Analysis of Genetic Variation of VP2 Gene in 6 FPV Strainsin China

In order to understand the variation of FPV strains in the Jinan area, Shandong China, the VP2 gene of 6 FPV strains was sequenced, and the analysis of the genetic relationship, evolution and main functional site variation was carried out. It was found that FPV-XY2, FPV-XY3 and FPV-XY6 were the same strain with 100% homology, and also close to FPV-XY1, and the homology between FPV-XY4 and FPV-XY5 was close. The homology between the reference strain and the test strain was over 99.3%. According to the evolutionary analysis, the genetic relationship among FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 was close, and the genetic relationship between FPV-XY4 and FPV-XY5 was close, and the result was similar to the homologous result. Compared with the VP2 amino acid sequence of the standard strain FPV-CU4, the VP2 protein of all the test strains changed from I to t on the 101st Amino acid, this may be the cause of immune failure in these 6 cases; the change of a to s in the 91st amino acid position of FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 may be the cause of enhanced virulence of FPV. This study provides a reference for exploring the epidemic law of FP in the Jinan area, the standard of FP treatment plan and the research and development of FPV subunit vaccine. the nucleotide sequence of standard strain. This study provides a theoretical basis for the prevention and control of FPV in Jinan, and provides a reference for the development of FPV subunit vaccines.

DOI: 10.4236/aim.2021. 114014 192 Advances in Microbiology culture in vitro is very similar to [1]. FPV is a single-stranded linear DNA virus with a diameter of about 25 nm and a full gene length of about 5000 bp. FPV currently reports only one serotype. FPV is mainly composed of two structural proteins and two non-structural proteins. The two structural proteins are VP1 and VP2, and the two non-structural proteins are NS1 and NS2. The VP1 protein is composed of 727 amino acids, and the VP2 protein is composed of 584 amino acids. The VP2 sequence is completely contained in the VP1 sequence [2]. There are 5 key amino acid positions in the VP2 protein, namely the 80th, 103rd, 323, 504, and 568 positions. They are related to the antigenicity, hemagglutination, and host specificity of the virus. The neutralizing antigen site of FPV is also located on the VP2 protein, which can well induce the body to produce antibodies, cause an immune response, and can also be used for preparing vaccines. The FPV-CU4 strain was first reported by Parrish, CR in the United States in 1990, and was later used as a standard strain in vaccine production and antibody production [3]. At present, it has been clinically found that some cats are still infected by FPV even after receiving the vaccine, and some cats have died.
There are opinions that there may be mutations in the current epidemic strains in Jinan. In order to understand the mutation of FPV in the Jinan area, Shandong Province, China, the study selected 6 cases of cats, all of which were received by a chain animal hospital in Jinan who were still infected with FPV after immunization and were treated and nursed by me. Among them, there were 3 dead cases. Stool samples were collected from them and their DNA was extracted. Use the PCR method to sequence its VP2 gene, select reference sequence and standard strain sequence for genetic analysis and genetic evolution analysis, and perform mutation analysis with reference to the nucleotide sequence of standard strain. This study provides a theoretical basis for the prevention and control of FPV in Jinan, and provides a reference for the development of FPV subunit vaccines.

Material
1) Samples among the 24 cases vaccinated with FPV, 6 cases were still infected with FPV after being immunized with the vaccine, and 3 cases died after treatment. As shown in Table 1 Stool samples were taken from these 6 cases and stored in a refrigerator at −20˚C.
2) Reagents: The viral DNA extraction kit was purchased from Tiangen Biotechnology, agarose was purchased from Haibo Biotechnology Co., Ltd., DNA Marker DL2000 was purchased from Takara Company, Premix was purchased from Takara Company, and nucleic acid dye was purchased from Beijing Soleibao Company.  6) Analysis of the main amino acid site variation of the VP2 gene:

3) Main instruments and equipment
The 6 strains of VP2 genome sequences obtained from the sequencing were processed for protein translation with Bioedit software, and the processed amino acid sequence was compared with the VP2 amino acid sequence of the standard strain FPV-CU4 Comparison and analysis of main amino acid positions.

1) Results of PCR product gel electrophoresis:
The PCR product was subjected to 1% agarose gel electrophoresis, and it was found that the amplified fragments of the 6 PCR products were all consistent with the expected amplified fragment size. The size of the target fragment was about 2200 bp, which were named respectively FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY4, FPV-XY5 and FPV-XY6.
2) Homology analysis of VP2 gene Comparing the nucleotide sequence of the VP2 gene of the 6 FPV strains obtained by sequencing with the VP2 reference sequence of the selected FPV, it is found (see Figure 1) that the homology be-

Discussion
In recent years, the rapid development of molecular biology technology has allowed veterinarians to detect the cause of diseases at the genetic level, making the diagnosis results of diseases more accurate and convincing. In this study, 6    From a large number of case investigations and antibody tests, it is found that most of the cats immunized with regular FPV immunization programs will produce higher antibodies, which can effectively reduce the incidence of FPV. Therefore, the Miao Sanduo vaccine of the United States can prevent FPV and play a better role. However, the problem of virus mutation cannot be ignored either.
This experiment provides a certain reference for the improvement and development of FPV monoclonal antibodies and subunit vaccines.