Corrigendum: Protection efficacy and the safety of the synergy between modified Bazhen powder and PRRSV modified-live virus vaccine against HP-PRRSV in piglets

1 IntroductionPorcine reproductive and respiratory syndrome (PRRS) is a severe and highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Since its outbreak in the 1980s (1), PRRS has led to significant economic losses in the global swine industry. In 2006, China recorded an outbreak of the highly pathogenic PRRSV (HP-PRRSV) with high morbidity and mortality (2). The PRRSV strain HuN4, isolated from pig farms in the central region of China, has been identified as one of the HP-PRRSV strains responsible for more than 50% of PRRSV infections and 20 to 100% of deaths in pig farms (3).Despite extensive research, a reliable treatment for PRRS remains elusive. Vaccination is critical in preventing PRRSV infection, with available options including inactivated and modified live vaccines (MLV). However, these vaccines often fail to provide comprehensive protection against the diverse strains found in the field and carry the risk of reverting to higher virulence (4). Moreover, commercial PRRSV MLVs can induce delayed and weak immune responses, including humoral and cell-mediated responses (5, 6). Although neutralizing antibodies (NA) are essential for fighting PRRSV, vaccination does not consistently result in high NA levels (7). Additionally, the risk of vaccine virus transmission to unvaccinated animals increases due to potential viremia and shedding post-vaccination (8). Hence, there is an urgent need to develop new strategies to improve the safety and efficacy of PRRSV vaccines for effective disease control.Traditional Chinese medicine (TCM) offers a range of health benefits, including reducing inflammation, inhibiting viruses (4), and supporting the immune system. The immunomodulatory strategies of TCMs with anti-PRRSV effect involve the regulation of cytokines production in host cells and inhibiting pathways that promote PRSSV pathogenesis (4). Thus, combining immunomodulating TCMs with vaccines helps to boost the body's defense system by increasing interferon expression and raising antibody levels (9-11). Based on this, we postulated that adding TCM, particularly modified Bazhen powder (MBP), to the PRRSV MLV vaccine could strengthen the immune response, attenuate PRRSV virulence, and lower the chances of transmission.Bazhen, a traditional Chinese herbal prescription comprising eight commonly used herbs, is reported to possess immunomodulatory effects, enhancing T lymphocyte proliferation and IFN-γ secretion (12, 13). Furthermore, Bazhen has shown efficacy in reducing oxidative stress and inflammatory cytokines. Geng et al. showed that adding MBP to sows' diets significantly enhanced production performance and improved health parameters in sows and weaned piglets (14). Despite these findings, the potential immunomodulatory effects of combining Bazhen with vaccines remain unexplored. This study elucidated how MBP combined with MLV can effectively control PRRSV prevalence in the swine industry.To sum up, our study revealed how the oral administration of MBP in combination with PRRSV MLV enhanced the immune response to vaccination in growing pigs and protected them against the HP-PRRSV challenge. We also analyzed the active ingredients of MBP absorbed into the bloodstream. The mechanism behind the potential immunopotentiating effects of combining MBP with vaccines will provide new strategies for enhancing vaccine efficacy and control of PRRSV in the swine industry.2 Materials and methods2.1 MBP FormulaAll herbs in the formulation were obtained from the Chinese herbal market in Harbin (Harbin, China), all the plant materials were identified by Professor Yanyan Liu (College of Animal Medicine, Northeast Agricultural University, Harbin, China) and preserved in the Animal Pharmacy Laboratory of, Northeast Agricultural University. They contained Astragalus root (Astragalus membranaceus), Atractylodes rhizome (Atractylodes lancea), Hoelen (Poria cocos Wolf), Glycyrrhizae Radix (Glycyrrihiza uralensis Fischer et DC.), Raix Rubra (Paeonia albiflora Pallas var. trichocarpa Bunge), Angelica root (Angelica sinensis), Rehmanniae Radix et Rhizoma (Rehmannia glutinosa Liboschitz), Ziziphus jujuba Mill (Ziziphus zizyphus), Ligustici Chuanxiong Rhizoma (Ligusticum chuanxiong Hortorum) and Maltiflower Knotweed (Fallopia multiflora (Thunb.) Harald).2.2 Viral Strains and Cells The commercial live attenuated vaccine HuN4-F112 and the HP-PRRSV HuN4 strain (GenBank accession number: EF635006) were provided by Professor Tian from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Marc-145 cells used in this study were obtained from the stocks of Harbin Pharmaceutical Group Bio-vaccine Co., Ltd. Marc-145 cells, a PRRSV-permissive cell line derived from the African green monkey kidney cell line MA-104, grown in Dulbecco’s minimum essential medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, CLARK) and 100 IU/mL penicillin and 100 µg/mL streptomycin at 37 ℃ with 5% CO2.2.3 Ethics approvalAll animal experiments were done following protocols approved by the Experimental Animal Ethical Committee of Northeast Agricultural University (NEAUEC202303113). Animals were housed in environmentally conducive and specific pathogen-free laboratories. They were fed ad libitum with commercial feed and had unrestricted access to water. They were made to acclimatize to the environment for at least one week before the experiment. The animals were not subjected to stress during the experiment.2.4 AnimalsA total of 30 landrace piglets (four weeks old) were housed in the animal house of the Harbin Pharmaceutical Group Bio-vaccine Co., Ltd. The piglets were confirmed to be free of PRRSV, PRV, PCV, and Mph. They were provided with adequate water and a standard diet throughout the study.2.5 Experimental proceduresThe piglets were randomly divided into six groups: a normal control group (Con), a PRRSV control group (PRRSV), a vaccine-only group (MVV), a group treated with vaccine and challenged with HuN4 (Vac), a group treated with MBP combined with vaccine (MBV), and a group treated with MBP-vaccine and challenged with HuN4 (MV). All piglets were fed a standard diet. The MBV and MV groups received supplementation with 0.02% MBP mixture from 7 days before vaccination (-7) to 28 days post-vaccination (dpv). The Vac, MVV, MBV, and MV groups were intramuscularly injected with the live attenuated HuN4-F112 vaccine (batch 2020006; Harbin Weike Biotechnology Co., Ltd.) at 0 dpv. At 28 dpv (0 days post-challenge (dpc)), all piglets, except those in the Con, MVV, and MBV groups, were challenged with HP-PRRSV HuN4-F2 (containing 3×104.5 TCID50) and sacrificed at 21 dpc. Experimental procedures are depicted in Fig. 1 and Table 1.2.6 Sample CollectionBlood samples were collected from the anterior vein to measure immunological factors, neutralizing antibodies, and PRRSV viral load. Additionally, a portion of the blood samples was collected at 28 dpv and used to analyze the presence of traditional Chinese medicine active ingredients. Nasal, oral, and anal swab samples were collected simultaneously to assess viral shedding and measure PRRSV viral load using quantitative RT-PCR. Biochemical estimates of PGE2, IFN-α, IL-6, and IL-1β in blood samples were conducted using ELISA kits across all experimental groups. At 21 dpc, tissue samples were taken from the lymph nodes, tonsils, spleen, lungs, liver, heart, kidneys, stomach, colon, and brain. A portion of the tissue was fixed in 4% paraformaldehyde for pathological analysis, and another portion was stored at – 80 ℃. For viral load testing, 1 g of tissue samples, 200 µL swab samples, and 200 µL serum samples were used for RNA extraction to measure the viral load in the various samples. The experimental set up and histopathological scoring are presented in Table 1 and Table 2, respectively.2.7 Clinical EvaluationAverage weight gain (WG) was calculated from 0 to 28 dpv and 0 to 21 dpc respectively. Daily rectal temperature and clinical signs were recorded from 0-21 dpc. The clinical scores were calculated as previously described (Table 3). Observations were conducted twice daily: once in the morning from 9:00 to 10:00 and once in the afternoon from 14:00 to 15:00. These observations were systematically recorded to ensure consistency and accuracy.2.8 Serum Neutralization (SN) AssaySera from piglets were heat-inactivated at 56 ℃ for 30 min before conducting serum neutralization (SN) assays. Then, DMEM was used as the diluent to prepare serial twofold dilutions of each serum sample. Subsequently, suspensions containing 100 TCID50 of PRRSV per 100 µL were formulated, and then 100 µL of the suspension was added to each serum dilution. And then, the serum-viral mixtures were incubated at 37 ℃ for 1 h. Subsequently, the plates were further incubated at 37°C in a humidified atmosphere with 5% CO2 for 7 days and monitored daily for cytopathic effects (CPE). Finally, the Reed-Muench method was used to calculate the neutralization titer of the serum. Each dilution was replicated four times.2.9 Quantitative Real-Time PCRThe standard working curve of PRRSV was created using a series of plasmid standards (102 - 109 copies). Briefly, total RNA was extracted using a Simple P Virus DNA/RNA Extraction Kit. Next, reverse transcription of total RNA into cDNA was done using a Reverse Transcription Kit. Then, TaqMan-based real-time fluorescence quantitative RT-PCR was used to detect RNA copies of PRRSV (15). Quantitative RT-PCR assays were performed using Agilent MX3000P QPCR equipment. All results were obtained from at least three replicates.2.10 Flow Cytometry AssayFlow cytometry (Bricyte E6, Shenzhen, China) was used to analyze the immunity of piglets by quantifying T-cell subsets in the peripheral blood. Three-color phenotyping using fluorochrome-conjugated monoclonal antibodies (Southern Biotech, United States) for CD4+ and CD8+ T cells was conducted for the immunophenotyping of lymphocytes. Samples were processed and evaluated. The ratios of CD3+, CD4+, CD8+, and CD4+/CD8+, were calculated to assess the health status of the immune system.2.11 Statistical analysis Data obtained were analyzed using SPSS 17.0 statistical software (IBM, USA), and results were presented as mean ± standard deviation (SD). The Student's t-test and One-way analysis of variance (ANOVA) was used to analyze group differences. P > 0.05 was considered as the level of statistical significance. Pictures were analyzed using GraphPad Prism 8.0.2 software.3 Result 3.1 MBP reduces the duration of viremia The average weight gain (WG) from 0-28 dpv was 9.55 kg, 10.16 kg, and 9.76 kg for Con, MVV, and MBV groups, respectively, with no significant differences observed, suggesting that MBP does not significantly affect the productive performance of vaccinated piglets. Given that pigs inoculated with MLV shed viruses, we investigated viremia and viral shedding post-vaccination with HuN4-F112. The results indicated that the combination of MBP and Vac significantly reduced the duration of viremia, although it had no significant effect on viral load (Fig. 2B), thereby enhancing vaccine safety without interfering with HuN4-F112 immunogenicity.3.2 MBP enhances cellular immunity Previous experiments indicated that MBP has immune-potentiating effects (14). Thus, we used flow cytometry to determine CD3+, CD4+, and CD8+ T cell subpopulations (Fig. 3A) to assess MBP's impact on cellular immunity. At 28 dpv, the MBV group showed a significant increase in CD3+ T subpopulations compared to the Con group (P < 0.05), with no difference compared to the MVV group (Fig. 3B). Notably, pigs immunized with HuN4-F112 exhibited significantly higher CD4+ T subpopulations at 14 and 28 dpv in both MVV and MBV groups compared to the Con group (P < 0.05) (Fig. 3C). CD8+T subpopulation generation remained unaffected (Fig. 3D). Additionally, the CD4+/ CD8+ T subpopulation ratio in MBV group was significantly higher at 14 dpv compared to the Con group (P < 0.05) (Fig. 3E). These results confirmed our earlier claim that using MBP in combination with vaccine acts as a potential immunomodulating agent against PRRSV, thus highlighting the need for further research to how this combination at a MBP combined with MLV enhances we a increase in antibody levels in the peripheral blood of both the MVV and MBV groups from 28 to (dpv). antibody levels were significantly higher in the MBV group than in the MVV group at and dpv (P < 0.05), an enhanced and more antibody response in the MBV However, there were no significant differences the MVV and MBV groups in neutralizing antibody (NA) we the levels of cytokines in immune system and responses, IFN-γ and The MBV group exhibited significantly higher levels of at 21 and 28 dpv and IFN-γ at 7 dpv compared to the MVV Furthermore, the MBV group showed significantly IFN-γ levels compared to the Con group at 28 MBP clinical the effect of we that combining MBP with vaccines clinical by The presence of clinical including high of and was in the PRRSV group throughout the (Fig. The PRRSV group had rectal from 1 to 14 dpc following the HP-PRRSV (Fig. 1 to dpc, the rectal temperature of the Vac group was higher than with pigs than Despite these no deaths were The MV group had a rectal temperature and from 1 to 20 dpc, with no pig deaths (Fig. Notably, at dpc, the Vac group exhibited a significantly higher rectal temperature than the MV clinical signs were in the PRRSV group than in the Vac and MV groups (Fig. In the duration of clinical from 20 dpc in the PRRSV group to dpc in the Vac group and dpc in the MV group (Fig. Despite this, no significant difference in the duration of clinical was the Vac group and the MV group throughout the experiment. The clinical scores of the PRRSV group were significantly 0.05) higher than those of the Vac and MV groups. And the Con group remained normal throughout the study (Fig. MBP not post-challenge viremia the duration We viremia groups viremia in all groups except the Con in all pigs in the PRRSV group (Fig. The presence of the PRRSV was in pigs in the PRRSV group at and 21 dpc, with a in the of pigs in the Vac and MV groups. The mean duration of viremia was days in the Vac group and days in the MV group (Fig. However, there was no significant difference in PRRSV copies the Vac and MV groups at (Fig. also analyzed viral shedding the duration from the (PRRSV), (Vac), and (Fig. However, oral shedding (PRRSV), (Vac), and (Fig. anal shedding was (PRRSV), (Vac), and (Fig. Viral shedding and dpc, with the PRRSV group the (Fig. the PRRSV group shedding of up to copies from copies from anal and copies from oral The PRRSV group consistently showed higher copies than the Vac and MV groups, with no significant difference the Vac and MV reduces viral load and assess and vaccine we the of pigs from each with HuN4-F112 reduced and tissue caused by PRRSV compared to the PRRSV group (Fig. QPCR analysis showed higher copies in of the PRRSV group than in the Vac and MV groups (Fig. Notably, the MV group had significantly lower viral in compared to the Vac group (Fig. and reduced were in and were in the of pigs in the PRRSV Then, with cell were in the and lymph with cell and tissue were in the Subsequently, severe and inflammatory cell were in the and The had significant and of inflammatory cells no were (Fig. Furthermore, and were in the Moreover, with cell proliferation was shown in the brain. The exhibited inflammatory Furthermore, was in the histopathological in the PRRSV group compared to the Vac and MV groups (Fig. inflammation, a in immune responses, and can to study revealed that the PRRSV significantly including (Fig. (Fig. (Fig. IL-1β (Fig. (Fig. and IFN-γ (Fig. remained throughout the experiment. Despite higher levels in the PRRSV cytokines remained immune research showed that PGE2, IL-6, and IL-1β levels in the group from dpc. However, these levels in the Vac and MV groups. at dpc, the Vac group had higher IL-1β levels than the MV suggesting MBP's potential to and and cellular by and in inhibiting However, significant increases in serum levels of these cytokines following the PRRSV to clinical in the PRRSV group (Fig. and This the of on and cellular immunity to the and potential in immunity. MBP its potential in enhancing and cellular as by significantly higher levels of and IFN-γ in the MV group at and 7 dpc (Fig. and respectively, compared to the Vac humoral immune response of piglets was by antibodies and NA using an ELISA and assays (Fig. and antibodies were in all piglets from dpc (Fig. antibody However, in the of and 7 antibody levels were significantly higher in the Vac and MV groups than in the PRRSV highlighting the efficacy of the HuN4-F112 vaccine in an antibody results at dpc (Fig. showed higher NA in the Vac and MV groups compared to the PRRSV with of and respectively, in the PRRSV group were NA at in the Vac group at 7 dpc, the MV and PRRSV groups of and at dpc, respectively. Notably, at dpc, NA in the MV group were higher than those in the Vac group (Fig. the of MBP in NA levels in pig serum for an our that the oral of MBP combined with vaccination significantly the inflammatory by the PRRSV in growing This effectively the expression of lungs, improved cellular and and high levels of to the of clinical and disease on an earlier study MBP's we on more comprehensive research to study the mechanism by MBP helps improve immune following PRRS Additionally, we the effects of combining MBP with vaccines against the HP-PRRSV HuN4 and analyzed the active ingredients in MBP that to its these we revealed that MBP is a potential that can attenuate PRRSV and vaccine shown in Fig. no significant difference was in viral load the MVV and MBV groups MBP's not affect MLV PRRSV MLV vaccines are for viral shedding confirmed by oral, and anal collected up to 28 days analysis of viral shedding revealed shedding in oral (Fig. This could be due to PRRSV in the tonsils, a and into oral Previous oral as a to serum for PRRSV in pigs our revealed the of blood and oral samples for a more of PRRSV Despite no significant difference in shedding duration from oral there was a in the duration of viremia in the MBV group post-vaccination compared to Given MBP's of anti-PRRSV we on its efficacy in the body's immune response, disease and PRRSV a critical in immune responses, with the CD3+ used to total T lymphocyte we CD4+ and CD8+ T cell subsets and the ratio in piglets from MVV and MBV groups (Fig. 3). were no significant differences in the of CD4+ and CD8+ T cells the ratio the MVV and MBV groups the study However, at 28 dpv, the MBV group showed a significantly higher of CD3+ T cells than the Con an increase in total T lymphocyte due to MBP Furthermore, the MBV group exhibited significantly higher levels of IFN-γ at 28 dpv, with CD3+ findings, suggesting MBP's in enhancing cellular immunity. Subsequently, we humoral immunity by the MVV and MBV groups using the and NA Although there was no significant difference in NA the MVV and MBV groups the MBV group consistently showed higher NA Additionally, during the of the the in the MBV group were higher than those in the MVV a effect of MBP on humoral immunity (Fig. with pigs commonly a high a that remains to control with vaccination In our we that piglets challenged with the HuN4 strain exhibited high more in the Vac group compared to the MV group (Fig. can be by various as IL-6, and possess inflammatory and These cytokines production to HP-PRRSV a inflammatory response, in an to its IL-1β host expression of IL-1β can a that to a inflammatory response, viral In this IL-1β and levels at dpc in the PRRSV group and dpc in the Vac and MV groups. Notably, at and dpc, IL-1β levels were significantly lower in the MV group compared to the Vac Previous to IL-1β the lower and duration of in the MV group compared to the Vac it was that levels of cytokines are with with histopathological as in of PRRSV by and that HP-PRRSV inflammation, in tissue and Hence, we used an improved of the method by to assess the and of pigs In our we MBP its effects on clinical and and compared the Vac and the MV groups and The for the MV group was to the of MBP to the inflammatory The and effects of MBP are due to the combined of its active ingredients the tissue of PRRSV, we viral in various We found a significant in viral load in the MV group compared to the Vac group (Fig. in are for PRRSV infection, viral presence and potential transmission oral and that MBP the of PRRSV in tonsils, inhibiting PRRSV Additionally, we investigated and oral viral shedding duration in piglets (Fig. differences were not the MBP group showed a reduced an of viral transmission oral and responses to PRRSV were once considered due to However, the of neutralizing antibodies (NA) against PRRSV, in protection against viral infections Animals with NA viremia, higher can viremia not viral and transmission study showed NA in the Vac and MV groups at 7 and dpc, respectively. However, viremia pigs with more higher NA levels for protection Although the on Marc-145 cells not in our results higher NA levels in the MV group (Fig. suggesting an enhanced immune IFN-α, and IFN-γ also significant in preventing PRRSV MBP to the of and IFN-γ levels (Fig. highlighting its in enhancing and cellular immunity against PRRSV, thereby piglets in to vaccination the oral administration of modified Bazhen powder and MLV vaccination a and effective immunological response, as by levels of antibody and viremia In following the HP-PRRSV it significantly the of and virus The oral administration of MBP also significantly IL-6, and IL-1β its potential to attenuate the inflammatory that PRRSV These results showed that MBP has the potential to as an oral vaccination safety and significant protection against PRRSV in Data will provide the supporting the of this without For further the of that the research was conducted in the of commercial that could be as a potential of and to and and on provided and and was in and analysis, and All to the and approved the was by the Research and of and the China Research of and