cherry strains were found to show clinical signs in only 50%of cats after infection,with clinical signs maintained for approximately 3 days and no virus excreted,whereas the FHV?TK m Cherry strain showed clinical signs in 100%of cats after infection,with clinical signs maintained for approximately 5 days and live virus The results of the immune efficacy assay showed that the FHV?TK m Cherry infection was not effective.Immunocompetence experiments showed that all FHV-1 deficient strains were able to achieve a neutralising antibody potency of approximately 1:32 or more by two immunisations.Only 50%and 25%of the cats in the FHV?g I/g E e GFP and FHV?g Ig E/TK e GFP-m Cherry groups,respectively,showed mild clinical signs after a strong viral attack.These results indicate that FHV?g I/g E e GFP and FHV?g Ig E/TK e GFP-m Cherry performed well in terms of clinical scoring,morbidity and safety,were free of viral excretion,were effective in reducing clinical signs in young cats,and had good safety and immunogenicity.3.Recombinant feline herpesviruses expressing FCV VP1 and FPV VP2 genes（FHV△g I/g E-FCV VP1 and FHV△g Ig E/TK FCV VP1-m Cherry-FPV VP2）were constructed using virulence gene deletion recombinant viruses as the backbone,respectively.Firstly,the two recombinant strains were purified in CRFK cells after 10 rounds of null spotting and passed for 20 consecutive generations.The recombinant fractions of FCV VP1 and FPV VP2 were identified without base mutations,and both successfully expressed the exogenous protein respectively,indicating that the two recombinant strains were successfully obtained and the recombinant viruses were genetically stable.The growth kinetics of the recombinant viruses were then evaluated and the results showed that the growth curves of the recombinant strains were generally consistent with those of the corresponding parental strains,and the differences in virus titres were not significant(10^7.17 TCID₅₀/0.1 m L and 10^5.83 TCID₅₀/0.1 m L,respectively).Finally,the safety and immunogenicity of the two recombinant viruses were evaluated.The safety test results showed that FHV?g I/g E-FCV VP1 and FHV?g Ig E/TK FCV VP1-m Cherry-FPV VP2 performed better in terms of clinical scores,morbidity and safety in the safety infection test,and there was no viral excretion.The FHV△g I/g E-FCV VP1 test group maintained clinical symptoms for approximately 4 days and the FHV△g Ig E/TK FCV VP1-m Cherry-FPV VP2 test group maintained clinical symptoms for approximately 3 days and all clinical symptoms disappeared at a later stage.The results of the immune potency assay showed that after a single nasal drip immunisation,neutralising antibodies against FHV and FCV were detected in both recombinant virus immunisation groups,and the secretory expression levels of cytokines IL-1β,IFN-α,TNF-α,IL-2,IL-4 and IFN-γwere significantly increased,indicating that both recombinant viruses could induce a strong cellular immune response.After attack with the strong FCV strain,the FHVΔg I/g E-FCV VP1 immune group performed better than the FHVΔg Ig E/TK FCV VP1-m Cherry-FPV VP2 immune group in terms of clinical scores and safety,and the short viral detoxification cycle was effective in reducing clinical signs in the test cats.In summary,the findings of this thesis are as follows:（1）three virulence gene deletion strains were successfully constructed,of which the double and triple deletion strains have good safety and stable transmission and can be used as excellent viral vectors;（2）two recombinant feline herpesviruses expressing FCV VP1 and FPV VP2（FHV?g I/g E-FCV VP1 and FHV?g Ig E/TK FCV VP1）were successfully constructed.g Ig E/TK FCV VP1-m Cherry-FPV VP2).The results of immune efficacy tests showed that both recombinant vaccines were able to induce both good cellular and humoral immune responses and protect cats against highly pathogenic FCV attack.The results of this study provide a solid basis for the development of a live triple vector vaccine against feline herpesvirus,culexvirus and feline microvirus.