It consists of 10 double-stranded RNA fragments of different sizes, three large ones are L 1 ~ L3, three are M4 ~ M6, and four small ones are S7 ~ S 10, with the code of 10 protein (VP 1 ~ VP7 and V7). Underwear shell consists of two major proteins VP3 and VP7 and three minor proteins VP 1, VP4 and VP6, among which VP6 is considered as the helicase of virus dsRNA [2]. The outer shell consists of two kinds of protein, VP2 and VP5, which will be removed when the virus passes through the cell membrane. In addition, there are at least three nonstructural proteins (NS 1, NS2 and NS3/3A) in infected cells. The virus has 9 serotypes with different antigenicity [3]. Although there is no evidence of intra-type variation in this field, it is generally believed that there are some cross relationships between serotypes, especially AHSV- 1 and 2, AHSV-3 and 7, AHSV-5 and 8, and AHSV-6 and 9. 2. 1 VP2 protein
VP2 protein, encoded by L2 gene, is the most important type-specific antigen of virus, which can react with virus neutralizing antibody together with VP5. The cDNA of nine serotype L2 genes of AHSV9 has the 5 ′-GTT and TAC-3 ′ terminal sequences of typical circular viruses. The molecular weight of protein encoded by the ORF of L2 gene is about 123ku. The variation range of VP2 protein sequence of 9 serotypes of AHSV is 47.6% ~ 765438 0.4%, which is the protein with the largest variation rate of AHSV [4].
BentleyL et al. [5] identified multiple antigenic regions of recombinant AHSV-3VP2 protein by phage display technology and various antiserum. Most antigen sites and neutralization sites are located between 252 and 488 amino acids. This region is not only different between virus serotypes, but also mostly hydrophilic amino acids, among which there are linear epitopes between 369aa~403aa [6]. In addition, the internal sequences of these antigenic sites are highly consistent among virus serotypes with cross reaction, so there is higher homology among virus serotypes with cross reaction. This can explain the serological cross-reaction between serotypes.
The major antigenic regions of AHSV-4VP2 protein are 199aa ~ 4 14aa, while 1aa ~ 199aa (N-terminal) and 414aa ~1060aa (n-terminal). AHSV-4VP2 has 15 antigenic sites, which are divided into two groups. One group covers 223 AA ~ 400 AA, including 12 antigen sites; The other group includes 568aa ~ 68 1aa, including the other three antigenic sites. The interaction between VP5 and VP2 can better display the neutralizing epitope of VP2, thus enhancing the immune response.
Three of the 15 sites can induce neutralizing antibodies against AHSV-4, of which two neutralizing epitopes are located at 32 1aa ~ 339aa and 3, respectively.
77aa~400aa[7]. The combination of these two epitopes can induce more effective neutralization reaction, but the titer of neutralizing antibody is relatively low, so it is unlikely to be used to produce synthetic vaccine of AHSV. This effect may be due to the conformational change caused by the binding of antibodies to one epitope, which makes it easier for other antibodies to bind to another epitope. Another reason may be that the neutralization reaction of these two sites has different functions in virus infection (such as virus adsorption to cells, virus translocation, etc.). ) or they form a continuous part of a relatively discontinuous epitope. ASHV-9VP2 also has two neutralizing epitopes [5]. At the same time, BTV also has neutralization epitopes in the same region, especially between 328-335 AA and 327-402 AA. Therefore, this region may be the main antigenic epitope and important neutralizing antibody target of cyclic virus. These epitopes can be on the surface of the virus capsid, because neutralizing epitopes usually bind to the virus surface to prevent the virus from binding to the receptor of the cell and being absorbed by the cell. In particular, these two neutralizing epitopes are located in the most hydrophobic region of protein, which is likely to exist on the surface of the virus. The 15 antigenic site in [7] was detected by ELISA, and it was found that antigenic sites 8, 1 1 and 12 could bind to antibodies, while antigenic sites 4, 6 and 15 could react with antibodies, but showed no neutralizing activity, especially at site 4, which was in the virus. Other sites do not react with antibodies, indicating that they may be embedded in the virus.
Internal arrangement of African horse plague virus
Although exposed to the virus surface, the surface exists in a conformation that is not recognized by antibodies.
2.2 VP5 protein
VP5 protein is encoded by M6 gene. The conserved sequence of the 5'- terminal noncoding region of M6 gene is 5'-gu uaa-3', which is also reflected in BTV and EHDV associated with AHSV. The conserved sequence of the 3'- terminal non-coding region is 5'-ACA UAC-3', with the only exception of AHSV-6, which has a cytosine nucleotide at the 3'- terminal, which is 5'-ACA uacc-3'. The molecular weight of protein encoded by M6 gene ORF is about 56.9ku.
The amino acid sequences of VP5 of AHSV-6, AHSV-4, BTV- 10 and EHDV- 1 are highly consistent [8]. The three conserved regions are located at the N-terminal (1aa ~ 123aa), the middle (192aa ~ 273aa) and the C-terminal (438aa ~ 49 1aa). These conserved regions may interact with VP2 or VP7, thus maintaining the stability of virus structure. As coat protein, the VP5 protein between ASHV serotypes is highly similar, while VP5 is surrounded by VP2 protein, so it cannot be neutralized with the host.
According to the hydrophobicity profile [8], VP5 protein is divided into two parts, the N-terminal region (1aa~220aa ~ 220aa) and the C-terminal region (about 280aa~505aa), and an alanine-glycine rich region (200aa~270aa) is in the middle as a hinge to connect the two parts. N-terminal is spiral and C-terminal is spherical. These regions play a role in stabilizing the molecular structure within and between protein. The important function of VP5 is to interact with the more conservative core protein and compensate the changes of VP2 protein. VP5 can indirectly affect the serotype of the virus, and its interaction with VP2 changes the conformation of VP2, thus causing changes in the serological characteristics of the virus.
The expression of VP5 alone or together with VP2*** can induce the production of AHSV specific neutralizing antibodies [9]. Among the 330 N-terminal residues, the most obvious immunodominant regions of VP5 protein are 15 1aa ~ 200aa and 83aa ~ 120aa, which are divided into eight antigenic sites with neutralizing epitopes of 85aa~92aa and179aa ~ respectively. The neutralizing epitope of the former is highly conserved in different cyclic viruses, and its monoclonal antibody can recognize the VP5 protein of BTV and EHDV.
2.3 VP7 protein and VP3 protein
VP7 protein and VP3 protein are encoded by S7 and L3 genes respectively, which are the main underwear coat proteins of the virus. VP7 is highly conserved in all serum of ASHV, which is the reason.
Structure of African horse plague antigen
Virus serogroup specific antigen [10]. MareeS et al [1 1] cloned and expressed VP3 and VP7 of ASHV-9 in insect cells with recombinant baculovirus. The expression level of VP7 is high, and it aggregates into unique crystals. The expression of VP3 and VP7*** can form core-like particles in cells. AHSVVP7 protein forms a planar hexagonal crystal in the cytoplasm of infected cells, while VP7 expressed by recombinant baculovirus forms a large discoid crystal, which can be seen under the optical microscope.
Under the electron microscope, the core-like particles formed by VP3 and VP7 proteins of AHSV are very similar to the empty core particles of ASHV. Different from the ASHV core, the core-like particles have a dark central region, showing a typical icosahedral symmetric structure. The outer layer is surrounded by a low electron density layer, which is nodular and extends outward to form nuclear particles with fluff appearance. The diameter of AHSV core particles is about 72nm, which is consistent with the diameter of BTV core particles under electron microscope during freezing, but slightly larger than that of ASHV core particles. It was modified by the reaction of VP7 monoclonal antibody with AHSV core particles. Under the electron microscope, it can be seen that there is a shadow around each particle, which is formed by the interaction between VP7 monoclonal antibody and VP7 shell.
Wade-Evans and others studied the application effect of AHSV-9VP7 as a subunit vaccine. Mice were immunized with purified AHSV-9VP7 crystals and challenged with AHSV-7. It was found that VP7 had a very good protective effect on mice. The protective effect of inoculation with denatured VP7 crystal or GST-VP7 fusion protein expressed in prokaryotic cells is weaker than that of inoculation with VP7 crystal, indicating that the conformation of VP7 protein plays an important role in the function of protein. Passive antibody produced by Balb/c mice immunized with AHSV-9VP7 can not protect newborn mice from infection with AHSV-7, which indicates that antibody may not be the only protective factor. The virus has three nonstructural proteins, namely NS 1, NS2 and NS3/NS3A. They are encoded by M5, S8 and S 10 genes respectively.
3. 1 NS 1 protein
NS 1 is highly conservative in AHSV. Huismans and Els found in 1979 that there are unique virus-specific microtubules in the cytoplasm of cells infected by cyclic virus.
Recombination process of African horse plague virus
Structure, which consists of NS 1 There are a lot of microtubules in BTV infected cells, which are mainly distributed near or around the nucleus. These morphological structures are attached to the intermediate filaments of cytoskeleton. Their role may be to transport mature virus particles from virus inclusion bodies to cell membrane, and then release the virus through the action of NS3, or as molecular chaperones to prevent the core particles from being assembled before the minor proteins (VP 1, VP4 and VP6) are correctly combined with the virus genome.
BTV and EHDV NS 1 expressed in insect cells by recombinant baculovirus all form microtubules. The microtubule diameter of NS 1 of BTV is 52.3nm, which is composed of helices formed by dimers of NS 1, and each helix has 22 dimers of NS 1. Maree and Huismans expressed AHSV-6NS 1 in insect cells with baculovirus, and analyzed NS 1 microtubules by sucrose gradient density centrifugal sedimentation. A large number of target fragments were collected in 200s-400s, which indicated that the expressed NS 1 protein existed in infected cells in the form of particles or polymers. Although NS 1 is the main protein component of these fragments, there are also some other protein with different numbers, which are presumed to be baculovirus protein.
In the 200 s ~ 400 s complex of NS 1, the average diameter of microtubules is 23 nm 2 nm, and the longest is 4 μ m. The unequal length may be due to the breakage during purification, which may also be the reason why the sedimentation coefficient of NS 1 is different in sucrose gradient. Under the electron microscope, the fine structure of AHSV microtubules is quite different from BTV and EHDV microtubules in appearance. AHSV microtubules have an internal structure in the form of fine network cross ripples. The central region of each microtube has alternating electron density, while the low density region represents the cavity of the microtube. Microtubules have smooth edges, clear boundaries and no visible subunit structure. There is no stepped or segmented appearance in the wider BTV microtubules (68nm) and EHDV microtubules (52nn). The hollow ring structure can represent the cross section of microtubules or very short segments of large microtubules. The diameter of these structures is the same as that of ASHV microtubules, and the cavity diameter is about 7nm.
There are also a few specific microtubules of baculovirus in the 200 s ~ 400 s fragment. These microtubules are obviously different from AHSV microtubules. They are larger (40 nanometers in diameter) and have different fine structures. AHSV microtubules can be recognized by antibodies. Microtubules were fixed on the grid and combined with ASHV-6 antiserum. Because the antibody binds to NS 1, there are shadows around NS 1 microtubules under electron microscope, but there are no microtubules of baculovirus.
NS 1 microtubule is unstable in 0.2mol/L and higher concentration of CaCl2 _ 2, and only amorphous protein aggregates can be seen. In 1mol/L NaCl, only a few microtubules can be seen, and their length is obviously shortened, and there are many annular forms. In buffer solution or between pH 8.0 and 8.5, the morphology of microtubules is also seriously affected, the length is reduced and the fine structure disappears. NS 1 microtubule can tolerate relatively low pH, but it is easier to degrade than BTV microtubule under alkaline conditions. Between pH 5.0 and 5.5, the length of microtubules decreases and the surface becomes uneven. At pH5.0 or lower, microtubules denature and NS 1 aggregate into amorphous protein aggregates.
3.2 NS3/NS3A protein
Compared with the high expression level of NS 1 and NS2, the two smallest nonstructural proteins, NS3 and NS3A, were synthesized in a small amount in infected cells. These two related egg whites are encoded by two identical overlapping open reading frames on the S 10 gene. The only difference between them is that the N-terminal of NS3 is 10 amino acids more than that of NS3A. NS3 exists on the cell membrane of infected cells, especially the release site of AHSV on Vero cell membrane, which indicates that NS3 is related to the morphogenesis and release of virus. Virus release may occur before cytopathic effect or cell lysis. AHSVNS3 expressed by recombinant baculovirus is a membrane-associated protein, and its localization does not depend on the existence of AHSV particles. In addition, when recombinant baculovirus expressed ASHVNS3, only 24ku of NS3 protein was synthesized, but no NS3A was synthesized.
The mutation rate of AHSVNS3 is second only to coat protein VP2[ 12] in AHSV protein. The mutation rate of NS3 genotype of ASHV-8 was 27.6%, while that of ASHV-4 was only 15%. The variation rate between types can be used to distinguish subtypes of homovirus. The difference of NS3 sequence can be used to distinguish the wild strain and attenuated live vaccine strain of the same serotype, and the variation rate of 2.3% ~ 9.7% is enough to distinguish them, especially when combined with phylogenetic analysis.
The phylogenetic study of ASHVNS3 divided NS3 protein into three different phylogenetic lineages, named α, β and γ[ 13]. NS3 genus α of AHSV-4, 5, 6, 8 and 9, NS3 genus β of ASHV-3, 7 and 8, and NS3 genus γ of ASHV-2. Although there are obvious differences among the three NS3 evolutionary groups, which evolutionary group a particular AHSVNS3 belongs to depends not only on the virus serotype. The gene rearrangement of AHSV can explain some large mutation rates. Gene rearrangement naturally occurs in viruses with segmented genomes, such as orthomyxovirus and reovirus. The mixed infection of zebra and horse with multiple serotypes may lead to the recombination of S 10 fragments between different serotypes. The serotype grouping of NS3 phenotypic group showed that those serotypes in the same NS3 evolutionary group were more likely to exchange S 10 genes.
The NS3 length of AHSV-3 to 9 is 2 17aa, while the NS3 protein length of ASHV-2 is 2 18aa. The conserved region of NS3 includes the methionine start codon of NS3A, a proline-rich region (22aa~34aa), a highly conserved region (43aa~92aa) and two hydrophobic domains (116aa ~137aa and/kloc-0) that can form transmembrane helices. These structural features are common in NS3 proteins of other cyclic viruses, including BTV. The two hydrophobic regions of BTVNS3 span the cell membrane [15]. BTVNS3 also has glycosylation sites, which can prevent the degradation of BTVNS3 [15]. AHSVNS3 is very different from BTVNS3, except for some conservative features. There is no evidence that glycosylation of AHSVNS3 plays an important role, because NS3 of some virus serotypes has no putative glycosylation sequence. BTVNS3 is expressed at a high level in baculovirus expression system, while AHSVNS3 is expressed at a low level, which can only be detected by western blot or radioactive labeling.
Most of the different amino acids of AHSVNS3 exist in three regions, namely, 43 amino acids at the N-terminal, 93aa ~ 153aa and 15 amino acids at the C-terminal. The regions with the largest variation (the variation rate is 82.4%) are between 136aa and 153aa. Except AHSV-2, all serotypes of ASHV have a conserved cysteine at 123aa, and the cysteine of ASHV-2NS3 is at 120aa, while the second cysteine (164aa) is conserved in all serotypes. In addition, there is a highly conserved N- tetradecyl motif (60aa~65aa or 59aa~64aa) in the anchoring region of the protein on the cell membrane. At the end of tetradecyl motif is a positively charged amino acid region. These two features are both in the highly conserved region of 43aa~92aa mentioned above. Compared with NS3 proteins of other cyclic viruses, the N- tetradecyl motif in the amino terminal region of all these proteins is highly conserved. Tetradecyl alkylation alone can not provide enough energy for protein to attach to phospholipid bilayer membrane. Membrane-associated/binding proteins of other viruses, such as Gag protein of human immunodeficiency virus type I (HIV- 1) and Src protein of Lauder sarcoma virus, contain a basic amino acid region, which can stabilize membrane interaction. There is a similar binary motif in NS3 protein of all cyclic viruses, which may be the membrane targeting signal of NS3 protein.
The two hydrophobic regions of ASHVNS3 are related to cytotoxicity. Any change of hydrophobic region will not change the targeting of protein to the membrane, but it can release their anchoring on the membrane, thus preventing their localization on the cell surface and eliminating their cytotoxicity. The cytotoxicity of AHSVNS3 expressed by baculovirus on insect cells (Sf9 cells) is achieved by changing the permeability of cell membrane.