Pseudorabies Virus HLJ Strain Suppresses Peripheral Blood T and B Lymphocytes in Mice

Pseudorabies, also called Aujeszky’s Disease (AD), is an acute, highly contagious and immunosuppressive swine disease caused by pseudorabies virus (PRV), which is characterized by nervous and respiratory symptoms. PRV belongs to the genus Varicellovirus in the family of Herpesviridae and its genome consists of a linear double-stranded DNA (Papageorgiou et al., 2017). PRV infect a wide variety of domestic animals, including swine, cattle, sheep, goats, cats, dogs and even in wildlife (Minamiguchi et al., 2019). It was recently reported that, the human acute encephalitis was caused by a novel PRV variant strain (Hou et al., 2022). However, swine is the only natural host of this virus because it’s the only animal species that survives after an acute primary infection, therefore, possing a treat to other livestock and crop farmers (Papageorgiou et al., 2017). In addition, PRV can also infect rodents, including mice and rats (Müller et al.,  2011). Therefore, as a susceptible animal, mice have been used as models to investigate PRV pathogenesis.

Lymphocytes, which play an important role in regulating the immune function of the body, are divided into T lymphocytes (CD3⁺) mainly involved in cellular immunity and B lymphocytes (CD19+) mainly involved in humoral immunity. T lymphocytes are divided into helper T lymphocytes (CD3⁺CD4⁺) and suppressive/cytotoxic T lymphocytes (CD3⁺CD8⁺). The latter two types of lymphocytes form a T cell network by inducing and restricting each other, which critically function in regulating cellular immune response and maintain immune balance. CD4⁺CD8⁺ double positive T cells refer to the simultaneous expression of CD4 immature T cells with CD8 antigen. They are widely distributed in many peripheral blood and immune organs of animals. They can be affected by mitogen, pathogen and toxic substances stimulation and proliferation.

Previous studies have suggested that PRV infection has an immunosuppressive effect on the host at the leukocyte level (Deng et al., 2022). However, whether PRV infection affects the cellular and humoral immunity remains unclear. Therefore, in this study, we aimed to detect the effect of PRV infection on peripheral blood T/B lymphocytes in mice through flow cytometry analysis. 

Cells, viruses and antibodies
 
PK 15 (Porcine kidney) cells were maintained in Dulbecco’s modified Eagle Medium (DMEM) containing 10% FBS (fetal bovine serum). PK 15 and DMEM were obtained from the laboratory of Tongren Polytechnic College. These cells were incubated in 37°C in a humidified 5% CO2 incubator. The PRV HLJ strain (Genbank accession number: MK080279.1) was kindly provided by Prof. Jingfei-Wang from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Antibodies against mouse CD3E PE-CY7 (552774), mouse CD4 APC, mouse CD8a FITC and FITC Rat Anti-Mouse CD19 (557398) antibodies were purchased from BD Pharmingen company.
 
Viral infection and titration detection
 
PK15 cells were infected with PRV at a multiplicity of infection (MOI) of 0.5. After incubation at 37°C for 1 h, the cells were washed three times with phosphate buffered saline to remove the unattached virus and then cultured in DMEM supplemented with 2% FBS at 37°C. PBS was purchased from Beijing Solarbio Science and Technology Company. Tissue culture infectious dose 50 (TCID₅₀) was used to detect viral titers according to the Reed and Muench formula (Reed and Muench, 1938).
 
Animal experiment
 
84 eight-week-old female BALB/c mice were bought from Experimental Animal Corporation of Dossy Biological Technology Company (Chengdu, Sichuan, China). After one week, the mice were divided randomly into control group (n=10) and PRV infected group (n=50) at 36 h, 48 h, 72 h and 96 h post-infection. Animals were kept at a room temperature of 22±3°C and a relative humidity of 50±10% with centralised ventilation conditioning through the HVAC system( Srinivasan et al., 2021). The mice in all groups were fed with a nutritionally balanced diet. Water was provided ad libitum. Mice was infected with the PRV-HLJ strain by subcutaneous injection at a dose of 1×10⁵ TCID₅₀ according to the method as previously described (Zang et al., 2015).

To determine the mortality rates of PRV-HLJ strain inoculated mice, another 24 eight-week-old female BALB/c mice were randomly divided into two groups and raised in separate isolator. The mice in the second group (n=6) were injected with 0.1 mL PBS as a negative control. The remaining mice served as the infection group and the PRV injection dose was 100 μl 1×10⁵ TCID₅₀. Signs of disease and death in all groups were observed within a week.
 
Determination the change of T/B lymphocytes by flow cytometry
 
The changes of peripheral blood T/B lymphocytes were detected by using a Z15 flow cytometry in Yangkester Co., Ltd. 100 μL anticoagulant was added in a flow tube and 1 μL of CD3, CD4 APC and CD8 antibody was added respectively and then stained in dark room at 4°C for 30 min. Another 100 μL anticoagulant is added in a new flow tube and 1 μL FITC labelled rat anti mouse CD 19 was added and stained in dark at 4°C for 30 min. Hemolysin working solution was used to lysis the red blood cell at room temperature for 10 min. After centrifuging twice at 250 g for 5 min, the precipitate was resuspended with 2 mL PBS (pH= 7.4). The sample was analyzed by a cytoflex flow cytometry and Kaluza 2.1 software was used to analyze the results.
 
Statistical data analysis
 
The data were represented as means ± standards deviations. One-way analysis of variance (ANOVA) in SPSS 22.0 software package (SPSS Inc., USA) was used to assess statistical significances between PRV–treated groups. All statistical analysis was performed by GraphPad Prism 6.0. A value of p<0.05 or p<0.01 was regarded as significant differences.

The survival rates of PRV-HLJ strain in mice were shown in Fig 1. At 48-hour post-infection (hpi) with the PRV-HLJ strain, the mice generally showed typical clinical neurological symptoms, such as pruritus. Mortality occurred within 60-96 hpi (Fig 1).



As shown in Fig 2(A-B) and Fig 3, the CD3+ and CD3⁺CD4⁺ T lymphocyte induced by PRV were determined by FCM method. The value of CD3⁺ T lymphocyte in mice was significantly decreased after 24 hpi (p<0.01) and remained at a low level until 72 hpi. Then the value of CD3⁺ T lymphocyte increased dramatically.





In addition, we detected the change of CD8⁺ T lymphocyte caused by PRV. It can be seen from Fig 4 that the CD8⁺ T lymphocyte was significantly lower after 24 hpi (p<0.01). The change of CD8⁺ T lymphocyte value was coincided with the CD4⁺ T lymphocyte. Furthermore, as shown in Table 1, the value of CD4⁺/ CD8⁺ was increased at 24 hpi.



In addition, B lymphocyte with CD19+ receptor was detected by FCM. It can be seen from Fig 2-E and 5 that CD19+ B lymphocyte at 24 hpi decreased significantly. The low volume of CD19+B lymphocytes could be observed for the first 12 h (p<0.01). Then CD19⁺ B lymphocyte increased significantly from 60 to 72 hpi. At 96 hpi, the volume of CD19⁺ B lymphocyte was lower compared to the early infection state (p<0.01).



The changes of the peripheral blood T/B lymphocyte volume caused by PRV are presented in Table 1. Pigs are one of the major domestic animals that have an important role in food security and socio-economic starting point (Das et al., 2022). AD caused by PRV is a common disease in many animals, especially in pigs (Zhao et al., 2023). AD is a devastating pig disease and an important viral disease in pigs all over the world, which is characterized by high incidence rate in piglets and reproductive problems in sows and boars (Pegu et al., 2016; Delva et al., 2022). Only some strains of PRV cause severe respiratory disease in older pigs (Cun et al., 2018). Although PRV elimination programs have been successfully implemented in domestic pigs in some countries (Müller et al.,  2011), infections frequently occur in the wild animals, especially in wild boars (Chiari et al., 2015; Pedersen et al., 2018). The uncontrollability of PRV in wild animals indicates a great potential risk for PRV transmission (Liu et al., 2022).

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In the past few years, scientists have conducted in-depth research on the immune mechanism during PRV infection. Previous clinical, immunological and virological data, strongly support the view of immunosuppression and viremia caused by PRV. According to that the virulence of PRV in pigs was related to that of mice (Minamiguchi et al., 2019). Mice may be use as a model which can give some ideas for PRV kinetics (Tang et al., 2017).

In this study, mice were infected with PRV and used to study the influence of the infection in the cellular adaptive immunity. The reference values of B and T cells and their sub-populations are very important to understand how the adaptive immune system is responding to virus infection. CD4⁺ and CD8⁺ T lymphocyte are the two kinds of T lymphocytes involved in cellular immunity (Arsenio, 2020). CD4⁺ and CD8⁺ T cells play an important role in the recognition and activation by binding antigens to histocompatibility (MHC) II and I molecules, respectively (Lee and Jang, 2022). CD8+ T cells play an important role in the adaptive immune response to intracellular viral infection. There is evidence indicating that CD4+ CD8⁺ T lymphocytes in peripheral blood have a pivotal role in preventing PRV infection (Ober et al., 1998). Previous study has shown that in vitro re-stimulation of peripheral blood lymphocytes of pigs infected with PRV induced a strong reaction of CD4+CD8 α+ and CD4-CD8 α⁺ T cells (de Bruin et al., 2000). In this study, we found that after PRV infection, the percentage of CD3⁺ T cells, CD3+CD4+ T cells and the ratio of CD4⁺/CD8⁺ in mice peripheral blood could be decreased in a time-dependent manner. According to Page et al., (1992), animals with high CD8⁺ T lymphocyte and low CD4⁺/CD8⁺ ratio can survive after PRV infection. In addition, animals with low CD8⁺ T lymphocyte and high CD4⁺/CD8⁺ ratio developed  severe disease and died. It maybe explains the mice death peak at 60-96 hpi PRV infection as shown in Fig 1.

CD19 molecule is a transmembrane glycoprotein of Ig superfamily, which is expressed by B cells in early pre-B phase and then is lost during plasma cell differentiation (Poe et al., 2001). CD19 provides a B cell line specific component, a receptor complex containing widely expressed molecular (Sworder et al., 2023). The density of CD19 on the cell surface was highly regulated during development and the expression levels of all mature conventional B cells in different peripheral lymphoid tissues were similar. In this work, we also investigated the dynamic change of CD19⁺ B lymphocyte in the peripheral blood after PRV infection. The results showed that CD19⁺ B lymphocyte decreased rapidly at 24 hpi and fluctuated until the end of this experiment. 

Qingyan Wang drafted the manuscript; Wei Sun and Samuel Kumi Okyere critically revised the manuscript; Shanshan Liu performed the animal experiment; Wei Sun and Qingyan Wang conceived this study and participated in the design of this work as well as data analysis. All authors gave final approval for publication and agreed to be responsible for the work performed.

This research was financially supported by Guizhou Science and Technology Planning Project (No. ZK[2024]-664).

The authors declare no competing interests.