一种非洲马瘟病毒VP6鉴别诊断ELISA的开发。

Development of an African horse sickness VP6 DIVA diagnostic ELISA.

作者信息

Tinarwo Munyaradzi, Dennis Susan J, Hitzeroth Inga I, Meyers Ann E, Rybicki Edward P, Mbewana Sandiswa

机构信息

Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa.

Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa.

出版信息

Virol J. 2025 Aug 12;22(1):276. doi: 10.1186/s12985-025-02898-1.

DOI:10.1186/s12985-025-02898-1

PMID:40796889

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12344834/

Abstract

BACKGROUND

African horse sickness (AHS) is a severe, noncontagious disease of equines caused by the African horse sickness virus (AHSV). The virus has nine serotypes and is transmitted by the midge. AHS is endemic in South Africa and other sub-Saharan African countries. Currently, the disease is managed using a live attenuated vaccine manufactured by Onderstepoort Biological Products (OBP). Although this vaccine has been in use for decades, it has several drawbacks, including the possibility of reversion to virulence, and it does not allow for the differentiation of infected horses from vaccinated horses (DIVA). Previously, our group developed recombinant AHSV serotype 4 and 5 virus-like particle (VLP) vaccine candidates in plants that elicited an immune response in guinea pigs and horses. In this research, we aimed to develop a diagnostic enzyme-linked immunosorbent assay (ELISA) using an AHSV-VP6 antigen expressed in plants, allowing for the differentiation of horses infected with the virus from those vaccinated with VLP vaccine candidates. For this DIVA ELISA, we utilized a robust, cost-effective, and easily scalable manufacturing process that employs transient expression of VP6 in .

RESULTS

AHSV-VP6 sequences from all nine serotypes were aligned to obtain a consensus sequence, which was then used to design the gene. The gene was successfully expressed in plants via -mediated infiltration. The VP6 protein was extracted from infiltrated leaves and purified. A purified yield of approximately 7.7 mg of recombinant VP6/kg fresh weight leaf material was obtained. The VP6 protein was also expressed in , yielding a purified product of 9.4 mg/L. Preliminary data revealed that AHSV-VP6 antigen expressed in both plants and could be used to differentiate between sera from infected horses and those vaccinated with the candidate AHSV4 and AHSV5 VLP vaccines. The plant-produced VP6 could detect more anti-VP6 antibodies than the produced VP6.

CONCLUSIONS

In this study, we expressed the AHSV-VP6 protein in plants, which enabled the differentiation of infected AHSV horse sera from those of horses vaccinated with the candidate VLP vaccines. To our knowledge, this is the first evidence of AHSV-VP6 expression in plants and the first demonstration of its diagnostic ability.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1186/s12985-025-02898-1.

摘要

背景

非洲马瘟（AHS）是由非洲马瘟病毒（AHSV）引起的马属动物的一种严重非传染性疾病。该病毒有9个血清型，通过蠓传播。AHS在南非和撒哈拉以南非洲其他国家呈地方流行性。目前，该病通过使用由翁德斯特普特生物制品公司（OBP）生产的减毒活疫苗进行防控。尽管这种疫苗已使用数十年，但它有几个缺点，包括可能恢复毒力，并且无法区分感染马和接种疫苗的马（DIVA）。此前，我们团队在植物中开发了重组AHSV血清型4和5病毒样颗粒（VLP）候选疫苗，在豚鼠和马中引发了免疫反应。在本研究中，我们旨在利用在植物中表达的AHSV - VP6抗原开发一种诊断性酶联免疫吸附测定（ELISA），以区分感染该病毒的马和接种VLP候选疫苗的马。对于这种DIVA ELISA，我们采用了一种强大、经济高效且易于扩展的生产工艺，该工艺利用VP6在……中的瞬时表达。

结果

对所有9个血清型的AHSV - VP6序列进行比对以获得共有序列，然后用于设计……基因。该……基因通过……介导的浸润在……植物中成功表达。从浸润的叶片中提取并纯化VP6蛋白。获得了约7.7 mg重组VP6/千克鲜重叶片材料的纯化产量。VP6蛋白也在……中表达，得到9.4 mg/L的纯化产物。初步数据表明，在植物和……中表达的AHSV - VP6抗原可用于区分感染马的血清和接种候选AHSV4和AHSV5 VLP疫苗的马的血清。植物产生的VP6比……产生的VP6能检测到更多的抗VP6抗体。