Outbreaks of infectious disease in shrimp pose a serious threat to shrimp agriculture worldwide. Shrimp lack adaptive immunity and depend only on innate immunity as a defense system against infectious disease. Toll-like receptors (TLR) are reported to play a critical role in the innate immune system. In this study, we identified a Toll-like receptor gene of a species of freshwater shrimp, Macrobrachium nipponense, designated MnToll, for the first time. The sequence of MnToll encoded 935 residues arranged as 10 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR CT) domain and a Toll/interleukin-1 receptor (TIR) domain and displayed 90% amino acid similarity to previously identified TLRs (Toll 1 and 2) of Macrobrachium rosenbergii. We additionally evaluated mRNA expression of MnToll in various tissues, including heart, gills, stomach, digestive gland, ventral nerve cord, antennal gland and muscle. Following infection with a viral pathogen, white spot syndrome virus (WSSV), MnToll expression was significantly upregulated between 12 and 72 h. Our data collectively suggest that the newly identified MnToll gene belongs to the TLR family in shrimp and is potentially involved in innate host defense, especially against WSSV.

Influenza viruses cause respiratory diseases in humans and animals with high morbidity and mortality rates. Conventional anti-influenza drugs are reported to exert side effects and newly emerging viral strains tend to develop resistance to these commonly used agents. Fritillaria thunbergii (FT) is traditionally used as an expectorant for controlling airway inflammatory disorders. Here, we evaluated the therapeutic effects of FT extracts against influenza virus type A (H1N1) infection in vitro, in ovo, and in vivo. In the post-treatment assay, FT extracts showed high CC50 (7,500 µg/ml), indicating low toxicity, and exerted moderate antiviral effects compared to oseltamivir (SI 50.6 vs. 222) in vitro. Antiviral activity tests in ovo revealed strong inhibitory effects of both FT extract and oseltamivir against H1N1 replication in embryonated eggs. Notably, at a treatment concentration of 150 mg/kg, only half the group administered oseltamivir survived whereas the FT group showed 100% survival, clearly demonstrating the low toxicity of FT extracts. Consistent with these findings, FT-administered mice showed a higher survival rate with lower body weight reduction relative to the oseltamivir group upon treatment 24 h after viral infection. Our collective results suggest that FT extracts exert antiviral effects against influenza H1N1 virus without inducing toxicity in vitro, in ovo or in vivo, thereby supporting the potential utility of FT extract as a novel candidate therapeutic drug or supplement against influenza.

Pigs are used as potential donor animals for xenotransplantation. However, porcine endogenous retrovirus (PERV), shown to infect both human and non-human primate (NHP) cells in vitro, presents a risk of transmission to humans in xenotransplantation. In this study, we analyzed PERV transmission in various organs after pig-to-NHP xenotransplantation. We utilized pig-to-NHP xenotransplant tissue samples obtained using two types of transgenic pigs from the National Institute of Animal Science (NIAS, Republic of Korea), and examined them for the existence of PERV genes in different organs via PCR and RT-PCR with specific primers. To determine PERV insertion into chromosomes, inverse PCR using PERV long terminal repeat (LTR) region-specific primers was conducted. The PERV gene was not detected in NHP organs in cardiac xenotransplantation but detected in NHP bladders in renal xenotransplantation. The insertion experiment confirmed that PERVs originate from porcine donor cells rather than integrated provirus in the NHP chromosome. We also demonstrate the presence of pig cells in the NHP bladder after renal xenotransplantation using specific-porcine mitochondrial DNA gene PCR. The PERV sequence was detected in the bladder of NHPs after renal xenotransplantation by porcine cell-microchimerism but did not integrate into the NHP chromosome.

BACKGROUND:The placenta is a reservoir enriched with growth factors, hormones, cytokines and minerals. While several beneficial effects of placenta extracts on wound healing, anti-aging and anti-inflammatory responses have been reported, relatively limited mechanistic exploration has been conducted to date. Here, we provide compelling evidence of anti-inflammatory and anti-oxidative activities of porcine placenta extracts (PPE) against contact dermatitis in vivo.METHODS:A contact dermatitis mouse model was established by sensitizing the dorsal skin of BALB/c mice using the contact allergen, 2,4-dinitrochlorobenzene (DNCB), and molecular consequences of topical application of PPE were investigated. PPEs were pre-sterilized via γ-irradiation, which is a milder but more effective way of sterilizing biomolecules relative to the conventional autoclaving method.RESULTS:DNCB-induced skin lesions displayed clear contact dermatitis-like symptoms and topical application of PPE dramatically alleviated both local and systemic inflammatory responses. Inflammatory epidermal thickening was completely abrogated and allergen-specific serum IgE levels significantly reduced in the presence of PPE. Moreover, anti-oxidative activities of PPE were observed both in vitro and in vivo, which may lead to attenuation of inflammatory responses. Prolonged treatment with PPE strongly inhibited production of DNCB-induced reactive oxygen species (ROS) and subsequently prevented oxidative degradation of hyaluronic acid (HA), which triggers innate inflammatory responses.CONCLUSION:Our findings supply valuable insights into the mechanisms underlying the anti-inflammatory effects of PPE and provide a functional basis for the clinical application of PPE in inflammatory diseases.

The emergence of oseltamivir-resistant variants of influenza virus has highlighted the necessity for the development of more effective novel antiviral drugs. To date, numerous researchers have focused on developing antiviral drugs using natural resources, such as traditional herbal medicines. Poncirus trifoliata is widely used in oriental medicine as a remedy for gastritis, dysentery, inflammation and digestive ulcers. In this study, we investigated the potential antiviral effect of the Poncirus trifoliata orange seed extract against influenza virus. An ethanol extract of Poncirus trifoliata seeds (PTex) inhibited the activity of influenza viruses, in particular, oseltamivir- resistant strains, in Madin-Darby canine kidney cells. In contrast to oseltamivir, PTex exerted a significant inhibitory effect on the cellular penetration pathway of the virus rather than HA receptor binding. The potent antiviral effect and novel working mechanism of PTex support its further development as an effective natural antiviral drug with a wide spectrum of activity against influenza and oseltamivir-resistant viruses.

PERV is a major virus concerning xenotransplantation study. However, the interesting part is that PERV is present in all kinds of pigs without pathogenicity and immune response. Furthermore, since pig cells have receptors for PERV, the gene delivery system using PERV envelope is highly likely to develop into an excellent viral vector in pigs. We developed a recombinant baculovirus with a modified surface for expressing the porcine endogenous retrovirus (PERV) envelope. Porcine reproductive and respiratory syndrome virus (PRRSV) infection is a severe concern in the porcine industry due to reproduction failure and respiratory symptoms. GP5 and M proteins are major immunogenic proteins of PRRSV. Using PERV-modified baculovirus (Ac mPERV) as a delivery vector, we constructed a dual antigen (GP5 and M)-encoding DNA vaccine system, Ac mPERV-C5/C6. Intramuscular immunization in mice and pigs, Ac mPERV-C5/C6 induced comparative high humoral and cellular immune responses. Our results support further development of Ac mPERV-C5/C6 as a potential PRRSV vaccine in the porcine industry. In addition, the Ac mPERV system may be applied to the generation of other effective DNA vaccines against porcine viral diseases.

Despite large economic losses attributable to white spot syndrome virus (WSSV), an infectious pathogen of penaeid shrimp and other crustaceans worldwide, no efficient vaccines or antiviral agents to control the virus are available at present. Here, we designed and constructed baculovirus-based vaccines delivering genes encoding the WSSV envelope proteins, VP28 and VP19. To enhance the immunogenicity of the baculovirus-based vaccine, we fused a Salmonella typhimurium flagellin 2 (FL2) gene with VP28 or VP19 gene. Both vaccine constructs elicited similar high titlers of anti-WSSV IgG after oral immunization in mice. The protective effect of oral vaccines upon WSSV challenge was observed in Macrobrachium nipponense. Bivalent vaccine displaying WSSV envelope proteins, VP19 and VP28, led to enhanced more than 10% survival protection against WSSV infection, compared to monovalent vaccine containing WSSV envelope protein, VP19 or VP28. Furthermore, a baculovirus-based WSSV vaccine fused with FL2 gene, Ac-VP28-ie1VP19FL2, efficiently protected mice against WSSV challenge (89.5% survival rate). In support of the efficacy of FL2 in our vaccine, we verified FL2 enhanced survival rate and induced the NF-κB gene in Palaemon paucidens. The collective results strongly suggest that our recombinant baculoviral system displaying WSSV envelope protein and delivering FL2-fused WSSV envelope gene effectively induced protective responses, supporting the utility of a potential new oral DNA vaccine against WSSV.

An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we reported the efficacy of influenza HA vaccination using a baculoviral DNA vaccine (AcHERV-HA). However, the immune response elicited by the AcHERV-HA vaccine, which only targets the HA antigen, was lower than that of the commercial killed vaccine. To overcome the limitations of this previous vaccine, we constructed a human endogenous retrovirus (HERV) envelope-coated, baculovirus-based, virus-like-particle (VLP)-forming DNA vaccine (termed AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). BALB/c mice immunized with AcHERV-VLP (1×10(7) FFU AcHERV-VLP, i.m.) and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than egg-based killed vaccines.

INTRODUCTION:The first identification of swine-originated influenza A/CA/04/2009 (pH1N1) as the cause of an outbreak of human influenza accelerated efforts to develop vaccines to prevent and control influenza viruses. The current norm in many countries is to prepare influenza vaccines using cell-based or egg-based killed vaccines, but it is difficult to elicit a sufficient immune response using this approach. To improve immune responses, researchers have examined the use of cytokines as vaccine adjuvants, and extensively investigated their functions as chemoattractants of immune cells and boosters of vaccine-mediated protection. Here, we evaluated the effect of Granulocyte-macrophage Colony-Stimulating Factor (GmCSF) as an influenza vaccine adjuvant in BALB/c mice.METHOD AND RESULTS:Female BALB/c mice were immunized with killed vaccine together with a murine GmCSF gene delivered by human endogenous retrovirus (HERV) envelope coated baculovirus (1 × 10(7) FFU AcHERV-GmCSF, i.m.) and were compared with mice immunized with the killed vaccine alone. On day 14, immunized mice were challenged with 10 median lethal dose of mouse adapted pH1N1 virus. The vaccination together with GmCSF treatment exerted a strong adjuvant effect on humoral and cellular immune responses. In addition, the vaccinated mice together with GmCSF were fully protected against infection by the lethal influenza pH1N1 virus.CONCLUSION:Thus, these results indicate that AcHERV-GmCSF is an effective molecular adjuvant that augments immune responses against influenza virus.

To confirm that Korean Food and Drug Administration (KFDA) guidelines are applicable to test the efficacy of mosquito repellents, these guidelines were used to test the efficacy and complete protection times (CPTs) of three representative mosquito repellents: N,N-diethyl-3-methylbenzamide (DEET), citronella, and fennel oil. The repellency of citronella oil decreased over time, from 97.9% at 0 h to 71.4% at 1 h and 57.7% at 2 h, as did the repellency of fennel oil, from 88.6% at 0 h to 61.2% at 1 h and 47.4% at 2 h. In contrast, the repellency of DEET remained over 90% for 6 h. The CPT of DEET (360 min) was much longer than the CPTs of citronella (10.5 min) and fennel oil (8.4 min). These results did not differ significantly from previous findings, and hence confirm that the KFDA guidelines are applicable for testing the efficacy of mosquito repellents.

Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×10(8) copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×10(9) copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×10(10) copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×10(9) copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses.

Cervical cancer is strongly associated with chronic human papillomavirus infections, among which HPV16 is the most common. Two commercial HPV vaccines, Gardasil and Cervarix are effective for preventing HPV infection, but cannot be used to treat existing HPV infections. Previously, we developed a human endogenous retrovirus (HERV)-enveloped recombinant baculovirus capable of delivering the L1 genes of HPV types 16, 18, and 58 (AcHERV-HP16/18/58L1, AcHERV-HPV). Intramuscular administration of AcHERVHPV vaccines induced a strong cellular immune response as well as a humoral immune response. In this study, to examine the therapeutic effect of AcHERV-HPV in a mouse model, we established an HPV16 L1 expressing tumor cell line. Compared to Cervarix, immunization with AcHERVHPV greatly enhanced HPV16 L1-specific cytotoxic T lymphocytes (CTL) in C57BL/6 mice. Although vaccination could not remove preexisting tumors, strong CTL activity retarded the growth of inoculated tumor cells. These results indicate that AcHERV-HPV could serve as a potential therapeutic DNA vaccine against concurrent infection with HPV 16, 18, and 58.

Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58.