猪伪狂犬病病毒（PRV）是一种甲型疱疹病毒。PRV感染是一种遍布世界的猪传染病，世界动物卫生组织（OIE）将其列为必须报告的疾病。此外，牛、羊、狗、猫、鸡、兔、豚鼠、浣熊、负鼠等其他动物也会被PRV感染并发展为疾病，对经济动物和野生动物的健康构成威胁。最重要的是，最近有报道称PRV可以感染人类，导致脑炎和死亡。病毒建立感染的第一步是进入宿主细胞，然而，PRV进入细胞的关键细节以及宿主因子如何参与这些过程在很大程度上是未知的。巨胞饮作用是一种肌动蛋白依赖的内吞过程，在细胞表面受体激活之后，非特异性摄取液体、溶质和一些颗粒物质。在过去的几年里，巨胞饮作用被认为是病毒或细菌进入宿主细胞的主要途径。参与巨胞饮作用的细胞因子因病毒而异，但通常包括酪氨酸激酶（TK）、蛋白激酶C（PKC）、p21活化激酶1（Pak1）、PI(3)K、Rho GTPase（Rac1或CDC42）、Na+/H+交换泵、动力蛋白和C端结合蛋白1（CtBP1）。

为了确定PRV进入细胞的潜在机制，本研究通过特异性抑制剂、siRNA沉默或显性负突变体干扰关键宿主因子的表达，并全面研究了它们在RPV进入BHK-21细胞过程中的作用。本研究发现PRV入胞既不依赖内体酸化，也不依赖网格蛋白或小窝蛋白介导的内吞作用，而干扰了巨胞饮入胞途径之后，PRV的入胞和基因表达都显著减少。通过视频观察得出肌动蛋白重排介导了PRV通过巨胞饮途径的入胞过程，进一步研究发现在巨胞饮作用过程中，cofilin磷酸化参与了肌动蛋白重排的调控，PAK1磷酸化也参与这一过程，并且cofilin受PAK1 - LIMK调控。利用PAK1将与其相互作用的蛋白进行免疫共沉淀，发现PAK1的上游是RAC1和CDC42。通过沉默和过表达CDC42和RAC1，发现RAC1可以控制片状伪足的形成，而CDC42可以控制丝状伪足的形成。而PRV可通过丝状伪足或片状伪足进入细胞，并且发现CDC42和RAC1在PRV内化过程中具有代偿作用。继续深入研究发现CDC42 RAC1的活化需要Src激酶的激活。后经过广泛的生物信息学分析和试验验证，发现PRV通过gD蛋白结合Nectin-1激活Src。因此，PRV通过巨胞饮途径进入BHK-21细胞的过程得到清晰而完整的描述，即这些过程是由病毒糖蛋白gD与细胞受体Nectin-1结合而启动的。gD与nectin-1结合后，Nectin-1 - Src - RAC1/CDC42 - PAK1 - LIMK - cofilin - actin级联信号通路被触发，导致细胞表面形成丝状伪足或片状伪足摄取PRV。其中，CDC42和RAC1具有代偿作用，并在PRV入胞过程中起关键作用。此外，本研究通过跟踪单个病毒粒子的入胞过程，计算出PRV平均入胞时间为132秒。最后，PRV入胞后形成的巨胞饮体的运输需要Rab5A的参与，而不需要Rab7A。本研究全面描述各种细胞因子在PRV巨胞饮途径中的作用，该结果将有助于了解PRV的入胞机制和巨胞饮途径的特征。

伪狂犬病（PR）和猪瘟（CSF）是具有重要经济价值的猪传染病。我国大多数养殖场都需要对这两种疾病进行免疫。鉴于PRV感染的细胞谱广、入胞时间短，基因缺失的伪狂犬病毒可用于开发有前景的、经济的多价活病毒载体疫苗。PRV具有150 kb的基因组，可以利用细菌人工染色体（BAC）技术进行反向遗传操作。通过将毒力基因（TK、gE和gI）敲除，可成为免疫原性较好的弱毒株。另外，PRV基因组中含有许多非必需基因，如US4、US7、US8、US9，外源基因可以插入到这些基因中而不影响病毒的体外和/或体内复制潜力，使其成为表达其他猪病外源抗原的合适载体。多价疫苗，尤其是表达外源蛋白的病毒载体疫苗，是对抗猪的各种合并感染疾病的有效策略，已有许多PRV表达猪瘟E2蛋白疫苗的报道，但目前尚无广泛应用于畜牧业的PRV和猪瘟二联基因工程疫苗。

E2蛋白位于CSFV包膜表面，参与病毒的感染过程，是猪瘟病毒的主要保护性抗原，可诱导产生中和抗体。已有报道PRV能表达一个截短的E2蛋白（1-338aa），但未见PRV能表达一个全长E2蛋白的报道。本研究构建了不同表达形式的E2蛋白，发现只有在去除E2跨膜区并加入信号肽的情况下，E2蛋白才能在体外表达。对E2跨膜区的分析发现，E2跨膜区的高疏水性是其无法表达的主要原因。通过突变一个氨基酸来降低其跨膜区的疏水性，发现E2的全长突变体（E2FL-muta3或E2FL-muta4）可以表达。表达的全长突变体E2也可定位于细胞膜上。表达E2FL-muta3或E2FL-muta4的PRV载体疫苗免疫小鼠后，可产生针对E2蛋白的特异性细胞免疫，产生的E2抗体水平明显高于表达截短E2的PRV载体疫苗。免疫后的家兔可同时防止PRV-ZJ2013的致死攻击和猪瘟引起的发热反应。这些结果提示rPRV-dgE/TK-E2FL-muta4是一种有前途的针对CSFV和PRV感染的二联疫苗候选株。

另外，本研究设计热稳定化的E2蛋白作为免疫原插入PRV基因组，成为二联活病毒载体疫苗。蛋白质被折叠的主要原因是获得其功能。这种功能意味着一种一般的蛋白质结构，它必须在在结构上相当稳定，同时具有高的灵活性，才能允许生物过程发生。因此，蛋白质的天然状态不一定是最稳定的状态。本研究旨在将E2蛋白作为免疫原，其中E2不需要具有变构、结合或其他功能，但E2必须具有更稳定的结构，不易被沉淀降解或聚集。E2蛋白有48个变异位点，每个变异位点有2-5个候选氨基酸，同时对48个变异位点进行了多氨基酸替换，获得稳定化的E2序列，其能量较原始序列降低了22个Rosetta能量单位（REUs）。构建表达稳定化E2蛋白的重组PRV，发现稳定化E2蛋白的分泌效率提高2.97倍，热稳定性提高10.5倍。小鼠免疫后抗体水平提高2倍，并且产生了针对1.1亚型和2.1d亚型E2的平衡抗体，PRV-ZJ2013对家兔的致死性攻击和CSFV引起的发热反应可以同时得到预防。这些结果表明，rPRV-muta/287aaE2也是一种有前景的预防CSFV和PRV感染的二联疫苗候选株。

Pseudorabies virus (PRV) is a type of herpes A virus. PRV infection is a worldwide swine infectious disease. The World Organization for Animal Health (OIE) has classified it as a mandatory reporting disease. In addition, other animals such as cattle, sheep, dogs, cats, chickens, rabbits, guinea pigs, raccoons, and opossums can be infected with PRV and develop the disease, posing a health threat to economic and wildlife animals. Most importantly, PRV has recently been reported to infect humans, causing encephalitis and death. The first step in establishing infection by the virus is entry into the host cell; however, the critical details of PRV entry and how host factors are involved in these processes are largely unknown. Macropinocytosis is an actin-dependent endocytic process that non-specifically takes up fluid, solutes and some particulate following activation of cell surface receptors. In the last few years, macropinocytosis has been considered as the main route of entry of viruses or bacteria into host cells. The cellular factor involved in macropinocytosis vary from virus to virus, but usually include tyrosine kinase (TK), protein kinase C (PKC), p21-activated kinase 1 (Pak1), PI(3)K, RhoGTPase (Rac1 or CDC42), Na+/H+ exchanger, kinesin and C-terminal binding protein 1 (CtBP1).

To determine the underlying mechanism of PRV entry into cells, we interfered with the expression of key host factors by specific inhibitors, siRNA silencing, or dominant negative mutants, and comprehensively investigated their role in PRV entry into BHK-21 cells. We found that PRV entry was neither dependent on endosomal acidification nor clathrin- or caveolin-mediated endocytosis. Interference with the macropinocytosis pathway significantly reduced PRV entry and gene expression. It was observed by video that actin rearrangement mediated the entry of PRV through the macropinocytosis pathway, further studies revealed that cofilin phosphorylation was involved in the regulation of actin rearrangement during the macropinocytosis, that PAK1 phosphorylation was also involved in this process, and that cofilin was regulated by PAK1 - LIMK. PAK1 was used to immunoprecipitate proteins that interact with it, and RAC1 and CDC42 were found to be upstream of PAK1. by silencing and overexpressing CDC42 and RAC1, RAC1 was found to control the formation of lamellar pseudopods, while CDC42 could control the formation of filamentous pseudopods. In contrast, PRV can enter cells through either filamentous pseudopods or lamellar pseudopods, and CDC42 and RAC1 were found to have a compensatory role in the internalization of PRV. Further studies revealed that the activation of CDC42 RAC1 required the activation of Src kinase. Next, after extensive bioinformatics analysis and experimental validation, it was found that the gD protein of PRV activates Src by binding to Nectin-1. Thus, the description of PRV entry into cells through the macropinocytosis pathway is clear and complete: these processes were initiated by the binding of viral glycoprotein gD to Nectin-1. Upon binding of gD to nectin-1, the nectin-1-Src-Rac1/CDC42-PAK1-LIMK-cofilin-actin signaling cascade is triggered, leading to the formation of filopodia or lamellipodia on the cell surface for PRV uptake. Among them, CDC42 and RAC1 have a compensatory role and play a key role in PRV entry into the cell. In addition, we calculated an average PRV entry time of 132 seconds by tracking the entry of individual virions. Last, Rab5A, but not Rab7A, is required for the trafficking of the macropinosome formed after PRV entry. This study comprehensively describes the role of various cellular factor in the PRV macropinocytosis pathway, and the results will help us to understand the entry mechanism of PRV and the characteristics of the macropinocytosis pathway.

Pseudorabies (PR) and swine fever (CSF) are important economic infectious diseases of pigs. Most pig farms in our country have been immunized against both diseases. Pseudorabies virus (PRV) with gene deletion can be used to develop promising and economical multivalent live virus vaccines. PRV has a 150 kb genome and can be reverse-genetically manipulated using bacterial artificial chromosome (BAC) technology. By knocking out the virulence genes (TK, gE and gI), the attenuated strain with better immunogenicity could be obtained. In addition, the PRV genome contains many non-essential genes, such as US4, US7, US8, US9, into which exogenous genes can be inserted without affecting the in vitro and/or in vivo replication potential of the virus, making it a suitable vector for expressing heterologous antigens from other porcine diseases. Multivalent vaccines, especially viral vector vaccines expressing foreign proteins, are effective strategies against various co-infectious diseases in pigs There have been many reports of PRV expressing CSFV E2 protein vaccines, but there is no PRV and CSFV dual genetic engineering vaccine widely used in animal husbandry.

The E2 protein is located on the envelope surface of CSFV and is involved in the infection process of the virus. It is the main protective antigen of CSFV and can induce neutralizing antibodies. It has been reported that PRV can express a truncated E2 protein (1-338aa), but no report has shown that PRV can express a full-length E2 protein. We constructed different expression forms of the E2 protein and found that the E2 protein could be expressed in vitro only when the E2 transmembrane region was removed and the signal peptide was added. Analysis of the E2 transmembrane region revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. Full-length E2 mutants (E2FL-muta3 or E2FL-muta4) were found to be expressed by mutating one amino acid to reduce the hydrophobicity of the transmembrane region. The expressed full-length mutant E2 was also localized to the cell membrane. Mice immunized with PRV vector vaccines expressing E2FL-muta3 or E2FL-muta4 elicited specific cellular immunity against E2 protein and produced significantly higher levels of E2 antibody than those immunized with PRV vector vaccines expressing truncated E2. The immunized rabbits were protected from both lethal challenge with PRV-ZJ2013 and febrile response induced by CSFV. These results suggest that rPRV-dgE/TK-E2FL-muta4 is a promising bivalent vaccine candidate strains against CSFV and PRV infection.

In addition, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The main reason proteins are folded is to gain their function. This function implies a general protein structure that must be structurally fairly stable and at the same time highly flexible to allow biological processes to occur. Thus, the native state of the protein is not necessarily the most stable state. This study aims to use the E2 protein as an immunogen, where E2 does not need to have allosteric, binding, or other functions, but E2 must have a more stable structure that is not easily degraded or aggregated by precipitation. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site. Multiple amino acid substitutions at 48 variant sites were performed, and the stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ2013 and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine candidate strains against CSFV and PRV infections.

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