Stability and integrity of self-assembled bovine parvovirus virus­like particles (BPV­VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment.

BACKGROUND: Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences. METHODS: In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism. RESULTS: Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs. CONCLUSIONS: In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

The program of antiviral agents inhibits virus infection.

Virus infections are the root cause of epidemics in the world. Vaccines and antiviral agents have been the two important methods to control viral diseases; in recent times, RNA-mediated therapeutics and prevention have received much attention. In this review, we provide an overview of the current information regarding the use of vaccines, antiviral agents, and RNA-mediated methods in controlling or preventing viral infections. We stress specifically on the potential of existing RNA-mediated methods in clinical applications.

Development of a New RT-PCR with Multiple Primers for Detecting Southern African Territories Foot-and-mouth Disease Viruses.

INTRODUCTION: The extremely high genetic variation and the continuously emerging variants of foot-and-mouth disease virus (FMDV) of Southern African Territory (SAT) serotypes including SAT1, SAT2, and SAT3 make it necessary to develop a new RT-PCR for general use for monitoring viruses based on the updated genome information. MATERIAL AND METHODS: A FMDV SAT-D8 one-step RT-PCR was established based on the 1D2A2B genes of the SAT serotype viruses with a multiplex primer set. FMDV A, O, C, and Asia 1 serotypes, other vesicular disease viruses, inactivated SAT viruses, and 125 bovine, ovine, caprine and porcine tissue samples collected from the Chinese mainland were included for evaluating the assay. RESULTS: The new RT-PCR was proven to be specific without cross-reactions with Eurasian FMDV, swine vesicular disease virus (SVDV), Seneca valley virus (SVV), or other common viral pathogens of cattle, sheep, goat, and pig. An around 257 bp-sized amplicon clearly appeared when the inactivated SAT viruses were detected. However, all 125 samples collected from FMDV-susceptible animals from the Chinese mainland which has not known SAT epidemics showed negative results. CONCLUSIONS: A FMDV SAT-D8 one-step RT-PCR is a promising method for primary screening for FMDV SAT serotypes.

Role of interleukin-17A in early graft rejection after orthotopic lung transplantation in mice.

BACKGROUND: The cellular and molecular mechanisms underlying lung allograft rejection remain poorly understood. We investigated the potential role of interleukin (IL)-17A in lung transplant rejection in a mouse model, because previous studies in clinical and rodent models have implicated IL-17A in both acute and chronic rejection. METHODS: To generate an orthotopic lung transplantation model, lungs from C57BL/6 or BALB/c mice were transplanted into C57BL/6 mice (isograft and allograft models, respectively). The effects of anti-IL-17A treatment in allograft recipients were investigated. The histological features and rejection status of isografts and allografts were assessed at 3, 7, and 28 days after transplantation, and differences in graft infiltrating cells and mRNA expression of relevant cytokines were quantified at 3 and 7 days after transplantation. RESULTS: As expected, isografts showed no obvious signs of rejection, whereas allografts exhibited minimal-to-mild rejection (grade A1-A2) by day 3 and moderate-to-severe rejection (grade A3-A4) by day 7, without evidence of obliterative bronchiolitis (OB). However, by 28 days, evidence of OB was observed in 67% (2/3) of allografts and severe rejection (grade A4) was observed in all. IL-17 mRNA expression in allografts was increased with rejection, and interferon (IFN)-γ and IL-6 mRNA expression levels followed a similar pattern. In contrast, IL-22 expression in allografts was only slightly increased. Antibody (Ab) neutralization of IL-17A diminished the signs of acute rejection at 7 days after transplantation in allografts, and this early protection was accompanied by a decrease in cellular stress according to histological evaluation, suggesting the involvement of IL-17A in the development of early post-transplantation lesions. CONCLUSIONS: Our data indicate that IL-17A is important in the pathophysiology of allograft rejection, and neutralization of IL-17A is a potential therapeutic strategy to preventing lung transplant rejection.

Relationship of long noncoding RNA and viruses.

Long noncoding (lnc)RNAs comprise a diverse group of transcripts including large intervening noncoding (linc)RNAs, natural antisense transcripts (NATs) and intronic lncRNAs. The functions and mechanisms of more than 200 lncRNAs have been studied in vitro and the results suggest that lncRNAs may be molecular markers of prognosis in cancer patients. Some lncRNAs can promote virus replication and allow escape from cytosolic surveillance to suppress antiviral immunity. For example, lncRNA can cause persistent infection by Theiler's virus, and microRNA (miR)-27a/b is important for efficient murine cytomegalovirus (MCMV) replication. The available evidence suggests that lncRNAs may be potential targets of novel antiviral drugs.

The effect of multiple evolutionary selections on synonymous codon usage of genes in the Mycoplasma bovis genome.

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains' genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.

The effects of the context-dependent codon usage bias on the structure of the nsp1α of porcine reproductive and respiratory syndrome virus.

The information about the crystal structure of porcine reproductive and respiratory syndrome virus (PRRSV) leader protease nsp1α is available to analyze the roles of tRNA abundance of pigs and codon usage of the nsp1 α gene in the formation of this protease. The effects of tRNA abundance of the pigs and the synonymous codon usage and the context-dependent codon bias (CDCB) of the nsp1 α on shaping the specific folding units (α-helix, ß-strand, and the coil) in the nsp1α were analyzed based on the structural information about this protease from protein data bank (PDB: 3IFU) and the nsp1 α of the 191 PRRSV strains. By mapping the overall tRNA abundance along the nsp1 α, we found that there is no link between the fluctuation of the overall tRNA abundance and the specific folding units in the nsp1α, and the low translation speed of ribosome caused by the tRNA abundance exists in the nsp1 α. The strong correlation between some synonymous codon usage and the specific folding units in the nsp1α was found, and the phenomenon of CDCB exists in the specific folding units of the nsp1α. These findings provide an insight into the roles of the synonymous codon usage and CDCB in the formation of PRRSV nsp1α structure.

The silent point mutations at the cleavage site of 2A/2B have no effect on the self-cleavage activity of 2A of foot-and-mouth disease virus.

The 2A region of the foot-and-mouth disease virus (FMDV) polyprotein is 18 amino acids in length, and 2A self-cleavage site (2A/2B) contains a conserved amino acid motif G2A/P2B. To investigate the synonymous codon usage for Glycine at the 2A/2B cleavage site of FMDV, 66 2A/2B1 nucleotide sequences were aligned and found that the synonymous codon usage of G2A is conserved and GGG was the most frequently used. To examine the role of synonymous codons for G2A in self-cleavage efficiency of 2A/2B, recombinant constructs which contains the chloramphenicol acetyltransferase protein (CAT) and enhanced green fluorescent protein (EGFP) linked by the FMDV 2A sequence with four synonymous codons for G2A were produced. The activities of all the F2As based plasmids were determined in CHO cells. The results showed that the synonymous codon usage patterns for G2A at the cleavage site (2A/2B) have no effect on the cleavage efficiency. This suggests that the synonymous codon usage of 2A peptide has no effect on the cleavage efficiency of FMDV 2A element.

A reverse transcription loop-mediated isothermal amplification assay to rapidly diagnose foot-and-mouth disease virus C.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription- PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.

The distribution of synonymous codon choice in the translation initiation region of dengue virus.

Dengue is the most common arthropod-borne viral (Arboviral) illness in humans. The genetic features concerning the codon usage of dengue virus (DENV) were analyzed by the relative synonymous codon usage, the effective number of codons and the codon adaptation index. The evolutionary distance between DENV and the natural hosts (Homo sapiens, Pan troglodytes, Aedes albopictus and Aedes aegypti) was estimated by a novel formula. Finally, the synonymous codon usage preference for the translation initiation region of this virus was also analyzed. The result indicates that the general trend of the 59 synonymous codon usage of the four genotypes of DENV are similar to each other, and this pattern has no link with the geographic distribution of the virus. The effect of codon usage pattern of Aedes albopictus and Aedes aegypti on the formation of codon usage of DENV is stronger than that of the two primates. Turning to the codon usage preference of the translation initiation region of this virus, some codons pairing to low tRNA copy numbers in the two primates have a stronger tendency to exist in the translation initiation region than those in the open reading frame of DENV. Although DENV, like other RNA viruses, has a high mutation to adapt its hosts, the regulatory features about the synonymous codon usage have been 'branded' on the translation initiation region of this virus in order to hijack the translational mechanisms of the hosts.

Potential roles of synonymous codon usage and tRNA concentration in hosts on the two initiation regions of foot-and-mouth disease virus RNA.

The open reading frame of foot-and-mouth disease virus (FMDV) contains two authentic initiation codons and the second initiation codon is often selected in high frequency. In the study, we analyzed the effects of the host-cell synonymous codon usage and the overall tRNA concentration in the hosts on the region flanked by the two initiation codons (termed as the region 1) and the same length starting from the second initiation codon (defined as the region 2). We find that low-usage codons of hosts are more selected in the region 1 than the region 2; no obvious usage bias of codon with high C/G content exists in the region 1, and the latter part (ranging from the 13th codon position to the 28th codon position) of the region 1 generally contains the codon sites with the generally lower tRNA concentration than the counterpart of the region 2. The low-usage codons of the hosts with high selection in the region 1 and the cluster codon position with low tRNA concentration in the region 1 may serve as potential factors in decreasing the translation rate of the region 1 caused by initiation from the first start codon of FMDV.

Clustering of low usage codons in the translation initiation region of hepatitis C virus.

The adaptation of the overall codon usage pattern of hepatitis C virus (HCV) to that of human is estimated by the synonymous codon usage value (RSCU). The synonymous codon usage biases for the translation initiation region (TIR) of this virus are also analyzed by calculation of usage fluctuation of each synonymous codon along the TIR (the first 30 codon sites of the whole coding sequence of HCV). As for the overall codon usage pattern of HCV, this virus has a significant tendency to delete the codons with CpG or TpA dinucleotides. Turning to the adaptation of the overall codon usage of HCV to that of human, over half part of codons has a similar usage pattern between this virus and human, suggesting that the host cellular environment of the overall codon usage pattern influences the formation of codon usage for HCV. In addition, there is no obvious phenomenon that the codons with relatively low energy tend to be highly selected in the TIR of HCV, suggesting that the synonymous codon usage patterns for the TIR of HCV might be not affected by the secondary structure of nucleotide sequence, however, the formation of synonymous codons usage in the TIR of HCV is influenced by the overall codon usage patterns of human to some degree.

An overview of control strategy and diagnostic technology for foot-and-mouth disease in China.

Foot-and-mouth disease (FMD) is one of most contagious animal diseases. It affects millions of cloven-hoofed animals and causes huge economic losses in many countries of the world. There are seven serotypes of which three (O, A and Asia 1) are endemic in China. Efficient control of FMD in China is crucial for the prevention and control of FMD in Asia and throughout the world. For the control of FMD, a powerful veterinary administration, a well-trained veterinary staff, a system of rapid and accurate diagnostic procedures and, in many countries, compulsory vaccination of susceptible animals are indispensable. This article strives to outline the Chinese animal disease control and prevention system, in particular for FMD, with the emphasis on diagnostic procedures applied in Chinese laboratories. In addition, new technologies for FMD diagnosis, which are currently in the phase of development or in the process of validation in Chinese laboratories, are described, such as lateral flow devices (LFD), Mab-based ELISAs, reverse transcription loop-mediated isothermal amplification (RT-LAMP) and gold nanopariticle immuno-PCR (GNP-IPCR).

The effects of the synonymous codon usage and tRNA abundance on protein folding of the 3C protease of foot-and-mouth disease virus.

The 3C protease of foot-and-mouth disease virus (FMDV) has a conserved amino acid sequence and is responsible for most cleavage in the viral polyprotein. The effects of the synonymous codon usage of FMDV 3C gene and tRNA abundance of the hosts on shaping different folding units (α-helix, ß-strand and the coil) in the 3C protease were analyzed based on the structural information of the FMDV 3C protease from Protein Data Bank (PDB: 2BHG) and 210 genes of 3C for all serotypes of FMDV. The strong correlation between some codons usage and the specific folding unit in the FMDV 3C protease is found. As for the effect of translation speed caused by tRNA abundance on the formation of folding units, the codon positions with lowly abundant tRNA scatter in the 3C gene and there is the obvious fluctuation of tRNA abundance locating in the transition boundaries from the ß-strand to the α-helix and the coil, respectively. However, codon positions with lowly abundant tRNA clustering into these boundaries are not found, suggesting that the adjustment of translation speed is likely also achieved by the single codon position with low tRNA abundance rather than a cluster. The observations can provide the information for insight into the role of the synonymous codon usage in the formation of 3C protease of FMDV and effect of the tRNA abundance of the hosts on this formation of protease.

A comparative analysis on the synonymous codon usage pattern in viral functional genes and their translational initiation region of ASFV.

The synonymous codon usage pattern of African swine fever virus (ASFV), the similarity degree of the synonymous codon usage between this virus and some organisms and the synonymous codon usage bias for the translation initiation region of viral functional genes in the whole genome of ASFV have been investigated by some simply statistical analyses. Although both GC12% (the GC content at the first and second codon positions) and GC3% (the GC content at the third codon position) of viral functional genes have a large fluctuation, the significant correlations between GC12 and GC3% and between GC3% and the first principal axis of principle component analysis on the relative synonymous codon usage of the viral functional genes imply that mutation pressure of ASFV plays an important role in the synonymous codon usage pattern. Turning to the synonymous codon usage of this virus, the codons with U/A end predominate in the synonymous codon family for the same amino acid and a weak codon usage bias in both leading and lagging strands suggests that strand compositional asymmetry does not take part in the formation of codon usage in ASFV. The interaction between the absolute codon usage bias and GC3% suggests that other selections take part in the formation of codon usage, except for the mutation pressure. It is noted that the similarity degree of codon usage between ASFV and soft tick is higher than that between the virus and the pig, suggesting that the soft tick plays a more important role than the pig in the codon usage pattern of ASFV. The translational initiation region of the viral functional genes generally have a strong tendency to select some synonymous codons with low GC content, suggesting that the synonymous codon usage bias caused by translation selection from the host takes part in modulating the translation initiation efficiency of ASFV functional genes.

The analysis of codon bias of foot-and-mouth disease virus and the adaptation of this virus to the hosts.

The synonymous codon usage patterns of open reading frame (ORF) in foot-and-mouth disease virus (FMDV), the similarity degree of the synonymous codon usage between this virus and the hosts and the genetic diversities of FMDV ORFs and the viral functional genes in viral ORF have been investigated by some simply statistical analyses. As for the synonymous codon usage of FMDV, some over-represented and under-represented codons have a similar usage in all seven serotypes. 33 out of 59 synonymous codons are similarly selected between FMDV ORF and the hosts. It is interesting that the overall codon usage pattern of the strains of serotype O isolated from pigs is different with that of strains of the same serotype isolated from non-pig origin, suggesting that the factor of pigs takes part in the formation of codon usage of FMDV serotype O. Projection of codon usage of nine viral functional genes onto the two-dimensional map represents that even though viral functional genes have various genetic diversities and each gene is not separated from each other based on seven serotypes, the codon usage patterns of VP2, 2C, 3A, 3C and 3D genes belonging to serotype O strains isolated from pigs are different with those of the same serotype strains from non-pig origin. In addition, the interaction between GC12% and GC3% of viral functional genes indicates that the codon usage patterns of VP1, VP2, 2B, 3A, 3C and 3D genes are influenced by mutation pressure from virus. Furthermore, distribution plots of ENC value vs. GC3% for viral function genes indicate that mutation pressure is not the only factor in the formation of codon usage of these genes. The results suggest that both the mutation pressure from virus and the translation selection from the hosts take part in the evolution process of viral functional genes of FMDV.

Comparative [corrected] codon usage between the three main viruses in pestivirus genus and their natural susceptible livestock.

Classical swine fever virus, bovine viral diarrhea virus (BVDV), and border disease virus can cause serious livestock diseases. The relative synonymous codon usage value, the "effective number of codons" (ENC), the ratio of K(s) value to K(a) value and the principle component analysis were employed to analyze the genetic characteristics of open reading frame (ORF) and the four genes (the N(pro), Erns, E1, E2 genes) of the three viruses and the relationship of codon usage pattern between each virus and its most common host. The amount of under-represented codons is larger than the amount of over-represented ones in ORFs or the four genes of the three viruses. The ENC value and the ratio of K(s)/K(a) for each gene show that mutation pressure plays a role in their evolutional processes. In addition, the evidence that selection from the natural host might influences the codon usage patterns of virus is found in the differences of codon usage patterns of ORF and Erns gene of BVDV strain ZM-95 isolated from domestic pig and those of the rest of BVDV strains isolated from cattle. These results indicate that although a strong mutation pressure from the three pestiviruses takes part in their evolutional processes by the alternation of synonymous codons, translation selection from the susceptible livestock on some genes should not be ignored. The codon usage pattern of the three pestiviruses is a result caused by the equilibrium of mutation pressure from virus and translation selection from its host.

Comparative analysis of ovine adenovirus 287 and human adenovirus 2 and 5 based on their codon usage.

Ovine adenovirus 287 (OAdV287) emerges as one of the most promising gene vectors resulting from its unique biological characteristics. To obtain a more detailed knowledge about the codon usage of OAdV287, a comparative study based on the codon usage of OAdV287 and the prototypes of human adenovirus serotypes 2 and 5 (HAdV2/5) was carried out. Some commonly used indices measuring the codon usage patterns, including effective number of codons, relative synonymous codon usage, and statistical methods, were adopted. Overall, OAdV287 had a more biased and conservative codon usage pattern than that of HAdV2/5. Both mutation pressure and natural selection played important roles in shaping the codon usage patterns of these three adenoviruses. All the preference codons of OAdV287 had A/U ends and were totally different from those of sheep and humans; however, the preference codons of HAdV2/5 mostly had G/C ends and were mostly coincident with those of sheep and humans. The codon usage analysis in this study supplies some clues for further comprehending the unique biological characteristics of OAdV287 as gene vectors.

The characteristic of codon usage pattern and its evolution of hepatitis C virus.

To give a new perspective on the codon usage of the hepatitis C virus (HCV) and the factors accounting for shaping the codon usage pattern of the virus, the relative synonymous codon usage (RSCU) values, aromaticity and hydrophobicity of each polyprotein of the virus, effective number of codons (ENC) values and nucleotide contents were calculated to implement a comparative analysis to evaluate the dynamics of the virus evolution. The RSCU values of each codon of 144 HCV ORFs indicated that all abundant codons were C/G-ended codons. The plots of principal component analysis based on sub-genotype of HCV indicated that sub-genotype 1a and 1b separated clearly on the axis of f2 suggesting that the codon usage bias between sub-genotype 1a and 1b strains was different. By comparing the codon usage between HCV and human cells, we found that the synonymous codon usage pattern of HCV was a mixture of coincidence and antagonism to that of host cells. The characteristics of the synonymous codon usage patterns and nucleotide contents of HCV, and the correlation analysis between GC(3s), GC(1,2s), GC% (ORF), GC% (5'-UTR), GC% (3'-UTR), aromaticity, hydrophobicity and ENC value, respectively, indicated that mutational pressure was the dominant factor accounting for the codon usage variation and selection pressure also accounted for HCV codon usage pattern.

An overview on ELISA techniques for FMD.

BACKGROUND: FMD is one of the major causes of economic loss of cloven-hoofed animals in the world today. The assessment of dominant genotype/lineage and prevalent trends and confirmation the presence of infection or vaccination not only provides scientific basis and first-hand information for appropriate control measure but also for disease eradication and regaining FMD free status following an outbreak. Although different biological and serological approaches are still applied to study this disease, ELISA test based on the distinct format, antigen type and specific antibody reinforce its predominance in different research areas of FMD, and this may replace the traditional methods in the near future. This review gives comprehensive insight on ELISA currently available for typing, antigenic analysis, vaccination status differentiation and surveillance vaccine purity and content at all stages of manufacture in FMDV. Besides, some viewpoint about the recent advances and trends of ELISA reagent for FMD are described here. METHODS: More than 100 studies regarding ELISA method available for FMD diagnosis, antigenic analysis and monitor were thoroughly reviewed. We investigated previous sagacious results of these tests on their sensitivity, specificity. RESULTS: We found that in all ELISA formats for FMD, antibody-trapping and competitive ELISAs have high specificity and RT-PCR (oligoprobing) ELISA has extra sensitivity. A panel of monoclonal antibodies to different sites or monoclonal antibody in combination of antiserum is the most suitable combination of antibodies in ELISA for FMD. Even though from its beginning, 3ABC is proven to be best performance in many studies, no single NSP can differentiate infected from vaccinated animals with complete confidence. Meanwhile, recombinant antigens and peptide derived from FMDV NPs, and NSPs have been developed for use as an alternative to the inactivated virus antigen for security. CONCLUSIONS: There is a need of target protein, which accurately determines the susceptible animal status based on the simple, fast and reliable routine laboratory test. A further alternative based on virus-like particle (VLP, also called empty capsids) in combination of high throughput antibody technique (Phage antibody library/antibody microarray) may be the powerful ELISA diagnostic reagents in future.