KR101361773B1 - Highly replicative and law pathogenicity recombinant influneza virus - Google Patents

Abstract

HA amino acid molecules and NA amino acid sequences associated with fetal high hyperplasia isolated from influenza viruses that are highly proliferative and low pathogenic; Recombinant influenza virus having said HA amino acid sequence coding polynucleotide and NA amino acid sequence coding polynucleotide associated with fetal high proliferation; A vaccine composition comprising the recombinant virus as an active ingredient; Influenza virus diagnostic composition comprising the recombinant virus as an active ingredient; And a method for increasing the fetal proliferation of influenza virus using the HA amino acid sequence and NA amino acid sequence.

Description

Highly Reproducible, Low Pathogenic Recombinant Influenza Virus

The present invention relates to HA amino acid molecules and / or NA amino acid sequences associated with chick embryonic hyperproliferation isolated from chick embryonic hyperproliferative low pathogenic influenza viruses; Recombinant influenza virus having said HA amino acid sequence coding polynucleotide and NA amino acid sequence coding polynucleotide associated with fetal high proliferation; A vaccine composition comprising the recombinant virus as an active ingredient; Influenza virus diagnostic composition comprising the recombinant virus as an active ingredient; And it relates to a method of increasing the fetal proliferation of influenza virus using the HA amino acid sequence and NA amino acid sequence.

Influenza viruses belong to an orthomyxovirus and have a genome of eight negative single-stranded RNA fragments in the genome, from which hemagglutinin (HA), neuraminidase (NA), nu Nucleoprotein (NP), matrix proteins 1 and 2 (matrix, M1, M2), polymerase units A, B1 and B2 (polymerase subunit A, B1 &B2; PA, PB1, PB2, respectively), and nonstructural Proteins 1 and 2 (nonstructural proteins 1 &2; NS1 and NS2, respectively) are made.

In the past, human influenza A vaccine was a unit vaccine in which HA and NA were purified by inactivating the rearrangement strain having excellent fetal proliferation with formalin. Prepared by mixed infection with A / Puerto Rico / 8/34 (PR8). Recent reverse genetics techniques are used to show excellent fetal proliferation and characteristics of PR8 virus, A / WSN / 33 (H1N1) virus, and A / Ann Arbor / 6/60 (H2N2) virus, which have been attenuated by low temperature adaptation. 8 genomes were cloned into a viral genome transcription vector and cloned into a vector expressing the PA, PB1, PB2, and NP coding portions, and 12 plasmids (Patent No. 0908757) were transfected into a cell line such as 293T to produce a desired virus. Alternatively, viral genome transcription occurs in one direction of the vector, and in the other direction, a recombinant virus is produced by trnasfection (patent 0862758) of eight plasmids in which mRNA is produced.

In general, HA and NA genes are amplified by a PCR method from a recent virus, cloned into a reverse genetic vector, transfection with the remaining six genes of PR8 to produce a vaccine strain and inactivated virus to prepare a deadly vaccine.

Influenza viruses are classified into low pathogenic avian influenza and high pathogenic influenza according to pathogenicity to chickens. Highly pathogenic influenza viruses are known as H5 and H7 subtype viruses. Known. In other words, HA1 and HA2 must be cleaved by protease to activate HA protein. If the basic amino acid is repeated at cleavage site, HA protein is produced by furin-like endogenous protease distributed in all organs. Because of this, viral proliferation occurs, and low-pathogenic influenza virus is activated only by exogenous proteases such as trypsin, so it is only activated in the respiratory or digestive organs, causing only local infection.

The highly pathogenic influenza virus is a deadly virus that causes not only significant damage to poultry, but also deaths in human infections. Especially in the case of H5N1 influenza virus, which was directly infected from birds to humans without adaptation in pigs, 18 people in Hong Kong in 1997 due to H5N1. The infection killed six of them, and later began in Asia in late 2003, spreading to Europe and Africa. A total of 566 confirmed cases of infection were reported to WHO from 2003 to October 10, 2011, of which 332 were The death rate is 58.7% and mortality has been reported every year since its first occurrence in Egypt, Indonesia and Cambodia (WHO / GIP).

The development of vaccine strains to cope with the threat of H5N1 virus has been made to attenuate the pathogenicity of the highly pathogenic H5N1 virus and to increase the proliferation in chicks and cell lines. The OIE recommends that the mortality rate of poultry embryos should be less than 10% during 3 days of inoculation in embryonated eggs under conditions suitable for influenza vaccines using poultry. It also means that pathogenicity should be low. Initially, studies have been conducted to develop low-pathogenic H5N1 virus isolated from the field as a deadly vaccine. However, due to its low viral titer and potential risk, reverse genetics techniques have been used to reverse the amino acid sequence of the HA protein cleavage site related to the pathogenicity of high-pathogenic H5N1 virus. Recombinant vaccine strains have been developed to have the remaining internal genes of viruses with good proliferative potential and low pathogenicity in HA and NA of H5N1 viruses substituted with the virus sequences, and in chick fetus cells. Thus produced recombinant viruses are low in pathogenicity, increased proliferative, but sometimes not proliferative, the development of technology for improving the proliferative potential of vaccine strains is required.

Therefore, the present inventors pass a low-proliferative H5N1 avian influenza virus A / Environment / Korea / SNU50-5-E2 / 2010 (H5N1) (hereinafter referred to as SNU50-5-E2) 18 times in embryonated eggs (chicken embryos) to give high proliferation. The H5N1 virus SNU50-5-E20 was established, and the entire genome sequences of SNU50-5-E2 and SNU50-5-E20 were analyzed to identify amino acid sequences showing differences between the two strains. The present invention was completed by establishing an amino acid sequence and an NA amino acid sequence.

Thus, one example of the present invention aims to provide HA amino acid molecules and NA amino acid sequences associated with fetal high proliferation.

Another example aims to provide a recombinant influenza virus having a HA amino acid molecule coding polynucleotide and a NA amino acid sequence coding polynucleotide associated with the fetal high proliferation.

Another example is to provide a vaccine composition against influenza virus comprising the recombinant influenza virus.

Another example is to provide an influenza virus diagnostic composition comprising the recombinant influenza virus as an active ingredient.

Another example aims to provide a method for increasing the fetal proliferation of influenza viruses by introducing HA amino acid molecule coding polynucleotides and NA amino acid sequence coding polynucleotides related to the fetal high proliferation into influenza viruses.

We established A / Environment / Korea / SNU50-5-E2 / 2010 (H5N1) (hereinafter referred to as SNU50), a low-proliferative H5N1 avian influenza virus isolated from wild algae to establish a high-proliferative H5N1 virus in chicks. -5-E2) was passaged 18 times in embryonated eggs to establish the SNU50-5-E20, a hyperproliferative H5N1 virus. To identify mutations associated with fetal hyperproliferation in established fetal high-proliferation SNU50-5-E20, the entire genome sequence was analyzed to identify amino acid sequences that differ between SNU50-5-E2 and SNU50-5-E20. In this study, the HA and NA genes associated with high proliferation of fetuses, which were mutated in SNU50-5-E20, were isolated.

The HA amino acid sequence of high-proliferative SNU50-5-E20 in poultry is amino acid 119 compared to the HA amino acid sequence of the parental strain SNU50-5-E2 (SEQ ID NO: 1), replacing tyrosine (Y) instead of histidine (H). Have glutamic acid (E) instead of lysine (K) with amino acid 177 (K119E) or proline (P) instead of leucine (L) with amino acid 333 (L333P), or amino acid 393 It was confirmed that the lysine (K) instead of arginine (R) (R393K), or two or more amino acid variations of these amino acid variations. In addition, the NA amino acid sequence of the highly proliferative SNU50-5-E20 in the fetus is compared to the NA amino acid sequence of the parent strain SNU50-5-E2 (SEQ ID NO: 17). It was confirmed that it has an (S369N) amino acid variation with).

Accordingly, one embodiment of the present invention provides a HA amino acid molecule associated with high proliferation of fetuses, or a composition for high fetus growth comprising the HA amino acid molecule. The HA amino acid molecule related to the high proliferation of poultry is, in the HA amino acid sequence of influenza virus, histidine (H) is substituted with tyrosine (Y) at position 119 (H119Y), and lysine (K) is glutamic acid at position 177 (E) substitution (K177E), leucine (L) is substituted with proline (P) at position 333 (L333P), and arginine (R) is substituted with lysine (K) at position 393 (R393K). It is characterized in that it comprises one or more amino acid variations selected from the group.

For example, the influenza virus includes all influenza viruses with HA and NA, for example H1N1, H2N1, H3N1, H4N1, H5N1, highly pathogenic H5N1, H6N1, H7N1, H8N1, H9N1, H10N1, H11N1, H12N1, H12N1 , H15N1, and H16N1 influenza virus may be at least one selected from the group consisting of, specifically, A / Environment / Korea / SNU50-5-E2 / 2010 (H5N1) (hereinafter, SNU50-5-E2).

In the case where the HA amino acid molecule associated with high fetality of the present invention is obtained from SNU50-5-E20, which is associated with SNU50-5-E2 18 times, the HA amino acid molecule associated with high fetality of the fetus is SNU50-5- Based on the HA amino acid sequence of E2 (SEQ ID NO: 1), replacing histidine (H) with tyrosine (Y) at position 119 (H119Y), replacing lysine (K) with glutamic acid (E) at position 177 (K177E) ), Replacing leucine (L) with proline (P) at position 333 (L333P), and arginine (R) with lysine (K) at position 393 (R393K). It may be to include a mutation.

Specifically, the HA amino acid molecule associated with fetal high proliferation may be one or more selected from the group consisting of:

The histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) (H119Y; SEQ ID NO: 2);

Lysine (K) at position 177 of SEQ ID NO: 1 substituted with glutamic acid (E) (K177E; SEQ ID NO: 3);

Leucine (L) at position 333 of SEQ ID NO: 1 substituted with proline (P) (L333P; SEQ ID NO: 4);

Arginine (R) at position 393 of SEQ ID NO: 1 substituted with lysine (K) (R393K; SEQ ID NO: 5);

Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) and lysine (K) at position 177 is replaced with glutamic acid (E) (H119Y / K177E; SEQ ID NO: 6);

Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) and leucine (L) at position 333 is replaced with proline (P) (H119Y / L333P; SEQ ID NO: 7);

Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) and arginine (R) at position 393 is substituted with lysine (K) (H119Y / R393K; SEQ ID NO: 8);

Lysine (K) at position 177 of SEQ ID NO: 1 is substituted with glutamic acid (E) and leucine (L) at position 333 is substituted with proline (P) (K177E / L333P; SEQ ID NO: 9);

Lysine (K) at position 177 of SEQ ID NO: 1 is substituted with glutamic acid (E) and arginine (R) at position 393 is substituted with lysine (K) (K177E / R393K; SEQ ID NO: 10);

Leucine (L) at position 333 of SEQ ID NO: 1 is substituted with proline (P) and arginine (R) at position 393 is substituted with lysine (K) (L333P / R393K; SEQ ID NO: 11);

Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y), lysine (K) at position 177 is substituted with glutamic acid (E), and leucine (L) at position 333 is proline (P). Substituted with (H119Y / K177E / L333P; SEQ ID NO: 12);

Histidine (H) at position 119 in SEQ ID NO: 1 is substituted with tyrosine (Y), lysine at position 177 is replaced with glutamic acid (E), and arginine (R) at position 393 is replaced with lysine (K). Substituted (H119Y / K177E / R393K; SEQ ID NO: 13);

Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y), leucine (L) at position 333 is substituted with proline (P), and arginine (R) at position 393 is lysine (K). Substituted with (H119Y / L333P / R393K; SEQ ID NO: 14);

Lysine (K) at position 177 of SEQ ID NO: 1 is substituted with glutamic acid (E), leucine (L) at position 333 is substituted with proline (P), and arginine (R) at position 393 is lysine (K). Substituted with (K177E / L333P / R393K; SEQ ID NO: 15); And

Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y), lysine (K) at position 177 is substituted with glutamic acid (E), and leucine (L) at position 333 is proline (P). Substituted with arginine (R) at position 393 with lysine (K) (H119Y / K177E / L333P / R393K; SEQ ID NO: 16).

Another example provides NA amino acid molecules associated with fetal high proliferation. The NA amino acid molecule associated with high proliferation of fetuses may be one in which serine (S) at position 369 is substituted with asparagine (N) in the NA amino acid sequence of influenza virus (S369N).

For example, the influenza virus includes all influenza viruses having HA and NA, and may be, for example, H5N1 influenza virus, specifically A / Environment / Korea / SNU50-5-E2 / 2010 (H5N1) (hereinafter referred to as SNU50-5). -E2).

When the NA amino acid molecule associated with high fetality of the present invention is obtained from SNU50-5-E20, which is 18 times linked to SNU50-5-E2, the NA amino acid molecule associated with high fetality of the fetus is SNU50-5- Based on the NA amino acid sequence of E2 (SEQ ID NO: 17), the serine (S) at position 369 may be substituted with asparagine (N) (S369N; SEQ ID NO: 18).

HA and / or NA amino acid sequence variations associated with fetal proliferation as described above have typically been identified in H5N1 influenza virus, but are not limited to H5N1 and can be applied to a variety of low-proliferative influenza viruses.

Another embodiment of the present invention provides a composition for enhancing fetal proliferation of influenza viruses comprising the HA amino acid molecule associated with high fetality and / or the NA amino acid molecule associated with high fetality.

In another embodiment of the present invention, in the HA amino acid sequence of influenza virus, histidine (H) is substituted with tyrosine (Y) at position 119 (H119Y), and lysine (K) is substituted with glutamic acid (E) at position 177. (K177E), substituted leucine (L) with proline (P) at position 333 (L333P), and substituted arginine (R) with lysine (K) at position 393 (R393K) An expression vector comprising an HA amino acid coding sequence associated with fetal hyperplasia comprising the above amino acid variation is provided. Another example provides an expression vector comprising an NA amino acid molecular coding gene associated with fetal hyperplasia in which serine (S) at position 369 of the NA amino acid of the influenza virus is substituted with asparagine (N). Another example provides an expression vector comprising the HA amino acid coding sequence associated with fetal high proliferation and an NA amino acid molecular coding gene associated with fetal high growth. In the expression vector of the present invention, the HA amino acid of the influenza virus may have an amino acid sequence of SEQ ID NO: 1, the HA amino acid associated with high proliferation of the fetus has a amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 16 Can be. In addition, the NA amino acid of the influenza virus may have an amino acid sequence of SEQ ID NO: 17, NA amino acid associated with the high proliferation of the fetus may have an amino acid sequence of SEQ ID NO: 18.

In addition, in order to confirm the relationship between the isolated HA gene (eg, the coding genes of SEQ ID NOs: 2 to 16) and NA gene (eg, the coding genes of SEQ ID NO: 18) of the isolated SNU50-5-E20, HA gene of SNU50-5-E2 (coding gene of SEQ ID NO: 1) and NA gene (coding gene of SEQ ID NO: 17), and A / Puerto Rico / 8/34 (H1N1) virus (hereinafter referred to as 'PR8 virus') Separated from a recombinant virus [rPR8-SNU50-5-E2 (HA-NA)] having the remaining six genes of the genes (including NC_002016 to NC_002023 genes), namely PA, PB1, PB2, NP, M, and NS genes. HA genes of SNU50-5-E20 (eg, coding genes of SEQ ID NOs: 2-16) and NA genes (eg, coding genes of SEQ ID NO: 18) and the remaining six genes of PR8 (PA, PB1, PB2, NP, M, and NS genes) for the production of recombinant virus [rPR8-SNU50-5-E20 (HA -NA)] having a comparison of 50% total concentration of fetal infection (EID ₅₀ / ml) And, it was confirmed the proliferative distinct difference between the two recombinant strains.

Therefore, another example of the present invention is that in the influenza virus, HA amino acid sequence is replaced with histidine (H) at position 119 by tyrosine (Y) (H119Y), and lysine (K) at position 177 is glutamic acid (E). Substituted (K177E), substituted leucine (L) at position 333 with proline (P) (L333P), and substituted arginine (R) at position 393 with lysine (K) (R393K). Provided are embryonic high proliferative and low pathogenic influenza virus variant strains comprising one or more amino acid variants. Another example provides a fetal high proliferative and low pathogenic influenza virus variant strain in which serine (S) at position 369 of the NA amino acid sequence is substituted with asparagine (N) for influenza virus (S369N). In another example, the HA amino acid sequence replaces histidine (H) with tyrosine (Y) at position 119 (H119Y) and replaces lysine (K) with glutamic acid (E) at position 177 (K177E), position 333 At least one amino acid variation selected from the group consisting of leucine (L) with proline (P) (L333P), and arginine (R) with lysine (K) at position 393 (R393K), and an NA amino acid Provided is a fetal high proliferative and low pathogenic influenza virus variant strain having an amino acid mutation in which serine (S) at position 369 of the sequence is substituted (S369N) with asparagine (N).

The fetal high proliferative and low pathogenic influenza virus mutant strain may be a recombinant influenza virus transformed with the expression vector described above.

In an embodiment, the influenza variant strain or recombinant influenza virus has a gene encoding the amino acid sequence of any one of SEQ ID NOS: 2-16 as an HA gene, and / or a gene encoding the amino acid sequence of SEQ ID NO: 18 as an NA gene. It may be. In one embodiment, the Genetic Resource Center of Biotechnology Research Institute, Daejeon, Korea, produced a recombinant strain having a gene encoding an amino acid sequence of SEQ ID NO: 16 as an HA gene, and a gene encoding an amino acid sequence of SEQ ID NO: 18 as an NA gene. Was deposited on November 3, 2011, and was given accession number KCTC 12046BP.

As a strain used as a parent strain of a mutant strain or a recombinant strain of the present invention, the PR8 virus, which is an influenza virus of the H1N1 strain, was used, but is not limited thereto, and applied to all influenza viruses such as low pathogenic influenza viruses used as vaccine strains, and HA and And / or by transducing the original HA and / or NA genes using the above-described mutations or by replacing the original HA and / or NA genes with the above-described expression vectors, the low-proliferative problem of chick embryos can be solved. As the parent strain of the mutant strain or recombinant strain, in addition to the PR8 virus, for example, A / WSN / 33 (H1N1) virus or A / Ann Arbor / 6/60 (H2N2), which has been attenuated by low temperature adaptation, may be used. .

In another example, the present invention provides a vaccine composition comprising the high proliferative and low pathogenic influenza virus mutant strain and / or the recombinant influenza virus strain as an active ingredient. The vaccine of the present invention is applicable not only to birds such as chickens and ducks, but also to mammals including pigs, dogs, humans and mice.

Another example of the present invention provides a method of enhancing proliferation of chick embryos by mutating the HA gene and / or NA gene of influenza virus.

The method comprises:

HA amino acid sequence of influenza virus replaces histidine (H) at position 119 with tyrosine (Y) (H119Y), lysine at position 177 (K) with glutamic acid (E) (K177E), and Louis at position 333 The HA gene was modified to include one or more amino acid mutations selected from the group consisting of replacing (L) with proline (P) (L333P) and arginine (R) at position 393 with lysine (K) (R393K). Mutating;

Mutating the NA amino acid sequence of the influenza virus to encode an NA protein having an amino acid variation in which serine (S) at position 369 is substituted with asparagine (N) (S369N); or

It may be to include both of the above steps.

The HA amino acid sequence of the influenza virus may be SEQ ID NO: 1, the NA amino acid sequence of the influenza virus may be SEQ ID NO: 17.

Thus, the method

Replacing the HA gene of the influenza virus with the coding gene of the amino acid sequence of any one of SEQ ID NOs: 2 to 16;

Replacing the NA gene of the influenza virus with the coding gene of the amino acid sequence of SEQ ID NO: 18; or

It may be to include both of the above steps.

Such genetic variation or recombination can increase the fetal proliferation of the influenza virus, thereby further enhancing its usefulness as a vaccine strain.

Another example of the present invention provides a composition for diagnosing influenza virus containing antisera against the high proliferative and low pathogenic influenza virus mutant strain and / or the recombinant influenza virus strain. For example, the fetal high proliferative and low pathogenic influenza virus variant strains and / or recombinant influenza virus strains incorporate the HA and / or NA genes of the fetal high proliferative variant of H5N1 (eg, SNU50-5-E20) into the PR8 influenza virus. It may be a composition for diagnosing influenza virus containing antisera against the rPR8-SNU50-5-E20 (HA-NA), a recombinant virus. Another example provides a kit for diagnosing avian influenza viruses comprising antiserum against the fetal hyperproliferative and low pathogenic influenza virus variant strains and / or recombinant influenza virus strains, such as rPR8-SNU50-5-E20 (HA-NA). do. The antiserum may be obtained from a bird or a mammal, and the patient to be diagnosed may be a bird or a mammal.

The present inventors inoculated the fecal suspension of wild algae in 2010 into the intimal cavity path of 10-day-old SPF embryonated eggs. 5-E2) was isolated and the virus was passaged 18 times in embryonated eggs to establish SNU50-5-E20, a highly proliferative H5N1 virus. As a result of measuring 50% fetal calf infection concentrations of SNU50-5-E2 and SNU50-5-E20, the titer difference was more than 100 times with 10 ^7.9 EID ₅₀ / ml and 10 ¹⁰ EID ₅₀ / ml, respectively. To identify mutations associated with fetal high proliferation, the entire genome sequences of SNU50-5-E2 and SNU50-5-E20 were analyzed to identify the amino acid sequences that differ between the two viruses. Amino acids 119, 177, 333, and 393 were changed from histidine to tyrosine (H119Y), lysine to glutamic acid (K177E), leucine to proline (L333P), arginine to lysine (R393K), and NA The 369 amino acid serine was transformed into asparagine (S369N), and no significant mutations were observed in the remaining genes such as PB2, PB1, NP, NS1, NS2.

In order to confirm whether antigenicity can be maintained by the amino acid mutations identified in the HA amino acid sequence of the mutant having enhanced fetal proliferation as described above, the HA5 amino acids of amino acids 119, 177, and 333 The location on the protein tertiary structure was confirmed (see FIG. 2). As a result, amino acid 177 was exposed at the end of HA protein, amino acid 119 was located at the junction of HA triplex, and amino acid 333 was located inside HA protein monoclonal. It is thought to affect the formation and structural stability of the triple complex and the monostructure tertiary structure rather than directly affecting it, but it has been shown to be a unique variation whose function has not been confirmed so far. That is, the HA amino acid mutations are independent of the epitope sites 190-Helix and 130-Loop, which determine the antigenicity of the HA protein, and are independent of all the amino acid positions known to affect the antigenicity of influenza viruses. As shown in FIG. 3, since the HA amino acid mutation does not affect the tertiary structure of the HA protein, the HA amino acid mutation as described above does not affect the antigenicity of the influenza virus, and the influenza virus whose mutated fetal proliferation is enhanced by the present invention is inherent. Can maintain antigenicity.

In addition, the NA amino acid sequence variation S369N is also a variation whose function has not been reported so far.

RPR8-SNU50-5-E2 (HA-NA) and SNU50-5 with HA and NA of SNU50-5-E2 and the remaining six genes of PR8 to confirm the proliferative relationship between HA and NA genes in embryonated eggs After a to have a -E20 of HA and NA manufactured rPR8-SNU50-5-E20 (HA- NA) with the rest of the PR8 6 genes compared to 50% total concentration of fetal infection (EID ₅₀ / ml) each of ^7.9 10 The difference in titer of 100 times or more was confirmed with EID ₅₀ / ml and 10 ¹⁰ EID ₅₀ / ml, and it was confirmed that the difference in fetal proliferation between SNU50-5-E2 and SNU50-5-E20 was due to HA and NA amino acid variation. Comparing the pathogenicity of -5-E20 and rPR8-SNU50-5-E20 (HA-NA) in chicks, SNU50-5-E20 was able to detect 100% mortality and bleeding of whole chicks within 3 days. Although rPR8-SNU50-5-E20 (HA-NA) was found to have no mortality, it was confirmed to be a viable vaccine line for OIE recommendations.

The present invention provides a recombinant PR8 virus having no or low virulence and excellent immunogenicity in order to solve the problem of pathogenicity in the production of a vaccine using the PR8 virus. Fetal) high productivity, mammalian high immunogenicity, and algae and mammalian pathogenicity, which are useful as vaccines as well as poisoned sods.

FIG. 1 shows wild algae, low-proliferative, low-pathogenic H5N1 virus (SNU50-5-E2), high-proliferative and low-pathogenic H5N1 virus (SNU50-5-E20), and increased virus proliferation by passing the virus for 20 generations in embryonated eggs; 50% fetal infection titer of rPR8-SNU50-5-E2 (HA-NA) and rPR8-SNU50-5-E20 (HA-NA), a PR8 recombinant virus with HA and NA genes It is a graph.
Figure 2 shows the location on the HA protein tertiary structure of the low pathogenic H5N1 virus fetal high proliferation related mutant amino acids.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One: Fetus Hyperproliferative Low pathogenic H5N1  Virus isolation

1-1. Virus isolation and passage

Fecal suspension from wild birds was inoculated in the urinary ganglia pathway of 10-day-old SPF embryonated eggs by A / Environment / Korea / SNU50-5-E2 / 2010 (H5N1), a low-proliferative and low-pathogenic H5N1 avian influenza virus. -E2), which can be easily obtained from Seoul National University Bird Research Center. Diluted SNU50-5-E2 with sterile phosphate buffer solution 1000 times, inoculated 200 μl of three SPF embryonated eggs (Sunrise Co., NY) by the urinary tract route and incubated at 37 ° C. After storage at 4 ° C, the urea was harvested and passaged 18 times in the same manner to establish SNU50-5-E20.

Each of the obtained strains was inoculated with 200 μl of three allantoic cavities of SPF embryonated eggs (Sunrise Co., NY), incubated at 37 ° C. for 3 days, and then harvested allantoic fluid to obtain flat platelets. Agglutination test was performed. 20 μl of the ureter solution and 20 μl of 0.1% chicken erythrocytes extracted from chickens hatched with SPF embryonated eggs (Sunrise Co., NY) were dipped onto a glass plate and mixed and subjected to plate hemagglutination test to store positive samples at −70 ° C. It was used for the experiment.

1.2. virus Potency  Measure

In order to measure the proliferation titer (50% embryo infection dose, EID ₅₀ / ml) in the chicks of the above-described viruses, each recombinant virus was diluted 10 ^-1 to 10 ^-9 with phosphate buffer solution, Five 10-11-day-old SPF embryonated eggs were inoculated with 100 μl of the urinary catheter pathway incubated for 3 days, followed by incubation for 3 days, followed by harvesting of the urinary fluid and confirming hemagglutination by red blood cells of the chickens. (EID ₅₀ / ml) was measured and the results are shown in FIG. As can be seen in Figure 1, the hemagglutination titer of SNU50-5 (E2) was 2 ^7, and the infectious titer of poultry showed 1x10 ⁷ EID ₅₀ / ml, whereas the hemagglutination titer of SNU50-5 (E20) was 2 ^10, and the infectious titer of the fetus was 1x10 ⁹ EID ₅₀ / ml, indicating that the proliferation of the embryo was markedly increased.

1-3. RNA  detach, Reverse transcriptase - Polymerase chain reaction , Sequencing and analysis

For RNA separation, using Viral Gene spin kit (iNtRON Co. Ltd., Seongnam, South Korea) from 150 µl of embryonic egg culture culture solution of SNU50-5-E2 and SNU50-5-E20 viruses obtained in 1-1 above RNA was isolated according to the method provided by QIAGEN's one-step RT-PCR kit and influenza virus genome amplification primers (PB2 amplification primers, Ba-PB2-1, Ba-PB2-2341R; PB1 amplification primers, BmPB1-1, Bm-PB1-2341R; PA amplification primers, Bm-PA-1, Bm-PA-2233R; HA amplification primers, Bm-HA-1, Bm-NS-890R; NP amplification primers, Bm -NP-1, Bm-NP1565R; NA amplification primers, Ba-NA-1, Ba-NA-1413R; M amplification primers, Bm-M-1, Bm-M-1027R; NS amplification primers, Bm- Eight genomes of the virus were amplified using NS-1; Bm-NS-890R; Hoffmann et al., Archives Virol, 2001). 4 μl of 5X one step RT buffer, 4 μl of dNTP (2.5 mM), 1 μl each of forward and reverse primers for each gene amplification (10 pmol / μl), 4 μl of one-step enzyme mix, After reacting at 50 ° C for 30 minutes with a PCR device (C-1000, Bio Rad Laboratories Inc., USA), the reaction mixture was heated at 95 ° C for 15 minutes and then reacted at 94 ° C for 20 seconds, The reaction was repeated 30 times for 30 seconds at -72 ° C for 7 minutes, followed by reaction at 72 ° C for 7 minutes. The obtained amplification product was electrophoresed on an agarose gel and purified using a gel extraction kit (QIAGEN Co., USA), and the base sequence was determined using an ABI3777 automatic base sequence analyzer (Macrogen, Seoul, Korea).

Coding sequences for each genome can be obtained from Bioedit (ver 7.0.5.3; Hall, TA 1999. Bio Bio: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98 / NT Nuc. Acids. Symp. Ser. 41: 95-98.) Was translated into amino acids, and the amino acid difference between SNU50-5-E2 and SNU50-5-E20 by multiplexing was identified and summarized in Table 1.

SNU50-5-E2 and SNU50-5-E20 Virus Amino Acid Sequence Differences SNU50-5 (E2) Location of Amino acid SNU50-5 (E20) HA H 119 Y K 177 E L 333 P R 393 K NA S 369 N

As can be seen in Table 1, the amino acid sequence showing the difference between the two viruses, the HA amino acid sequence, 119, 177, 333, 393 amino acid from histidine to tyrosine (H119Y), lysine to glutamic acid (K177E), leucine to proline (L333P), arginine to lysine (R393K), respectively, and the amino acid sequence of the 337 amino acid serine was found to be asparagine (S369N).

The amino acids 238 (226, H3 numbering) and 240 (228, H3 numbering), which determine avian-to-mammal receptor binding capacity, were amino acids that showed affinity for algal receptors, and the amino acid deficiency in the NA stalk region to balance HA activity It was not observed (Table 2).

* H3 numbering

The positions of amino acids 119, 177, and 333 identified as mutations in HA of SNU50-5-E20 are shown in FIG. 2. As can be seen in Figure 2, amino acid 177 was exposed to the outer end of the HA protein, amino acid 119 is located at each junction of the HA triple complex, amino acid 333 is inside the HA protein unit Because it is located, it is thought to affect the formation and structural stability of the triple complex and monomer tertiary structure rather than directly affecting the binding capacity of the HA protein, but it is a unique variation that has not been confirmed so far. S369N, a variation of NA, is also a variation that has not been reported so far.

Comparing the amino acid sequence of the HA protein, which plays the most important role in the defense of influenza viruses, SNU50-5-E20 is a human-derived H5N1 virus, A / Viet Nam / 1203/2004 (H5N1) (GenBank Accession No. AY651334) and A /. 93% matched with Hong Kong / 213/2003 (H5N1) (GenBank reg. No.EF541401) and 90% matched with A / swine / Korea / C13 / 2008 (H5N2) (GenBank reg. No. Showed more than% homology.

Example  2: SNU50 -5 HA  And NA  Having a gene PR8  Recombinant Virus Construction and Characterization

2-1. Production of recombinant virus

For the production of recombinant influenza virus, Dr. Hoffman's Reverse Genetic Vector System (Patent No. 0862758) was used.

Specifically, HA and NA genes of the SNU50-5-E2 and SNU50-5-E20 viruses amplified in Example 1-2 were cloned into a TOPO-TA cloning vector (Invitrogen Co. USA), and the M13F / M13R primer (M13F, 5'-GTAAAACGACGGCCAG-3 '(SEQ ID NO: 19); M13R, 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 20)) was determined and BsmBI (NEB, Inc, USA) 20 U / 2 μl, 10 X 2 μl of buffer, pHW2000 plasmid (Korean Patent Registration No. 0862758, obtained from the National Veterinary Research and Quarantine Service with the consent of Dr. Webster and Dr. Hoffman from St. Jude Children's Research Hospital), 10 μg / 3 μl, sterile tertiary distilled water 13 μl The mixture was incubated at 50 ° C. for 1.5 hours, followed by electrophoresis on agarose gel, and the DNA was purified by Gel extraction kit (QIAGEN Co.).

top10 competent cell, Invitrogen Co. USA)에 transformation 한 후 염기서열을 분석하여 정확한 클론을 선발하여 플라스미드를 추출하였다. Also T4 DNA ligase (1,000,000 U / ㎕ , NEB Inc, USA) in the vector pHW2000 6㎕ BsmBI was treated in the same manner as described above were purified by electrophoresis on an agarose gel with respect to the NS 2㎕ pHW2000 1㎕, 1 μl of 10 × reaction buffer was added and allowed to stand at room temperature for 2 hours, followed by Escherichia coli , OneShot

top10 competent cell, Invitrogen Co. USA) and the nucleotide sequence was analyzed to select the correct clone and to extract the plasmid.

Six plasmids corresponding to the PB1, PB2, PA, NP, M, and NS genes of PR8 of the Hoffman vector system (pHW191-PB2, pHW192-PB1, pHW193-PA from Dr. Webster of St. Jude Children's Research Hospital) pHW195-NP, pHW197-M, and pHW195-NS, reverse genetics plasmids (Vaccine 2002, 20: 3165-3170; pHW2000 (see Patent No. 10-0862758)) and each virus prepared above (SNU50-5-E2) In order to construct recombinant virus with HA and NA plasmids of SNU50-5-E20 virus, 293T cells (Life Resource Center, KCTC) were loaded with 5% (v / v) FBS in a 6-well cell culture vessel. 2 × ¹⁰ 6/2 ml of MEM (GIBCO BRL) medium was added to each well and added for 3-4 hours, after which the medium was removed and 2 ml of Opti-MEM medium (Invitrogen Co. USA) was added.

All 8 plasmids prepared above were placed in an amount of 300ng each in one 1.5ml tube, the Opti-MEM medium was added to make a final 25µl, and 6µl of plus reagent (Invitrogen Co. USA) was added to another 1.5ml tube. And 69 μl of Opti-MEM medium were mixed and then added to the 1.5 ml tube containing the plasmid, followed by reaction at room temperature for 15 minutes.

While waiting, 4 μl of lipofectamine (Invitrogen Co) and 96 μl of Opti-MEM were mixed and reacted for 15 minutes, and then 100 μl was added to the tube containing the plasmid and further reacted for 15 minutes. The resulting reaction product was added to 100 μl each well containing the 293T cells. 6-well culture vessel was incubated at 5% CO ₂ , 37 ° C. for 20 hours, and then 10 μg (2.5 μg / 4 μl) of trypsin was added per well, and the supernatant was harvested after 24 hours to produce 10-11 days old SPF embryos ( Sunrise Co., NY) was inoculated with 200 ul of the harvested stock solution by the uremic cavity pathway. After the inoculated embryos were incubated at 37 ° C. for 3 days, the ureter was harvested to confirm hemagglutination.

By extracting RNA in the same manner as described above 1-3, amplifying HA and NA genes, confirming the nucleotide sequence, and confirming the specific sequence of SNU50-5-E2 and SNU50-5-E20. Recombinant virus having a coding polynucleotide of SEQ ID NO: 1) and an NA (coding polynucleotide of SEQ ID NO: 17) was encoded by HA of rPR8-SNU50-5-E2 (HA-NA), SNU50-5-E20 (SEQ ID NO: 16). Recombinant virus with polynucleotides) and NA (coding polynucleotide of SEQ ID NO: 18) was named rPR8-SNU50-5-E20 (HA-NA). Among them, rPR8-SNU50-5-E20 (HA-NA) was deposited on November 3, 2011 to the Genetic Resources Center (KCTC), located in Daejeon, and received the accession number KCTC 12046BP.

The hemagglutination titer of these recombinant viruses was measured, and the virus propagated in embryonated eggs by diluting 100-fold was stored at -70 ° C and used for the experiment.

2-2. virus Potency  Measure

In order to measure the proliferative titer (50% embryo infection dose, EID ₅₀ / ml) in the embryos of the recombinant viruses prepared above, each recombinant virus was diluted 10 ^-1 to 10 ^-9 with phosphate buffer solution. Inoculate 100 µl of each 10-11-day-old SPF embryonated egg into the ureteric pathway and incubate for 3 days. After harvesting the urea solution, check for hemagglutination by red blood cells of the chickens, according to the Reed-Muench formula. The result of measuring the titer (EID ₅₀ / ml) is shown in FIG.

As can be seen in Figure 1, the hemagglutination titer of SNU50-5 (E2) was 2 ^7, and the infectious titer of poultry showed 1x10 ^7.1 EID ₅₀ / ml, whereas the hemagglutination titer of SNU50-5 (E20) was 2 ^10, and the infectious titer of poultry was 1x10 ^9.5 EID ₅₀ / ml.

2-3. Fetus  Pathogenic measurement

The recombinant virus was diluted 10 ^-3 with a phosphate buffer solution and inoculated into the umbilical cord at an age of 10 to 11 days in a SPF broth (Sunrise Co., NY) in an amount of 200 쨉 l, cultured at 37 째 C for 3 days, And 3 days after storage at 4 ° C for 12-24 hours. The lesions were observed in the fetuses and the lesions such as hemorrhage or redness. The obtained results are shown in Table 3.

virus Number of development Number of deaths after our inoculation 1 day 2 days 3 days SNU50-5 (E20) 5 3 2 0 rPR8-SNU50-5-E20 (HA-NA) 5 0 0 0

As can be seen in Table 3, SNU50-5 (E20) showed 100% mortality of mortality within 2 days of inoculation, but rPR8-SNU50-5-E20 (HA-NA) all died of mortality for 3 days. It did not occur at all and was evaluated as a vaccine strain that meets OIE recommendations.

Korea Biotechnology Research Institute KCTC12046 20111103

Attach an electronic file to a sequence list

Claims (22)

delete delete delete HA amino acid molecule of the influenza virus, consisting of an amino acid sequence selected from the group consisting of:
The histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) (H119Y; SEQ ID NO: 2);
Lysine (K) at position 177 of SEQ ID NO: 1 substituted with glutamic acid (E) (K177E; SEQ ID NO: 3);
Leucine (L) at position 333 of SEQ ID NO: 1 substituted with proline (P) (L333P; SEQ ID NO: 4);
Arginine (R) at position 393 of SEQ ID NO: 1 substituted with lysine (K) (R393K; SEQ ID NO: 5);
Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) and lysine (K) at position 177 is replaced with glutamic acid (E) (H119Y / K177E; SEQ ID NO: 6);
Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) and leucine (L) at position 333 is replaced with proline (P) (H119Y / L333P; SEQ ID NO: 7);
Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y) and arginine (R) at position 393 is substituted with lysine (K) (H119Y / R393K; SEQ ID NO: 8);
Lysine (K) at position 177 of SEQ ID NO: 1 is substituted with glutamic acid (E) and leucine (L) at position 333 is substituted with proline (P) (K177E / L333P; SEQ ID NO: 9);
Lysine (K) at position 177 of SEQ ID NO: 1 is substituted with glutamic acid (E) and arginine (R) at position 393 is substituted with lysine (K) (K177E / R393K; SEQ ID NO: 10);
Leucine (L) at position 333 of SEQ ID NO: 1 is substituted with proline (P) and arginine (R) at position 393 is substituted with lysine (K) (L333P / R393K; SEQ ID NO: 11);
Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y), lysine (K) at position 177 is substituted with glutamic acid (E), and leucine (L) at position 333 is proline (P). Substituted with (H119Y / K177E / L333P; SEQ ID NO: 12);
Histidine (H) at position 119 in SEQ ID NO: 1 is substituted with tyrosine (Y), lysine at position 177 is replaced with glutamic acid (E), and arginine (R) at position 393 is replaced with lysine (K). Substituted (H119Y / K177E / R393K; SEQ ID NO: 13);
Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y), leucine (L) at position 333 is substituted with proline (P), and arginine (R) at position 393 is lysine (K). Substituted with (H119Y / L333P / R393K; SEQ ID NO: 14);
Lysine (K) at position 177 of SEQ ID NO: 1 is substituted with glutamic acid (E), leucine (L) at position 333 is substituted with proline (P), and arginine (R) at position 393 is lysine (K). Substituted with (K177E / L333P / R393K; SEQ ID NO: 15); And
Histidine (H) at position 119 of SEQ ID NO: 1 is substituted with tyrosine (Y), lysine (K) at position 177 is substituted with glutamic acid (E), and leucine (L) at position 333 is proline (P). Substituted with arginine (R) at position 393 with lysine (K) (H119Y / K177E / L333P / R393K; SEQ ID NO: 16). delete The amino acid molecule of claim 4, wherein the influenza virus is H5N1 influenza virus.
delete delete A composition for enhancing fetal proliferation of influenza viruses, comprising the amino acid molecule of claim 4.
The composition of claim 9, further comprising an amino acid molecule consisting of the amino acid sequence of SEQ ID NO: 18.
An expression vector comprising the coding gene of the amino acid molecule of claim 4.
A fetal high proliferative and low pathogenic influenza virus variant strain comprising the amino acid molecule of claim 4.
The strain of claim 12, wherein the strain further comprises an amino acid molecule consisting of the amino acid sequence of SEQ ID NO: 18. 14.
delete delete The influenza virus variant strain according to claim 13, which is Accession No. KCTC 12046BP.
A vaccine composition against influenza virus, comprising the strain of any one of claims 12, 13 and 16.
delete delete A composition for diagnosing influenza virus containing antisera against a strain of any one of claims 12, 13 and 16.
The diagnostic composition of claim 20, wherein the antisera are derived from birds or mammals.
The diagnostic composition of claim 20, wherein the diagnostic subject is a bird or a mammal.

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