HEK293 and MARC-145 cells were grown in DMEM containing 10% fetal bovine serum (FBS) and 1% streptomycin. The NADC34-like PRRSV strain was separated in this laboratory. The virus titer was 1 × 10^3.5 TCID₅₀/ml.

In order to amplify the GP3 and GP5 genes of MADC34-like, PCR primers were designed (Table 1). Gene synthesis after optimization design (Sangon Biotech (Shanghai)). Three recombinant adenoviruses (pac-Ad5-34-GP3, pac-Ad5-34-GP5, and pac-Ad5-34-GP35) were constructed, and the sequence encoding the G4S flexible linker was inserted into the adenovirus expression plasmid pac-Ad5-34-GP35 between the GP3 and GP5 genes.

The successfully packaged recombinant adenovirus was used to inoculate a six-well plate containing 70% HEK293 cells. When the cells showed CPE, they were recovered and sonicated, and the supernatant was recovered after centrifugation and separated by 10% SDS-PAGE. The protein is transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA) and blocked (5% BSA), the membrane is combined with PRRSV-specific GP5 antibody (diluted 1:100 in PBS containing 0.05% Tween-80, PBST), incubated at 37°C for 2 h, washed the membrane, and then incubated with goat anti-rabbit IgG horseradish peroxide (HRP) secondary antibody at 37°C for 1 h. The results were observed using GE Healthcare Bio-Sciences AB.

Thirty-five weaned piglets of 4–5 weeks were randomly divided into seven groups, each with 5 pigs. From the first to the sixth group, they were immunized with recombinant adenovirus pac-Ad5-34-GP3, pac-Ad5-34-GP5 and pac-Ad5-34-GP35, as well as the control group HP-PRRSV commercial vaccine, ad-wt (adenovirus wild virus) and PBS negative control, the immunization dose was shown in Table 2; booster immunization on the 21st day; 35 days after immunization, NADC34 strain challenge protection test (Table 2).

The pig serum was tested for specific antibodies. The inactivated standard antigen of GP5 subtype was coated overnight in a 96-well plate (expression and purification in the laboratory). After blocking with 5% skim milk for 2 h, the samples were washed three times with PBST buffer for 5 min each time; added the sample to be tested (5 μL/well) to the ELISA plate, incubated at 37°C for 2 h, and washed three times with PBST buffer for 5 min each time; 1:1000 diluted HRP-labeled goat anti-mouse IgG secondary antibody (100 μL/well) to the ELISA plate was added, incubated at 37°C for 2 h, washed three times with PBST buffer, 5 min each time; TMB chromogenic substrate (100 μL/well) was added and protected from light and room temperature for 10 min; stop solution was added to the ELISA plate to stop the reaction (50 μL/well) and measured the OD value at 450 nm absorbance.

The porcine serum was inactivated in a 56°C water bath for 30 min and centrifuged at 3000 rpm for 10 min; DMEM culture medium was used to dilute the inactivated serum with a 2-fold gradient and added it to a 96-well plate, with three replicate wells for each gradient; 200 TCID₅₀ NADC34-like was added to each well virus solution, set up positive and negative controls. After incubation at 37°C for 2 h, 3 × 103 MARC-145 cells were added to each well and were cultured at 37°C and 5% CO₂ for 5–7 days to observe the cell CPE phenomenon. Spearman–Karber calculation was made.

Serum IL-4 and IFN-γ was detected according to the manufacturer’s instructions (ELISA Ready-SET-Go!^®, eBioscience, San Diego, CA, USA).

On the 35th day after immunization, the vaccine immunization experimental group and the control group were subjected to a PRRSV challenge protection test. The challenge titer was 1.0 × 10³ TCID₅₀/mL, and each piglet was injected with 2 mL into the neck muscle. Pig rectal temperatures were measured daily during challenge; 14 days after the challenge, the piglet’s heart, liver, spleen, lung, kidney, submandibular lymph node, and inguinal lymph node were ground to extract RNA, and a reverse transcription kit (TaKaRa Biotechnology, Dalian, China) was used according to the manufacturer’s instructions. Reverse transcriptase nested polymerase chain reaction (RT-PCR) was used to amplify the PRRSV ORF7 gene, and a quantitative fluorescence detection method (absolute quantification) was established to detect the PRRSV viral load in blood and tissues. Referring to the sequence of the PRRSV ORF7 gene, primers 5′-ATGGCCAGCCAGTCAATCA-3′ and 5′-TCGCCCTAATTGAATAGGTG-3′ were used to design primers. The reaction was carried out at 95°C for 5 min, followed by 35 cycles of 95°C for 45 s and 53°C for 45 s.

The dissected lung tissues were fixed with 4% formalin solution, and the fixed samples were dehydrated and coated with paraffin and then sectioned and stained (Servicebio®, Wuhan, China).

All data were analyzed using GraphPad Prism software (version 6.0) and expressed as mean ± SD. The cytokine data were evaluated by one-way repeated-measures analysis of variance. The difference was considered significant (p < 0.05).

Recombinant adenovirus pac-Ad5-34-GP5 and pac-Ad5-34-GP35 can detect GP5 and GP3-GP5 protein expression after infection of HEK293 cells were 23 kDa and 52 kDa, respectively. The control group did not express the target protein (Figure 1).

After immunization, blood was collected every week to detect PRRSV GP5-specific antibodies. The results showed that the recombinant adenovirus vaccine pac-Ad5-34-GP3 experimental group, adenovirus wild virus control group, and PBS control group did not produce GP5-specific antibodies. Adenovirus vaccines pac-Ad5-34-GP5 and pac-Ad5-34-GP35 experimental group and commercial vaccine control group produced corresponding GP5-specific antibodies, but the difference between the groups was not significant (p > 0.05). On the 35th day after immunization, the recombinant adenovirus vaccine pac-Ad5-34-GP5 experimental group had the highest GP5 antibody level than other experimental groups (Figure 2).

Serums were tested for PRRSV-neutralizing antibodies at different time points after immunization. The results showed that neutralizing antibodies appeared in the recombinant adenovirus vaccine experimental group on the 28th day after immunization, but the adenovirus wild virus control group and the PBS control group never produced neutralizing antibodies. On the 35th day after immunization, the commercial vaccine immunization experimental group has a higher neutralizing titer than other immunization groups, and the difference between the groups is not significant (p > 0.05; Figure 3), indicating that the recombinant adenovirus vaccine can produce certain neutralizing antibodies in pigs (Table 3).

The serum of piglets were tested for IFN-γ and IL-4 cytokines at 14 and 35 days after immunization.

The results showed that the expression level of cytokine IFN-γ in the immune group increased at 35dpi compared with 14dpi, which may be the reason for the booster immunity. The expression level of IFN-γ cytokine in the vaccine experimental group was higher than that in the control group; 35 days after immunization, the expression levels of IFN-γ cytokine of the pac-Ad5-34-GP3 and pac-Ad5-34-GP5 experimental groups were 1.99, 1.97 times and 1.42, 1.43 times that of the PBS control group and the adenovirus wild virus control group, respectively, the difference was significant (p < 0.05); the IFN-γ cytokine expression levels in the pac-Ad5-34-GP35 experimental group were 1.82 times and 1.31 times higher than those in the PBS control group and the adenovirus wild-virus control group, respectively. The differences were significant (p < 0.05; Figure 4A).

The IL-4 cytokine expression levels in the pac-Ad5-34-GP3 and pac-Ad5-34-GP5 experimental group were 1.52, 1.59 times and 1.60, 1.92 times higher than those in the PBS control group and the adenovirus wild-virus control group, respectively. The differences were significant (p < 0.05). The IL-4 cytokine expression levels in the pac-Ad5-34-GP35 experimental group were 1.30 times and 1.36 times higher than those in the PBS control group and the adenovirus wild-virus control group, respectively. The differences were significant (p < 0.05). Compared with the experimental groups of pac-Ad5-34-GP3, pac-Ad5-34-GP3, and pac-Ad5-34-GP35, the difference was not significant (p > 0.05; Figure 4B). The increase in IFN-γ cytokine content indicates that piglets can effectively secrete Th1 cytokines, the level of cellular immunity is enhanced, and PRRSV infection can be inhibited. The increase in IL-4 cytokine expression level indicates that piglets can effectively secrete Th2 cytokine, which has a certain effect on the enhancement of humoral immunity.

The body temperature of the NADC34 strain changes after the challenge; 3 days after the challenge, the body temperature of the adenovirus control group began to rise and reached 40.4°C; the body temperature of the PBS control group started to rise on the 6-7th day, reaching 40.3°C and 40.4°C, and then began to decrease it tends to be normal. The body temperature of the adenovirus control group and the PBS control group after the challenge was slightly higher than the other vaccine-immunized groups, and the other experimental groups did not experience any increase in body temperature (Figure 5). On the 14th day after the challenge, the piglets were subjected to necropsy and the main tissues and organs were analyzed for viral load. The results showed that the experimental group had a lower viral load in the tissues and organs and also lower than the adenovirus control group and the PBS control group in terms of viremia. According to the viremia, vaccine immunity compared with the control group showed that the group decreased by approximately 2.5 titers (Figure 6). It shows that the recombinant adenovirus vaccine group has a better protection effect against the virus.

After 14 days of challenge, the piglets were dissected and the lungs were examined for histopathology. Clinical manifestations: pac-Ad5-34-GP3, pac-Ad5-34-GP5, and pac-Ad5-34-GP35 experimental groups did not show clinical symptoms such as loss of appetite and mild cough. The adenovirus wild virus control group and the PBS control group showed general clinical symptoms after challenge, including difficulty breathing and coughing. Histopathological analysis showed that the pac-Ad5-34-GP3, pac-Ad5-34-GP5, and pac-Ad5-34-GP35 experimental groups showed local alveolar wall thickening, accompanied by a small amount of lymphocyte infiltration, and a small amount of lymphocyte infiltration and showed PRRSV lung pathology score level 2. The adenovirus wild virus control group and the PBS control group showed more alveolar wall thickening, accompanied by more lymphocyte infiltration and large alveolar cavity expansion and showed PRRSV lung pathology score level 4 (Figure 7).