Unveiling Hypothalamic Molecular Signatures via Retrograde Viral Tracing and Single-Cell Transcriptomics.

Despite the importance of hypothalamic neurocircuits in regulating homeostatic and survival-related behaviors, our understanding of the intrinsic molecular identities of neural components involved in these complex multi-synaptic interactions remains limited. In this study, we constructed a Cre recombinase-dependent pseudorabies virus (PRVs) capable of crossing synapses, coupled with transcriptome analysis of single upstream neurons post-infection. By utilizing this retrograde nuclear Connect-seq (nuConnect-seq) approach, we generated a single nuclei RNA-seq (snRNA-seq) dataset of 1,533 cells derived from the hypothalamus of CRH-IRES-Cre (CRH-Cre) mice. To ensure the technical validity of our nuConnect-seq dataset, we employed a label transfer technique against an integrated reference dataset of postnatal mouse hypothalamus comprising 152,524 QC-passed cells. The uniqueness of our approach lies in the integration of diverse datasets for validation, providing a more nuanced diversity of hypothalamic cell types. The presented validated dataset may deepen our understanding of hypothalamic neurocircuits and underscore the essential role of comprehensive integrated transcriptomic data for technical validity.

Membrane fusion, potential threats, and natural antiviral drugs of pseudorabies virus.

Pseudorabies virus (PrV) can infect several animals and causes severe economic losses in the swine industry. Recently, human encephalitis or endophthalmitis caused by PrV infection has been frequently reported in China. Thus, PrV can infect animals and is becoming a potential threat to human health. Although vaccines and drugs are the main strategies to prevent and treat PrV outbreaks, there is no specific drug, and the emergence of new PrV variants has reduced the effectiveness of classical vaccines. Therefore, it is challenging to eradicate PrV. In the present review, the membrane fusion process of PrV entering target cells, which is conducive to revealing new therapeutic and vaccine strategies for PrV, is presented and discussed. The current and potential PrV pathways of infection in humans are analyzed, and it is hypothesized that PrV may become a zoonotic agent. The efficacy of chemically synthesized drugs for treating PrV infections in animals and humans is unsatisfactory. In contrast, multiple extracts of traditional Chinese medicine (TCM) have shown anti-PRV activity, exerting its effects in different phases of the PrV life-cycle and suggesting that TCM compounds may have great potential against PrV. Overall, this review provides insights into developing effective anti-PrV drugs and emphasizes that human PrV infection should receive more attention.

Antiviral effect of an extract from Kaempferia galanga L. rhizome in mice infected with pseudorabies virus.

Pseudorabies virus (PrV) is one of the most important herpesviruses which can cause severe diseases in many mammals and some avian species. In recent years, repeated outbreaks of pseudorabies worldwide indicated an urgent need for new control measures. The results described in this study demonstrated that an extract prepared from the rhizome of Kaempferia galanga L (Kge), which consisted of flavonoids (2.82%), saccharides (61.37%), phenols (1.22%) and saponins (3.10%), possessed a potent anti-PrV activity. In PK-15 cells, Kge treatment inhibited PrV-induced cell death by more than 90% at a dose of 200 µg/mL. The 50% inhibitory concentration (IC50) was 55.85 µg/mL. In the PrV-infected mice treated with Kge, the survival rate was up to 60% at day 6 post-infection, while the infected mice without Kge treatment all died. The virus titers in the brains of the Kge-treated infected mice were significantly reduced. Kge treatment also alleviated the severity of the PrV-induced lesions in the heart, liver, spleen, lung and kidney. Kge exhibited immune-regulating activity through the regulation of cytokines (IFN-α, IFN-ß, IL-4, IL-6 and TNF-α) in the serum of PrV-infected mice, suggesting that one possible mechanism of anti-PrV activity was through the regulation of immune function. These results suggested that Kge could be a promising drug candidate for treating PrV infections.

Proteomic Analysis of Vero Cells Infected with Pseudorabies Virus.

Suid herpesvirus 1 (SuHV-1), known as pseudorabies virus (PRV), is one of the most devastating swine pathogens in China, particularly the sudden occurrence of PRV variants in 2011. The higher pathogenicity and cross-species transmission potential of the newly emerged variants caused not only colossal economic losses, but also threatened public health. To uncover the underlying pathogenesis of PRV variants, Tandem Mass Tag (TMT)-based proteomic analysis was performed to quantitatively screen the differentially expressed cellular proteins in PRV-infected Vero cells. A total of 7072 proteins were identified and 960 proteins were significantly regulated: specifically 89 upregulated and 871 downregulated. To make it more credible, the expression of XRCC5 and XRCC6 was verified by western blot and RT-qPCR, and the results dovetailed with the proteomic data. The differentially expressed proteins were involved in various biological processes and signaling pathways, such as chaperonin-containing T-complex, NIK/NF-κB signaling pathway, DNA damage response, and negative regulation of G2/M transition of mitotic cell cycle. Taken together, our data holistically outline the interactions between PRV and host cells, and our results may shed light on the pathogenesis of PRV variants and provide clues for pseudorabies prevention.

Anatomical Evidence for the Neural Connection from the Emotional Brain to Autonomic Innervation in the Anterior Chamber Structures of the Eye.

OBJECTIVE: Previous studies have shown that the autonomic nervous system (ANS), which can be affected by emotions, is important in the occurrence or progression of glaucoma. The autonomic innervation distributed in the anterior chamber (AC) structures might play an efferent role in the neural regulation of intraocular pressure (IOP). This study aimed to investigate the anatomic neural connection from the emotional brain to autonomic innervation in the AC. METHODS: A retrograde trans-multisynaptic pseudorabies virus encoded with an enhanced green fluorescent protein (PRV531) and non-trans-synaptic tracer FAST Dil were injected into the right eye of mice, respectively. Fluorescent localization in the emotional brain and preganglionic nuclei was studied. Five and a half days after PRV531 injection into the right AC, fluorescent signals were observed in several emotional brain regions, including the amygdala, agranular insular cortex, lateral septal nuclei, periaqueductal gray, and hypothalamus. Autonomic preganglionic nuclei, including Edinger-Westphal nucleus, superior salivatory nucleus, and intermediolateral nucleus, were labeled using PRV531. RESULTS: The sensory trigeminal nuclei were not labeled using PRV531. The fluorescence signals in the nuclei mentioned above showed bilateral distribution, primarily on the ipsilateral side. Seven days after injecting FAST Dil into the AC, we observed no FAST Dil-labeled neurons in the central nervous system. CONCLUSION: Our results indicate a neural connection from the emotional brain to autonomic innervation in the AC, which provides anatomical support for the emotional influence of IOP via the ANS.

Anti-inflammatory effects of Flos Lonicerae Japonicae Water Extract are regulated by the STAT/NF-κB pathway and HO-1 expression in Virus-infected RAW264.7 cells.

This study examined the effect of the Flos Lonicerae Japonicae water extract (FLJWE), chlorogenic acid, and luteolin on pseudorabies virus (PRV)-induced inflammation in RAW264.7 cells and elucidated related molecular mechanisms. The results revealed that FLJWE and luteolin, but not chlorogenic acid, inhibited the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines in PRV-infected RAW 264.7 cells. We found that the FLJWE and luteolin suppressed nuclear factor (NF)-κB activation by inhibiting the phosphorylation of signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3, respectively). Moreover, the FLJWE significantly upregulated the expression of pNrf2 and its downstream target gene heme oxygenase-1 (HO-1). Our data indicated that FLJWE and luteolin reduced the expression of proinflammatory mediators and inflammatory cytokines, such as COX-2 and iNOS, through the suppression of the JAK/STAT1/3-dependent NF-κB pathway and the induction of HO-1 expression in PRV-infected RAW264.7 cells. The findings indicate that the FLJWE can be used as a potential antiviral agent.

Current Status and Challenge of Pseudorabies Virus Infection in China.

Pseudorabies (PR), also called Aujeszky's disease, is a highly infectious disease caused by pseudorabies virus (PRV). Without specific host tropism, PRV can infect a wide variety of mammals, including pig, sheep, cattle, etc., thereby causing severe clinical symptoms and acute death. PRV was firstly reported in China in 1950s, while outbreaks of emerging PRV variants have been documented in partial regions since 2011, leading to significant economic losses in swine industry. Although scientists have been devoting to the design of diagnostic approaches and the development of vaccines during the past years, PR remains a vital infectious disease widely prevalent in Chinese pig industry. Especially, its potential threat to human health has also attracted the worldwide attention. In this review, we will provide a summary of current understanding of PRV in China, mainly focusing on PRV history, the existing diagnosis methods, PRV prevalence in pig population and other susceptible mammals, molecular characteristics, and the available vaccines against its infection. Additionally, promising agents including traditional Chinese herbal medicines and novel inhibitors that may be employed to treat this viral infection, are also discussed.

Isatis root polysaccharide promotes maturation and secretory function of monocyte-derived dendritic cells.

BACKGROUND: Pseudorabies virus (PRV) is an animal virus that is globally responsible for the high economic losses in the swine industry. Isatis root is a traditional Chinese medicinal herb that possesses immune-enhancing and antiviral properties. However, the molecular mechanisms underlying the effects of the active component of the isatis root polysaccharide (IRPS) extract on immature dendritic cells remain elusive. METHODS: In this study, we investigated the molecular changes in primary porcine peripheral blood monocyte-derived dendritic cells (MoDCs) during PRV infection, using enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription-polymerase chain reaction. Additionally, we studied the effect of IRPS on PRV-infected DCs. RESULTS: The results showed that IRPS stimulated the maturation of MoDCs, induced IL-12 secretion, and downregulated IL-6 expression. CONCLUSIONS: Collectively, these results suggest that IRPS is a promising candidate for promoting maturation of DCs and enhancing their secretory potential after PRV infection.

[Use of Transsynaptic Viral Tracers for Observing Neural Circuit Control of Physiological Responses].

Central neural circuits in the brain receive and integrate environmental and internal information to enable the animals to execute appropriate behaviors and physiological responses. Communication between the brain and peripheral organs via peripheral neural circuits maintains energy homeostasis in the body. Therefore it is important to investigate the anatomical organization of central and peripheral neural circuits for elucidating the mechanisms of energy homeostasis. Transsynaptic viral tracers can travel through connected neurons via synaptic connections and have been used to delineate the anatomical organization of neural circuits with specific functions. Herein, I review our recent studies investigating neural circuits and their involvement in physiological changes using transsynaptic tracers.

Antiviral activities of Radix isatidis polysaccharide against pseudorabies virus in swine testicle cells.

BACKGROUND: Radix isatidis has been used in China and other Asian countries for its antiviral and anti-inflammatory effects for thousands of years. However, the antiviral effect of Radix isatidis polysaccharide against pseudorabies virus (PRV) is still unknown. METHODS: The polysaccharide were isolated from extract of the roots of Radix isatidis. MTT assays were used to determine the preventive effect, inhibitory effect and antiviral effect of Radix isatidis polysaccharide on PRV in vitro. RESULTS: This study found that different concentrations of polysaccharides from this plant can inhibit PRV replication by 14.674-30.840%, prevent infection at rates of 6.668-14.923%, and kill this virus at rates of 32.214-67.422%. CONCLUSION: These results broaden the understanding of this traditional Chinese herb and provide a theoretical basis for further research. Moreover, Radix isatidis polysaccharide could be used for antiviral therapy.

Talaromyolides A-D and Talaromytin: Polycyclic Meroterpenoids from the Fungus Talaromyces sp. CX11.

Talaromyolides A-D (1-4) and talaromytin (5) were isolated from a marine fungus Talaromyces sp. CX11. Their structures were unambiguously determined by nuclear magnetic resonance (NMR), mass spectrometry, X-ray crystallography experiments, and time-dependent density functional theory electronic circular dichroism calculations. Talaromyolides A and D represent two novel carbon skeletons. Talaromytin exhibits two slowly interconverting conformers in DMSO-d6 and CH3OH-d4 that were studied by temperature-dependent NMR experiments. Talaromyolide D exhibits potent antiviral activity against pseudorabies virus (PRV) with a CC50 value of 3.35 µM.

Virucidal activity of Garcinia parvifolia leaf extracts in animal cell culture.

BACKGROUND: Garcinia species contain bioactive compounds such as flavonoids, xanthones, triterpernoids, and benzophenones with antibacterial, antifungal, anti-inflammatory, and antioxidant activities. In addition, many of these compounds show interesting biological properties such as anti-human immunodeficiency virus activity. Garcinia parvifolia is used in traditional medicine. Currently, the antiviral activity of G. parvifolia is not known. METHODS: This study was conducted to determine the effects of ethyl acetate (45 L Ea), ethanol (45 L Et), and hexane (45 L H) leaf extracts of G. parvifolia on the infectivity of pseudorabies virus (PrV) in Vero cells. The antiviral effects of the extracts were determined by cytopathic effect (CPE), inhibition, attachment, and virucidal assays. RESULTS: The 50% cytotoxicity concentration (CC50) values obtained were 237.5, 555.0, and < 1.25 µg/mL for 45 L Ea, 45 L Et, and 45 L H, respectively. The 45 L Ea showed the greatest viral inhibition potency of 75% at 125 µg/mL. Both 45 L Ea and 45 l Et caused 100% residual viral inhibition at 250 µg/mL. The selectivity index values for 45 L Ea, 45 L Et, and 45 L H were 2.65, 1.75, and 0.10 showing that 45 L Ea had the greatest antiviral activity among the three extracts. CONCLUSION: This study showed that ethyl acetate is the best solvent to be used to obtain extract from G. parvifolia leaves with potent antiviral activities.

Ginseng stem-leaf saponins in combination with selenium enhance immune responses to an attenuated pseudorabies virus vaccine.

Pseudorabies, a herpesvirus infection, is mainly controlled by using attenuated live vaccines. In this study, the effect of ginseng stem and leaf saponins (GSLS) in combination with selenium (Se; in the form of sodium selenite) on vaccination against attenuated pseudorabies virus (aPrV) was evaluated. It was found that GSLS and Se have an adjuvant effect and that a combination of GSLS and Se stimulates significantly enhanced immune responses than does GSLS or Se alone. Following oral administration of GSLS, mice immunized with an attenuated PrV vaccine diluted in Se-containing physiological saline solution (PSS) provoked a significantly stronger gB-specific serum antibodies response (IgG, IgG1 and IgG2a), enhanced lymphocyte proliferation and cytolytic activity of NK cells, along with higher production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) by splenocytes. Notably, the combination of GSLS and Se conferred a much higher resistance to fPrV challenge after immunization of the mice with aPrV vaccine. This study offers convincing experimental evidence that an injection of Se with oral GSLS is a promising adjuvant combination that improves the efficacy of vaccination against PrV and deserves further study regarding improvement of responses to other animal vaccines.

Dunaliella salina alga extract inhibits the production of interleukin-6, nitric oxide, and reactive oxygen species by regulating nuclear factor-κB/Janus kinase/signal transducer and activator of transcription in virus-infected RAW264.7 cells.

Recent investigations have demonstrated that carotenoid extract of Dunaliella salina alga (Alga) contains abundant ß-carotene and has good anti-inflammatory activities. Murine macrophage (RAW264.7 cells) was used to establish as an in vitro model of pseudorabies virus-induced reactive oxygen species (ROS) response. In this study, antioxidant activities of Alga were measured based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, trolox equivalent antioxidant capacity assays, reducing power, and virus-induced ROS formation in RAW264.7 cells. Anti-inflammatory activities of Alga were assessed by its ability to inhibit the production of interleukin-6 and nitric oxide (NO) using enzyme-linked immunosorbent assay, then the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was investigated by measuring the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (p50 and p65), JAK, STAT-1/3, and suppressor of cytokine signaling 3 (SOCS3) by Western blotting. In addition, Alga inhibited virus replication by plaque assay. Our results showed that the Alga had high antioxidant activity, significantly reduced the virus-induced accumulation of ROS, and inhibited the levels of nitric oxide and interleukin-6. Further studies revealed that Alga also downregulated the gene and protein expressions of iNOS, COX-2, nuclear factor-κB (p50 and p65), and the JAK/STAT pathway. The inhibitory effects of Alga were similar to pretreatment with specific inhibitors of JAK and STAT-3 in pseudorabies virus -infected RAW264.7 cells. Alga enhanced the expression of SOCS3 to suppress the activity of the JAK/STAT signaling pathway in pseudorabies virus-infected RAW264.7 cells. In addition, Alga has decreased viral replication (p < 0.005) at an early stage. Therefore, our results demonstrate that Alga inhibits ROS, interleukin6, and nitric oxide production via suppression of the JAK/STAT pathways and enhanced the expression of SOCS3 in virus-infected RAW264.7 cells.

Improved immune response to an attenuated pseudorabies virus vaccine by ginseng stem-leaf saponins (GSLS) in combination with thimerosal (TS).

Vaccination using attenuated vaccines remains an important method to control animal infectious diseases. The present study evaluated ginseng stem-leaf saponins (GSLS) and thimerosal (TS) for their adjuvant effect on an attenuated pseudorabies virus (aPrV) vaccine in mice. Compared to the group immunized with aPrV alone, the co-inoculation of GSLS and/or TS induced a higher antibody response. Particularly, when administered together with GSLS-TS, the aPrV vaccine provoked a higher serum gB-specific antibody, IgG1 and IgG2a levels, lymphocyte proliferative responses, as well as production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) from lymphocytes, and more importantly provided an enhanced cytotoxicity of NK cells and protection against virulent field pseudorabies virus challenge. Additionally, the increased expression of miR-132, miR-146a, miR-147 and miR-155 was found in murine macrophages cultured with GSLS and/or TS. These data suggest that GSLS-TS as adjuvant improve the efficacy of aPrV vaccine in mouse model and have potential for the development of attenuated viral vaccines.

Antiviral and virucidal activities of Duabanga grandiflora leaf extract against Pseudorabies virus in vitro.

BACKGROUND: Duabanga grandiflora or known in Malaysia as Berembang Bukit, Megawasih, or Pedada Bukit, is a native plant of the Southeast Asian countries. In this study, the anti-viral properties of D. grandiflora were investigated. METHODS: The D. grandiflora leaf extracts were obtained with ethyl acetate, hexane, and ethanol as solvents and labelled 37 leaf ethyl acetate (37 L EA), 37 leaf hexane (37 L H), 37 leaf ethanol (37 L ET), respectively. The cytotoxicity of the extracts on Vero cells were determined by the 3-(4,5-Diamethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. RESULTS: Among extracts, 37 L EA was most cytotoxic to Vero cells, followed by 37 L H and 37 L ET, with CC50 of 218, 833, and >1000 µg/mL, respectively. The cytopathic effect (CPE) and plaque reduction, inhibition, and virucidal assays and the selective index (SI) were employed to determine the effect of the extracts on infectivity and replication of pseudorabies virus (PrV) in Vero cells. The D. grandiflora leaf extracts showed dose-dependent antiviral activities, with higher activities at high doses. The 37 L ET and 37 L EA showed anti-viral effects through plaque formation and viral replication inhibitions, and virucidal property. The SI of the 37 L ET and 37 L EA by the viral replication inhibition assay was 8.3 and 1.9, respectively, and by the CPE reduction assay, 6.7 and 2.9, respectively. CONCLUSION: Ethanol is the best solvent for the preparation of D. grandiflora leaf extract as an antiviral agent.

Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds.

A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.

Luteolin inhibits viral-induced inflammatory response in RAW264.7 cells via suppression of STAT1/3 dependent NF-κB and activation of HO-1.

Luteolin is a common dietary flavonoid present in Chinese herbal medicines that has been reported to have important anti-inflammatory properties. Previous studies have shown that luteolin is an anti-inflammatory and anti-oxidative agent. In this study, the anti-virus inflammatory capacity of luteolin and its molecular mechanisms of action were analyzed. The cytotoxic effects of luteolin were assessed in the presence or absence of pseudorabies virus (PRV) via LDH and MTT assays. The results showed that luteolin (<10µM) had no toxic effects and there were tendencies toward higher cell survival. In PRV-infected RAW264.7 cells, luteolin potently inhibited the production of NO, iNOS, COX-2 and inflammatory cytokine production. Luteolin did not inhibit the phosphorylation of ERK 1/2, p38, and JNK 1/2 either. We found that PRV-induced NF-κB activation is regulated through inhibition of STAT1and STAT3 phosphorylation in response to luteolin. Additionally, luteolin caused the induction of HO-1 via upregulation of Nrf2, both of which are involved in the secretion of proinflammatory mediators. The blockade of HO-1 expression with SnPP, a HO-1 inhibitor, attenuated HO-1 induction by luteolin and thus mitigated its anti-inflammatory effects during PRV-infected RAW264.7 cells. Taken together, our data indicate that luteolin diminishes the proinflammatory mediators NO, inflammatory cytokines and the expression of their regulatory genes, iNOS and COX-2, in PRV-infected RAW264.7 cells by inhibiting STAT1/3 dependent NF-κB activation and inducing Nrf2mediated HO-1 expression.

Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity.

3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 µg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 µg) and 47% (10 µg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

Immune response in pigs treated with therapeutic doses of enrofloxacin at the time of vaccination against Aujeszky's disease.

The effect of treatment with enrofloxacin was studied on the postvaccinal immune response in pigs. Forty pigs were used (control not vaccinated (C), control vaccinated (CV), vaccinated, received enrofloxacin (ENRO)). From day -1 to day 3 pigs from ENRO group received enrofloxacin at the recommended dose. Pigs from ENRO and CV groups were vaccinated twice against Aujeszky's disease virus (ADV). There was a significant delay in the production of humoral response of enrofloxacin dosed pigs when compared with CV group. Moreover, in ENRO group the significant decrease in IFN-γ production and significantly lower values of stimulation index after ADV restimulation was noted, as compared with CV group. The secretion of IL-6, IL-10 and TNF-α by PBMC after recall stimulation was also affected in ENRO group. The results indicate that enrofloxacin, in addition to its antimicrobial properties, possess significant immunomodulatory effects and may alter the immune response to vaccines.