CN112522213B - Separated H3N2 influenza virus strain and application thereof - Google Patents

Abstract

The invention discloses a separated H3N2 influenza virus strain and application thereof, wherein the amino acid sequence of HA protein of the virus is shown as SEQ ID No.2, and the amino acid sequence of NA protein is shown as SEQ ID No. 3. The invention aims to provide a replication-enhanced H3N2 influenza virus CP15, wherein the virus skeleton is completely derived from a parent A/Canine/Jiangsu/06/2010 virus strain, amino acid mutations of K156E and R201K respectively appear only in HA and NA proteins, and a serum antibody of an anti-CP 15 virus HAs certain neutralizing activity on CP15 and a parent JS/10, so that a foundation is laid for applying the virus strain to the prevention and treatment of Canine influenza.

Description

Separated H3N2 influenza virus strain and application thereof

Technical Field

The invention belongs to the technical field of animal virology, and particularly relates to a separated H3N2 influenza virus strain and application thereof.

Background

Influenza A Virus (IAV) has become a significant threat to world public health and global economic development. Influenza viruses are enveloped viruses belonging to the family orthomyxoviridae, single-stranded negative-stranded segmented RNA viruses. The virus is easy to generate genetic variation and evolution, and particularly shows antigen drift and antigen transfer of virus spike glycoprotein Hemagglutinin (HA) and Neuraminidase (NA). Currently, IAVs already have 18 HA subtypes (H1 to H18) and 11 NA subtypes (N1 to N11).

The natural host for IAVs is avian, but certain IAV lineages may infect other different hosts, including humans, pigs, mink, seals, horses, cats, and dogs. Equine derived H3N8 Canine Influenza Virus (CIV) was first reported in 2004 to cause an epidemic of canine respiratory disease in florida. In 2007, avian-derived H3N2 subtype CIV was isolated in korea, and now H3N2 subtype CIV has become the most prevalent subtype in asia. In addition, occasionally, cases of canine infection with other IAV subtypes have been reported, including H5N1, H5N2, H3N1, H1N1, and H6N 1.

In 2009, a new H1N1 virus (pandemic H1N1, pH1N 1) emerged in mexico and the united states and rapidly spread to other countries. Most cases of infection and transmission of the virus occur mainly in humans, but occasionally transmission cases have been reported in animals (e.g., pigs, ferrets, dogs, cats, and turkeys). This spread of pH1N1 beyond its major host barrier raises concerns about the emergence of non-human hosts and recombinant strains of influenza virus. Reports in the literature (Sun et al, 2014.A clinical summary of canine H3N2, pandemic H1N1/09and human seascone H3N2 underfluwenses in dogs in China. Vet. Microbiol.168, 193-196.) indicate that canines may be hosts for human seasonal H3N2 and pH1N1 influenza viruses. Serological investigations carried out in china from 2011 to 2014 show that canines may develop co-infections of pH1N1 and canine H3N2 (canineH 3N2, cH3N 2). Subsequently, 23 strains of the pH1N1 and cH3N2 recombinant viruses naturally co-infected in dogs were identified in korea. All data indicate that dogs have the potential to act as "mixing vessels" for influenza virus.

As a close partner for humans, dogs have many opportunities to come into contact with human IAVs. Therefore, it is necessary to evaluate the possibility of gene mutation or recombination occurring when cH3N2 and pH1N1 influenza viruses are co-infected in Madin-Darby canine kidney (MDCK) cells, which is very necessary for the control of canine influenza and has important significance in public health.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made keeping in mind the above problems and/or problems occurring in the prior art.

The invention aims to provide a replication-enhanced H3N2 influenza virus CP15, wherein the virus skeleton is completely derived from a parent A/Canine/Jiangsu/06/2010 (JS/10) virus strain, only amino acid mutations of K156E and R201K respectively appear in HA and NA proteins, and a serum antibody of an anti-CP 15 virus HAs certain neutralizing activity on the CP15 and the parent JS/10, so that a foundation is laid for applying the virus strain to the prevention and treatment of Canine influenza.

In order to solve the technical problems, the invention provides the following technical scheme: an isolated H3N2 influenza virus strain, wherein the HA protein of the virus HAs an amino acid sequence shown as SEQ ID No.2, and the NA protein HAs an amino acid sequence shown as SEQ ID No. 3.

As a preferred embodiment of the isolated influenza H3N2 virus strain of the present invention, wherein: the homology of the virus and influenza virus A/Canine/Jiangsu/06/2010 is 100% except HA protein and NA protein.

It is another object of the present invention to provide a cell infected with the influenza H3N2 virus as described above.

As a preferred embodiment of the cell of the present invention, there is provided a cell wherein: the cells are canine MDCK cells.

It is another object of the present invention to provide a vaccine comprising the influenza H3N2 virus as described above.

In some preferred embodiments, the vaccine is a water-in-oil emulsion having an aqueous phase and an oil phase.

In some preferred embodiments, the vaccine is an oil-in-water emulsion having an aqueous phase and an oil phase.

The vaccines are administered in a manner compatible with the dosage formulation, and in amounts such as a therapeutically effective amount and an immunogenically effective amount. The amount administered will depend on the subject being treated, the ability of the subject's immune system to synthesize antibodies, and the degree of protection desired. The exact amount of active ingredient to be administered will depend on the judgment of the practitioner, and will vary from individual to individual. Suitable protocols for initial administration and booster inoculations may also vary.

The invention also aims to provide the application of the vaccine in preparing products for preventing and/or treating animal diseases caused by influenza viruses; the influenza virus is an H3N2 influenza virus.

It is another object of the present invention to provide the use of the H3N2 influenza virus strain as described above for the preparation of a vaccine.

The invention also aims to provide application of the H3N2 influenza virus strain in preparing an antigen reagent for influenza diagnosis.

The invention also aims to provide the application of the H3N2 influenza virus strain in preparing a positive serum reagent for influenza diagnosis.

Another object of the present invention is to provide the use of the H3N2 influenza virus strain as described above for the preparation of an antiserum agent for the treatment of influenza.

Above, it will be understood by those skilled in the art that viruses that are substantially genetically identical to the isolated H3N2 influenza virus strain, or that are substantially identical to the isolated H3N2 influenza virus strain in base substitution, or that vaccines comprising viruses that are substantially genetically identical to the isolated H3N2 influenza virus strain are also within the scope of the present application. As used herein, the term "substantially genetically identical" refers to viral particles in which the nucleic acid sequences of their genomes, or the amino acid sequences produced by their genomes, exhibit at least 98% or at least 99% sequence homology.

Compared with the prior art, the invention has the following beneficial effects: the CP15 virus strain of the present invention showed a faster plaque forming ability, and its genome sequencing results showed that the backbone of CP15 was entirely derived from the parent JS/10, but amino acid mutations of K156E and R201K occurred on the HA and NA proteins, respectively. Mice were inoculated intranasally with an equal amount of CP15 or JS/10 virus, and the results of in vivo experiments in mice showed that CP15 also replicates more strongly than JS/10, but the lung lesions produced therefrom were significantly lower than those caused by JS/10. CP15 serum antibodies showed not only neutralizing activity against CP15 in the cross-neutralization assay, but also good neutralizing titers against its parental JS/10. These results indicate that CP15 strains with higher replication capacity are ideal candidates for the development of efficient CIV vaccines.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive labor. Wherein:

FIG. 1 shows the results of the replication kinetics of CP15 and JS/10 in MDCK cells; wherein, (A) is CP15 plaque form; (B) shows a plaque form of JS/10.

FIG. 2 is a comparison of the growth curves for CP15 and JS/10.

FIG. 3 shows the body weight changes of JS/10 and CP15 infected mice, respectively.

FIG. 4 shows the viral load in the major organs and fecal samples at 6dpi and 8dpi after JS/10 and CP15 infection, respectively, in mice; wherein (A) is the viral load at 6 dpi; (B) viral load at 8 dpi.

FIG. 5 is a graph of TNF-. Alpha.and IFN-. Gamma.mRNA transcript levels in lung tissue following CP15 and JS/10 infection in mice; wherein (A) is the transcription level of TNF-alpha mRNA; (B) IFN-. Gamma.mRNA transcript levels.

FIG. 6 is gross pathology and histopathology results of mouse lung tissue at 6dpi after CP15 and JS/10 infection in mice; wherein (A) is gross pathology in JS/10 infected group, (B) is gross pathology in CP15 infected group, (C) is gross pathology in PBS group; (D) histopathological results for the JS/10 infected group; (E) histopathological results for the CP15 infected group; (F) histopathological results for the PBS group.

FIG. 7 is a comparison of the lung injury scores of the JS/10 infected group, CP15 infected group, and PBS group in FIG. 6.

FIG. 8 is the results of measurement of HI antibody titers of serum antibodies at days 2, 4, 6, 8, 10 and 14 after CP15 and JS/10 infection of mice.

FIG. 9 shows the results of measurement of neutralizing antibody titers at day 14 after CP15 and JS/10 infection in mice.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The following examples employ strains and cells as follows:

H3N2 subtype Canine influenza strains cH3N2, A/Canine/Jiangsu/06/2010 (JS/10), isolated and stored by the laboratory, genBank accession numbers JN247616 to JN247623.

Epidemic influenza H1N1 virus, A/Jiangsu/1/2009 (pH 1N 1/09), was supplied by the Jiangsu province disease prevention and control center with GenBank accession numbers ACU00159 through ACU00166 and ACS34705.

Canine kidney cells (MDCK) were purchased from ATCC in the USA and stored in the laboratory.

Example 1

(1) Co-infection plaque formation assay

MDCK cells were cultured as monolayers in T25 cell flasks. JS/10 and pH1N1/09 were inoculated to the cells at a multiplicity of infection (MOI) of 0.01, and 1h after infection, the cells were washed 3 times with Phosphate Buffer Saline (PBS),culture was continued for 72h with serum-free DMEM. The newly produced virus in the culture supernatant was screened and purified by a plaque formation assay using MDCK cells. In general, the supernatant is diluted in 10-fold gradient and MDCK cells are seeded after dilution. After 1h of virus adsorption at 37 ℃, the cells were washed three times with PBS, covered with DMEM (Sigma, st. Louis, USA) containing 1% low melting agarose, inverted and placed in 5% co₂Culturing at 37 deg.C for 72h in incubator to form plaques after virus replication, selecting single plaques (progeny virus) and purifying twice.

(2) Sequencing analysis of progeny viruses

Viral RNA was extracted from infected MDCK cell culture supernatants using a viral nucleic acid extraction kit (Geneaid, taiwan, china). The cDNA was reverse transcribed using the PrimeScriptTM 1st Strand cDNA Synthesis Kit (TaKaRa, dalian, china) using the Uni12 primer, cDNA was obtained by PCR amplification of HA, NA, M, NP, NS, PA, PB1, and PB2 genes using specific primers for the eight gene fragments of influenza virus designed according to the literature (Hoffmann et al, 2001.Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol.146, 2275-2289.), PCR products were purified by gel extraction Kit (Omega, norcross, USA), cloned into pMD19-T vector (TaKaRa), screened by resistance plates, positive cloned into STAR, and nucleotide and amino acid sequences were compared by DNA 7.0 software.

The Uni12 primer sequence is as follows:

Uni12：5’-AGCAAAAGCAGG-3’，SEQ ID NO.1。

JS/10 and pH1N1/09 are infected together for 72h, MDCK cell culture supernatant is collected for plaque forming test, 24 progeny virus strains CP 1-CP 24 are selected, and HA and NA genes are amplified and identified. Sequence analysis indicated that all strains were of the H3N2 subtype. A progeny virus named CP15 grows faster than other synchronously cultured viruses, larger plaques are formed, sequence analysis shows that 6 genes (M, NP, NS, PA, PB1 and PB 2) of CP15 have 100% of homology with JS/10, but two amino acid changes of mutation from K (Lys) to E (Glu) at 156 th position of HA protein receptor binding site and mutation from R (Arg) at 201 th position of NA protein antigen binding site to K (Lys) are generated.

The HA protein amino acid sequence of the virus is shown as SEQ ID No.2, and the NA protein amino acid sequence of the virus is shown as SEQ ID No. 3.

Example 2

Growth kinetics of viruses on MDCK cells

MDCK cells were seeded in 12-well plates to form monolayers, and selected progeny viruses were seeded onto the cells at an MOI of 0.01. After virus adsorption at 37 ℃ for 1h, cells were washed 3 times with PBS, cultured in serum-free DMEM containing TPCK-treated trypsin (Sigma), and culture supernatants were harvested at 12h, 24h, 36h, 48h, 60h, and 72h post-infection, respectively. The number of virions in the supernatant was determined by the plaque formation assay described above, and the viral titer was expressed as plaque-forming units (PFU).

To determine whether the CP15 virus replicated faster than its parental strain JS/10, we tested two strains of virus for their ability to grow in MDCK cells by plaque assay. Inoculating virus with MOI of 0.01 to MDCK cells, infecting for 1h, discarding infection solution, covering with mixed solution of 2 × DMEM and 2% agar, culturing for 36h to observe the plaque formation of two strains of virus, fixing cells with formaldehyde, and staining with 1% crystal violet. Culture supernatants were collected 12, 24, 36, 48, 60, 72h post-infection and virus replication kinetics were determined by plaque formation assay and expressed as log10 PFU/mL. The results are shown in FIGS. 1 and 2. FIG. 1 shows the results of the replication kinetics of CP15 and JS/10 in MDCK cells; wherein, (A) is CP15 plaque form; (B) shows a plaque form of JS/10. Fig. 2 compares the growth curves for CP15 and JS/10, P <0.05, illustrating the significant difference between the CP15 and JS/10 groups.

As shown in fig. 1, CP15 has a remarkably improved plaque-forming ability as compared to JS/10. At each time point of 12-72 h after infection, the virus content of CP15 is slightly higher than that of JS/10, especially at 36h, and the difference reaches a remarkable level (P)<0.05 As shown in fig. 2). At this time, CP15 and JS/10 viral titers almost peaked, the former (10)^7.75PFU/mL) is the latter (10)^6.75PFU/mL).

Example 3

Passage of progeny virus and analysis of genetic characteristics

The selected progeny virus is continuously passaged in MDCK cells for 20 times to verify the genetic stability. The Hemagglutination (HA) assay was used to detect the presence of influenza virus in the cell culture supernatant harvested each generation, in triplicate for each sample. And selecting the 5 th (P5), 10 th (P10) and 20 th (P20) generation viruses and the primary CP15 (P0) for genetic stability analysis. Extracting virus RNA in cell culture supernatant by using a virus nucleic acid extraction kit (Geneaid), carrying out cDNA synthesis and fragment amplification on the 8 gene fragments by using an RT-PCR method, purifying PCR products by using an agarose gel purification kit (TaKaRa), cloning the PCR products into a pMD19-T vector (TaKaRa), and carrying out sequencing analysis. Meanwhile, the P0, P5, P10 and P20 viruses were inoculated at an MOI of 0.01 to MDCK cells, and virus proliferation and titer were determined using a plaque formation assay.

CP15 virus was serially passaged in MDCK cells for 20 times, and the passaged virus markers were P1-P20. HA activity and plaque-forming ability on MDCK cells were examined for P0, P5, P10 and P20 viruses. The results showed that the HA titers of the four viruses were 2⁹、2⁹、2⁸And 2⁹(ii) a The virus titer was 1.36X 10 respectively⁸、9.56×10⁷、9.57×10⁷And 1.21X 10⁸PFU/mL. These data indicate that the mutant virus maintains stable replication capacity in MDCK cells after passage. Meanwhile, sequence analysis confirmed that no mutation occurred during passage, indicating that CP15 virus had genetic stability in MDCK cells for at least 20 serial passages.

Example 4

(1) Animal testing

To further determine whether there was a difference in pathogenicity and replicability between the selected progeny virus and its parent strain JS/10, we performed challenge experiments on mice. Prior to the development of this study, we obtained approval by the animal ethics Committee of Nanjing university of agriculture [ license number: SYXK (SU)]All animal experiments were performed following the guidelines of the chinese animal welfare committee. BALB/c mice (18-25g, 48 days old, female) were purchased from the animal laboratories, yangzhou university.75 mice were randomly divided into 3 groups (CP 15 group, JS/10 group and PBS group), and 25 mice were each group. Two groups of virus (CP 15 and JS/10 group) mice were vaccinated nasally with 70 μ L of 10 titer⁷TCID₅₀Reference is made to the literature (Xie et al, monoclonal antibody specific to HA2 glycooptide protectants from H3N2 underfluenza virus infection. Ve. Res.46, 33.), and control (PBS) mice are inoculated with the same volume of PBS via the nasal cavity. Body weight and clinical symptoms were recorded for at least 14 days. On days 6 and 8 post viral infection, 5 mice per group were randomly selected for euthanasia. Samples of heart, liver, spleen, lung, kidney, brain, intestine, etc. are collected, and feces are collected. The tissue or feces was homogenized with PBS at a ratio of 1 (g/mL), centrifuged, and the supernatant was collected to extract viral RNA. And (3) detecting virus RNA by nested PCR (polymerase chain reaction), and representing tissue distribution and feces detoxification conditions. Two pairs of conserved primers for amplifying the M gene of influenza A virus were designed according to the literature (Ellis and Zambon,2001.Combined PCR-heterologous specificity for detection and differentiation of infection of influenza A viruses from differential and specific species, J. Clin. Microbiol.39, 4097-4102.).

The primer sequences used were as follows:

first round PCR primer

sense-primer F1:5’-CCGTCAGGCCCCCTCAAAGC-3’，SEQ ID No.4；

Antisense primer R1:5 'AGGCGATCAAGAATCCACAAA-3' and SEQ ID No.5.

Second round primer

sense-primer F2:5’-GTGCCCAGTGAGCGAGGAC-3’，SEQ ID No.6；

Antisense primer R2:5 'ATCTCCAT GGCCTCTGCT-3', SEQ ID No.7.

The weight change of the JS/10 and CP15 infected mice, respectively, was monitored, and FIG. 3 is the weight change of the JS/10 and CP15 infected mice, respectively, and the percentage of weight change at the indicated time points was calculated relative to the initial weight.

Test results show that the CP15 and JS/10 mice have clinical symptoms of depression, decreased activity, messy hair and the like on day 2 after virus infection, and the JS/10 mice are more obvious than the CP15 mice. The JS/10 infected mice lost weight to a higher extent compared to the CP15 group, especially in the range of 2-8 dpi (P <0.001 or P < 0.05), with no significant difference between the CP15 group and the PBS group.

Notably, the two strains showed some differences in mouse stool detoxification and tissue distribution. At 6dpi, the CP15 infected mice with lung (5/5), spleen (5/5), heart (5/5), brain (5/5), kidney (5/5), liver (5/5), intestine (5/5) and feces (4/5) virus RNA has the highest positive rate. In JS/10 infected mice, viral RNA was detected in lung (5/5), spleen (2/5), heart (2/5), brain (3/5), kidney (3/5), liver (4/5) and intestine (4/5). At 8dpi, mice vaccinated with CP15 or JS/10 were able to detect viral RNA only in a few tissues, except the gut and feces. The number of viral RNA positive organs was greatly reduced at 8dpi compared to 6dpi, and the data are shown in Table 1. The control group did not detect viral RNA in all organs and feces.

TABLE 1 detection of viral RNA in tissue and fecal samples collected from CP15 or JS/10 vaccinated mice

(2) Real-time fluorescent quantitative PCR (qRT-PCR) for detecting virus load

And (3) detecting the viral RNA load by adopting an absolute quantitative qRT-PCR method. Total RNA from the above samples was reverse transcribed into cDNA using Uni12 primers. Primers were designed for specific regions of the M gene as follows:

M-F：5’-TCTATCGTCCCATCAGGC-3’，SEQ ID No.8；

M-R：5’-GGTCTTGTC TTTAGCCATTC-3’，SEQ ID No.9；

and (3) probe: 5-.

Plasmid standards were prepared using pMD19-T vector (TaKaRa) containing the corresponding target gene M of the virus, the initial concentration of the plasmid standards was adjusted to 100 ng/. Mu.L, and 10 g/. Mu.L with deionized water^-4～10^-1110-fold serial dilutions were performed with cDNA samples for fluorescent quantitative detection using AceQ qPCR probe Master Mix (Vazyme, nanjing, china). Drawing a standard curve to obtain the difference between the cycle threshold (Ct) and the sample concentrationThe sample copy number is calculated according to the formula, the copy number/g tissue (copy number/g) = [6.02 x 10 ]²³X (concentration. Times.10)^-6ng/μL)]/(full DNA length. Times.660).

To determine whether there was a difference in the replication capacity of CP15 and JS/10 in vivo, we examined the RNA load of the virus in tissues and feces of infected mice at two time points of 6 and 8 dpi. Each group was euthanized by randomly selecting 5 mice. Viral load in log per gram of sample₁₀RNA copy number. The results are shown in FIG. 4, which is the viral load in the major organs and fecal samples at 6dpi and 8dpi after the mice were infected with JS/10 and CP15, respectively; wherein (A) is the viral load at 6 dpi; (B) viral load at 8 dpi. On day 6 after challenge, the heart, liver, lung, and brain viral loads were essentially the same for CP15 and JS/10 mice, approximately 10^5.5The copy/g, small intestine and fecal virus titers were low, about 10 each^5.0Copy/g and 10^4.8Copy/g. While in spleen and kidney tissues, the RNA load was significantly higher in CP15 group than in JS/10 group (P)<0.05). At day 8 after challenge, viral RNA loads were reduced in both groups of mouse tissues compared to day 6. RNA loads were essentially identical, approximately 10, in heart, spleen, kidney and brain tissue^4.8Copy/g. However, in lung tissue, the RNA load was significantly higher in CP15 group than in JS/10 group (P)<0.05)。

Example 5

Cytokine assays

To assess the expression level of inflammatory cytokines in the lungs of challenge mice, we collected lung tissue from three groups of mice on days 2, 4, 6, 10 and 14 post-infection, respectively. 1g of fresh tissue homogenate is placed in 1ml of TRK lysis buffer (Omega), 3 000g of the homogenate is centrifuged for 5min, the supernatant is collected, total RNA is extracted by total RNA Kit I (Omega), cDNA is obtained by reverse transcription of PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa), the mRNA levels of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) are detected by qRT-PCR, and beta-actin housekeeping gene is used as an internal reference. qRT-PCR was performed using SYBR Premix Ex TaqTM kit (Vazyme) using the comparative cycle threshold method (2)^-△△CT) mRNA levels were analyzed, reference (Livak and Schmitgen, 2001.Analysis of relat)ive gene expression data usingreal-time quantitative PCR and the 2(T)(-Delta Delta C)method.Methods.25,402-408.)。

The primer sequences are as follows:

TNF-α-F：5’-AAGCCTGTAGCCCACGTCGTA-3’，SEQ ID No.11；

TNF-α-R：5’-GGCACCACTAGTTGGTTGTCTTTG-3’，SEQ ID No.12；

IFN-γ-F：5’-CGGCACAGTCATTGAAAGCCTA-3’，SEQ ID No.13；

IFN-γ-R：5’-GTTGCTGATGGCCTGATTGTC-3’，SEQ ID No.14；

β-actin-F：5’-TGACAGGATGCAGAAGGAGA-3’，SEQ ID No.15；

β-actin-R：5’-GCTGGAAGGTGGACAGTGAG-3’，SEQ ID No.16。

to evaluate the effect of two strains of virus on the production of inflammatory cytokines in mice, we examined the levels of TNF-. Alpha.and IFN-. Gamma.transcription in lung tissue. FIG. 5 is the levels of TNF- α and IFN- γ mRNA transcription in lung tissue following CP15 and JS/10 infection in mice; wherein (A) is the transcription level of TNF-alpha mRNA; (B) IFN-. Gamma.mRNA transcript levels. By using a comparison cyclic threshold method (2)^-△△CT) The mRNA level is measured. * P<0.05，**P<0.01, or<0.001 means that the difference between groups was statistically significant. As shown in FIG. 5 (A), JS/10 and CP15 infection both resulted in significantly elevated TNF- α mRNA levels compared to the PBS group. On day 6 after infection, the mRNA expression level of TNF-. Alpha.was significantly higher in JS/10 group than in CP15 group (P)<0.001). IFN-gamma mRNA expression levels were also significantly elevated following infection with both viruses. As shown in FIG. 5 (B), on days 4 and 6 after infection, the expression level of IFN-. Gamma.in CP15 group was significantly higher than that in JS/10 group (P)<0.01)。

Example 6

Histopathological analysis

The lung lesions of the mice are most obvious 6d after virus infection in the process of dissecting the mice, so that lung pathological sections with 6dpi are prepared. The sections were fixed overnight in 10% neutral formalin, paraffin-embedded after fixation, sectioned, stained with hematoxylin and eosin, and histopathological examination of the sections was performed. Lesion scores were classified on a scale of 0 to 3 according to pathological features, as described in the literature (Alymova et al, 2011.Immunopathogenic and antibacterial effects of H3N2 underfluenza A viruses PB1-F2 map to amino acid residues 62,75,79, and 82.J. Virol.85, 12324-12333.).

After the mice are attacked by the virus, the lung lesions of the mice are most obvious on the 6 th day after the virus infection in the process of the dissection, and pathological sections are prepared for the lung on the day. FIG. 6 is gross pathology and histopathology results of mouse lung tissue at 6dpi after CP15 and JS/10 infection in mice; wherein (A) is gross pathology in JS/10 infected group, (B) is gross pathology in CP15 infected group, (C) is gross pathology in PBS group; (D) histopathological results for the JS/10 infected group; (E) histopathological results for the CP15 infected group; (F) histopathological results for the PBS group; the image magnifications of (D), (E) and (F) were 100.

Pathological results show that: JS/10 infected mice showed severe right lung lobe materialization as shown in fig. 6A. In contrast to the JS/10 group, mice infected with CP15 developed slight macroscopic lesions with small lung bleeds and little edema, as shown in FIG. 6B. The surfaces of the lung lobes of the PBS group were smooth and moist with no signs of disease, as shown in fig. 6C. Histopathological examination of the JS/10 group showed alveolar structure destruction, alveolar septal thickening, and inflammatory cell infiltration in the lungs, as shown in FIG. 6D. The alveolar structure of the CP15 group was intact, but inflammatory cells and erythrocyte infiltration were clearly visible in the alveoli, as shown in FIG. 6E. The lung tissue and alveolar structure of the mice in the PBS group were normal, and there were no inflammatory cells and no infiltration of erythrocytes in the alveoli, as shown in FIG. 6F.

Fig. 7 compares lung injury scores for JS/10-infected, CP 15-infected, and PBS groups, with P <0.05, or P <0.001 indicating that there was statistical significance of the differences between the groups. The lung lesion scores of the two groups of virus mice were significantly increased compared to the PBS group (P <0.001 or P < 0.05), and the lung lesion score of the CP15 group was significantly lower than that of the JS/10 group (P < 0.05).

Example 7

To assess the level of antibody produced in mice after infection with the virus and to determine whether the antibodies have cross-protective effects, we performed a Hemagglutination Inhibition (HI) assay and a neutralization assay.

HI test method: serum samples diluted 2-fold in series were incubated with an equal volume of 4 hemagglutinated units of virus in 96-well microtiter plates at 37 ℃ for 30 minutes. Then, freshly prepared 1% chicken red blood cells were added to the test wells, mixed well and incubated at 37 ℃ for 30 minutes. HI titers were expressed as the reciprocal log2 of the highest dilution of serum when erythrocytes were not agglutinated at all.

Micro neutralization test: according to the HI results, the serum sample on day 14 with the highest HI titer was diluted continuously 10 times and then mixed with an equal volume of 200TCID₅₀The virus of (4) was incubated at 37 ℃ for 1h. The mixture was added to MDCK cells and cultured at 37 ℃ for 72h. The neutralization titer was expressed as the reciprocal of the highest dilution serum with 50% neutralization of CP15 or JS/10 virus in MDCK cells.

Serum was collected every 2 days after the mice were infected with CP15 and JS/10 via nasal cavity and the HI titer thereof was determined. FIG. 8 is a graph showing the results of measurement of HI antibody titers of serum antibodies at days 2, 4, 6, 8, 10 and 14 after CP15 and JS/10 infection of mice, which are expressed as the reciprocal log2 of the highest dilution factor of serum antibodies when hen erythrocytes (CRBC) were detected to be completely non-agglutinated. FIG. 9 is a graph showing the results of measurement of neutralizing antibody titer at day 14 after CP15 and JS/10 infection of mice, the neutralizing titer being expressed as the reciprocal of the highest dilution serum with 50% neutralization rate of CP15 or JS/10 virus in MDCK cells. * P <0.05, P <0.01 or P <0.001 indicates a significant difference between the groups.

As shown in FIG. 8, HI titers of two virus serum antibodies are gradually increased from 2 to 14dpi, and HI levels of the 8 to 14dpi and CP15 group antibodies are significantly higher than those of the JS/10 group (P <0.001 or P < 0.05). To determine whether the antisera produced after infection of the mice with the virus could neutralize the virus infection in MDCK cells, day 14 serum antibodies were subjected to a cell microneutralization assay. Our data show that there is no significant difference in the level of neutralizing antibodies for specific antisera against different strains, but there is a significant difference in the neutralizing titers of different serum antibodies against the same strain (P <0.01 or P < 0.001), as shown in figure 9. Serum antibodies produced by the mice in CP15 group had stronger neutralizing properties than those of JS/10 group.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

<110> Nanjing university of agriculture

<120> an isolated H3N2 influenza virus strain and uses thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 3

<211> 12

<212> DNA

<213> Uni12 primer (Artificial Sequence)

<400> 3

agcaaaagca gg 12

<210> 2

<211> 550

<212> PRT

<213> HA protein of CP15 (Artificial Sequence)

<400> 2

Gln Asn Leu Pro Gly Asn Glu Asn Asn Ala Ala Thr Leu Cys Leu Gly

1 5 10 15

His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asp Asp

20 25 30

Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Asn Ser Ser Thr

35 40 45

Gly Lys Ile Cys Asn Asn Pro His Lys Ile Leu Asp Gly Arg Asp Cys

50 55 60

Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Val Phe Gln

65 70 75 80

Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Asn Ala Phe Ser Asn

85 90 95

Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val

100 105 110

Thr Ser Ser Gly Thr Leu Glu Phe Ile Thr Glu Gly Phe Thr Trp Ala

115 120 125

Gly Val Thr Gln Asn Gly Gly Ser Gly Ala Cys Lys Arg Gly Pro Ala

130 135 140

Asn Gly Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Thr

145 150 155 160

Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asn Asn Phe Asp Lys

165 170 175

Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asn Gln Glu Gln Thr

180 185 190

Ser Leu Tyr Ile Gln Ala Ser Gly Arg Val Thr Val Ser Thr Arg Arg

195 200 205

Ser Lys Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg

210 215 220

Gly Gln Ser Gly Arg Ile Ser Val Tyr Trp Thr Ile Val Lys Pro Gly

225 230 235 240

Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly

245 250 255

Tyr Phe Lys Met His Ile Gly Lys Ser Ser Ile Met Arg Ser Asp Ala

260 265 270

Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile

275 280 285

Pro Asn Glu Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala

290 295 300

Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met

305 310 315 320

Arg Asn Val Pro Glu Arg Gln Thr Arg Gly Leu Leu Gly Ala Ile Ala

325 330 335

Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly

340 345 350

Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys

355 360 365

Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val

370 375 380

Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser

385 390 395 400

Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Arg Tyr Val Glu Asp Thr

405 410 415

Lys Val Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu

420 425 430

Asn Gln Asn Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe

435 440 445

Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn

450 455 460

Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser

465 470 475 480

Ile Arg Asn Gly Thr Tyr Asp His Asn Ile Tyr Arg Asp Glu Ala Val

485 490 495

Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys

500 505 510

Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys

515 520 525

Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Arg Gly Asn Ile

530 535 540

Arg Cys Asn Ile Cys Ile

545 550

<210> 3

<211> 471

<212> PRT

<213> NA protein of CP15 (Artificial Sequence)

<400> 3

Met Asn Pro Asn Gln Lys Ile Ile Ala Ile Gly Ser Val Ser Leu Thr

1 5 10 15

Ile Ala Thr Val Cys Phe Leu Leu Gln Ile Ala Ile Leu Ala Thr Thr

20 25 30

Val Thr Leu Tyr Phe Lys Gln Asn Glu Cys Asn Ile Pro Ser Asn Ser

35 40 45

Gln Val Val Pro Tyr Lys Pro Ile Ile Ile Glu Arg Asn Ile Thr Glu

50 55 60

Val Val Tyr Leu Asn Asn Thr Thr Ile Glu Lys Glu Lys Glu Ile Cys

65 70 75 80

Ser Val Val Leu Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Gln Ile

85 90 95

Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala

100 105 110

Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro

115 120 125

Ser Lys Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asn Asn

130 135 140

Lys His Ser Asn Gly Thr Ile His Asp Arg Ile Ser His Arg Thr Leu

145 150 155 160

Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val

165 170 175

Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu

180 185 190

His Val Cys Val Thr Gly Asp Asp Lys Asn Ala Thr Ala Ser Phe Val

195 200 205

Tyr Asn Gly Met Leu Val Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile

210 215 220

Leu Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr

225 230 235 240

Val Val Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile

245 250 255

Leu Phe Ile Arg Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly

260 265 270

Ser Ala Gln His Ile Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Asn

275 280 285

Val Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val

290 295 300

Ile Asp Ile Asn Met Ala Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys

305 310 315 320

Ser Gly Leu Val Gly Asp Thr Pro Arg Asn Asp Asp Ser Ser Ser Ser

325 330 335

Ser Asn Cys Arg Asp Pro Asn Asn Glu Arg Gly Asn Pro Gly Val Lys

340 345 350

Gly Trp Ala Phe Asp Asn Glu Asn Asp Val Trp Met Gly Arg Thr Ile

355 360 365

Ser Lys Asp Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly

370 375 380

Trp Thr Thr Ala Asn Ser Lys Ser Gln Val Asn Arg Gln Val Ile Val

385 390 395 400

Asp Asn Asn Asn Trp Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly

405 410 415

Lys Ser Cys Val Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Gly

420 425 430

Pro Gln Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe

435 440 445

Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala

450 455 460

Asn Ile Asn Phe Met Pro Ile

465 470

<210> 4

<211> 20

<212> DNA

<213> sense-primer F1(Artificial Sequence)

<400> 4

ccgtcaggcc ccctcaaagc 20

<210> 5

<211> 20

<212> DNA

<213> antisense primer R1 (Artificial Sequence)

<400> 5

aggcgatcaa gaatccacaa 20

<210> 6

<211> 19

<212> DNA

<213> sense-primer F2(Artificial Sequence)

<400> 6

gtgcccagtg agcgaggac 19

<210> 7

<211> 18

<212> DNA

<213> antisense primer R2 (Artificial Sequence)

<400> 7

atctccatgg cctctgct 18

<210> 8

<211> 18

<212> DNA

<213> M-F(Artificial Sequence)

<400> 8

tctatcgtcc catcaggc 18

<210> 9

<211> 20

<212> DNA

<213> M-R(Artificial Sequence)

<400> 9

ggtcttgtct ttagccattc 20

<210> 10

<211> 24

<212> DNA

<213> Probe (Artificial Sequence)

<400> 10

tgcagtcctc gctcactggg cacg 24

<210> 11

<211> 21

<212> DNA

<213> TNF-α- F(Artificial Sequence)

<400> 11

aagcctgtag cccacgtcgt a 21

<210> 12

<211> 24

<212> DNA

<213> TNF-α-R(Artificial Sequence)

<400> 12

ggcaccacta gttggttgtc tttg 24

<210> 13

<211> 22

<212> DNA

<213> IFN-γ-F(Artificial Sequence)

<400> 13

cggcacagtc attgaaagcc ta 22

<210> 14

<211> 21

<212> DNA

<213> IFN-γ-R(Artificial Sequence)

<400> 14

gttgctgatg gcctgattgt c 21

<210> 15

<211> 20

<212> DNA

<213> β-actin-F(Artificial Sequence)

<400> 15

tgacaggatg cagaaggaga 20

<210> 16

<211> 20

<212> DNA

<213> β-actin-R(Artificial Sequence)

<400> 16

gctggaaggt ggacagtgag 20

Claims (2)

1.An HA protein of an H3N2 influenza virus, characterized in that: the amino acid sequence of the HA protein is shown in SEQ ID No. 2.

2. An NA protein of H3N2 influenza virus, which is characterized in that: the amino acid sequence of the NA protein is shown as SEQ ID No. 3.

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