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The K418 chimeric virus of porcine reproductive and respiratory syndrome virus (PRRSV) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of PRRSV, FL12, with the same region obtained from a Korean dominant field strain, LMY. The K418 reached 10(6) TCID50/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. The chimeric clone pK418 can be used as a practical tool for further studying the molecular characteristics of PRRSV proteins through genetic manipulation. Furthermore, successful construction of the K418 will allow for the development of customized vaccine candidates against PRRSV, which has evolved rapidly in Korea.

article i nfo We have previously reported that nasally administered Lactobacillus fermentum CJL-112 (CJL-112) efficiently im- provesresistanceagainst lethal influenza infection in both mice and chicken. The aim of the presentstudy was to understand the underlying mechanismsof the significant anti-influenza activity of this lactobacilli strain.In vitro, co-culturing of the chicken macrophage cell line HD-11 with CJL-112 significantly increased nitric oxide (NO) production. In vivo, CJL-112 was nasally administered to BALB/c mice for 21 days prior to influenza A/NWS/33 (H1N1) virus (IFV) infection. Significant up-regulation of T-helper 1 (Th1) cytokines (IL-2, IFN-γ) was observed, while the levels of T-helper 2 (Th2) cytokines (IL-4, IL-5, IL-10) was either reduced or unchanged than that in control mice were. Furthermore, IgA and specifi ca nti-influenza IgA levels increased significantly in the treated mice than those in untreated mice. Therefore, CJL-112 likely protects the mice against lethal IFV infection via stimulation of macrophages, activation of Th1 and augmentation of IgA production, when directly delivered into the respiratory tract. © 2013 Published by Elsevier B.V.

Introduction/Background The aim of this study was to investigate the correlations between the modified Rankin Scale (mRS) grades and Korean versions of the MBI (K-MBI) scores in assessing the residual functional status of stroke survivors. Material and method The Korean versions of the MBI and mRS scales were administered to 5759 ischemic stroke patients at 3 months after onset of stroke. The sensitivity and specificity were calculated at all possible K-MBI score cutoffs for each mRS grade in order to obtain the optimally corresponding K-MBI scores and mRS grades. Receiver operator characteristic (ROC) curves and the area under the curve (AUC) was calculated. Results The K-MBI cutoff points with the highest sum of sensitivity and specificity were 100 (sensitivity 0.940; specificity 0.612), 98 (sensitivity 0.904; specificity 0.838), 94 (sensitivity 0.885; specificity 0.937), 78 (sensitivity 0.946; specificity, 0.973), and 55 (sensitivity 937; specificity 0.986) for mRS grades 0, 1, 2, 3, and 4, respectively. The AUC for the ROC curve was 0.791 for mRS grade 0, 0.919 for mRS grade 1, 0.970 for mRS grade 2, 0.0 for mRS grade 3, and 0.991 for mRS grade 4. Conclusion The K-MBI cutoff score ranges for representing mRS grades were variable. mRS grades 0, 1, and 2 had narrow K-MBI score ranges, while mRS grades 3, 4, and 5 showed broad K-MBI score ranges. mRS grade seemed to sensitively differentiate mild residual disability of stroke survivors, whereas K-MBI can provide more specific information of the functional status of stroke survivors with moderate to severe residual impairment.

Attenuated live infectious laryngotracheitis (ILT) virus (ILTV) vaccines have been used to prevent and control the outbreak of ILT worldwide. Recent studies using high-throughput sequencing technology have increased the number of complete genome sequences of ILTVs, enabling comparative genome analysis. Although 37 complete genome sequences of ILTV, including vaccine strains, have been reported, the complete genome sequence of any field strain of ILTV in South Korea is yet to be published. In this study, we determined and analyzed the complete genome sequences of three virulent Korean field strains of ILTV (40798/10/Ko, 0206/14/Ko, and 30678/14/Ko). Two of the Korean field strains (40798/10/Ko and 0206/14/Ko) displayed fewer non-synonymous single nucleotide polymorphisms than those of the Serva vaccine strain, indicating that these Korean field strains of ILTV most likely originated from the vaccine strain. The third ILTV strain, 307678/14/Ko, had two regions in the genome showing recombination between the Serva vaccine-like strain and the Australian A20 vaccine-like strain. Comparative genome analysis of ILTV using the Korean field strains with variable virulence can shed light on the recent trend of the emergence of virulent ILTV strains in the field. A few amino acid changes in the genome of ILTV vaccines could enhance the virulence in the vaccine strain, and natural recombination should be considered one of the major risks for the generation of revertant strains of ILTV under field conditions.

Matrix 2 protein ectodomain (M2e) of influenza virus appears to be a promising vaccine candidate because its sequence is highly conserved among virus strains. However, M2e is too meager to induce a strong immune response by itself. Several approaches are being used to increase the antigenicity of M2e. In an effort to enhance the M2e-specific immune response, we generated a TAT-conjugated M2e recombinant protein. Seven-week-old BALB/c mice were divided into three groups and transcutaneously immunized with 100 μg TAT-8×M2e (TAT conjugated with eight copies of M2e) and 8×M2e (eight copies of M2e) proteins on days 1, 15 and 29. The control mice were injected with PBS on the same days. Antibody titers specific for M2e were measured using indirect ELISA. Mice immunized with the TAT-8×M2e and 8×M2e proteins developed almost the same levels of M2e-specific total IgG and IgG1 antibodies. However, a higher level of M2e-specific IgG2a was observed in mice immunized with TAT-8×M2e than in those immunized with 8×M2e. These results suggest that TAT has an adjuvant effect that induces a Th1-type immune response. Therefore, the TAT-M2e vaccine can be applied to animals as a new influenza vaccine for enhancement of Th1-type immune responses.

The glycoprotein 3 (GP3) of type II porcine reproductive and respiratory syndrome virus has the characteristic domains of a membrane protein. However, this protein has been reported to be retained in the endoplasmic reticulum (ER) rather than transported to the plasma membrane of the cell. In this study, we performed confocal laser scanning microscopy analysis of variants of GP3 and found that the signal sequence of the GP3 led to confinement of GP3 in the ER, while the functional ortransmembrane domain did not affect its localization. Based on these results, we concluded that the signal sequence of GP3 contains the ER retention signal, which might play an important role in assembly of viral proteins.

Infectious bronchitis virus (IBV) infections cause great economic losses to the poultry industry worldwide. IBVs continuously evolve by developing mutations in antigenic sites; therefore, an IBV vaccine that provides broad cross-protection can be a highly relevant and practical method in IBV control strategies. Although some IBV vaccine strains are known to provide protection against multiple IBV serotypes, in general commercially available IBV vaccine strains provide protection against antigenically related viruses but not distinct heterologous viruses. In the present study we characterized the Korean variant IBV K40/09 strain with regard to its immunogenicity and protective efficacy against seven currently circulating IBV serotypes. Three-week-old specific-pathogen-free chickens were intraocularly immunized with the IBV K40/09 strain at 10(3.5) 50% egg infective dose (EID50). Three weeks after immunization all the birds were challenged with seven different strains at 10(4.5) EID50. Chickens immunized with the IBV K40/09 strain showed significantly high levels of protection against all challenge viruses at the trachea and kidney levels. Our results suggest that IBV K40/09 could be useful to ensure IBV vaccine effectiveness owing to its cross-protective ability. Therefore, the IBV K40/09 strain merits consideration as a vaccine candidate to prevent infection as well as the spread of new IBV strains and many IBV variants that have been reported worldwide.

PURPOSE Porcine proliferative enteropathy (PPE) is known as one of the most important risk factors causing economic losses in swine industry worldwide. This study was conducted to evaluate the efficacy of a commercial oral attenuated Lawsonia intracellularis vaccine (Enterisol Ileitis) against PPE under a commercial pig farm condition in Korea. MATERIALS AND METHODS Thirty two-day-old 672 piglets were randomly allocated into vaccinated and control groups. All piglets in the vaccinated group were inoculated with a commercial attenuated L. intracellularis vaccine as following the manufacturer's instruction. Body weights of all pigs in both groups were measured on the vaccination day and 6, 14, and 20 weeks post vaccination and an average daily weight gain (ADWG) was calculated. Health status was observed biweekly during the whole trial. RESULTS The vaccinated group showed significantly higher body weight (p

Korea experienced its fifth Foot-and-Mouth Disease (FMD) outbreak (type O) since 1934 from November 2010 to April 2011. The Korean government initiated emergency vaccination for all FMD-susceptible domestic animals in December 2010 using type O FMD vaccines. Starting in September 2011, trivalent FMD vaccines (types O, A, and Asia1) were used for the vaccination of all animals. This study was performed to identify the appropriate time for FMD vaccination in growing pigs when vaccination is applied only once (at either 8 weeks or 12 weeks of age). Seroprevalences from growing pigs under different vaccination regimens (once or twice) were also studied. A total of 526 growing pigs on 7 farms were used in this study. This study showed that the vaccination of growing pigs at both 8 and 12 weeks of age resulted in higher seroprevalences (97.5% in type O, 92.3% in type A and 99.4% in type Asia1) than did a single vaccination at 8 weeks of age (86.7% in type O, 88.0% in type A and 93.0% in type Asia1) (P

The extensive morbidity and mortality caused by influenza A viruses worldwide prompts the need for a deeper understanding of the host immune response and novel therapeutic and/or prophylactic interventions. In this study, we assessed the sublingual route as an effective means of delivering probiotics against influenza virus in mice. In addition, IgA levels, NK cell activity, T cell activation, and cytokine profiles in the lungs were examined to understand the mechanism underlying this protective effect. Sublingual administration of Lactobacillus rhamnosus provided enhanced protection against influenza virus infection by enhancing mucosal secretory IgA production, and T and NK cell activity. Moreover, interleukin (IL)-12 levels in the lungs increased significantly. Conversely, IL-6 and tumor necrosis factor alpha levels in the lungs decreased significantly. On the basis of these promising findings, we propose that the sublingual mucosal route is an attractive alternative to mucosal routes for administering probiotics against influenza virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces reproductive failure in sows and respiratory problems in pigs of all ages. Live attenuated and inactivated vaccines are used on swine farms to control PRRSV. However, their protective efficacy against field strains of PRRSV remains questionable. New vaccines have been developed to improve the efficacy of these traditional vaccines. In this study, virus-like particles (VLPs) composed of the GP5 and M proteins of PRRSV were developed, and the capacity of the VLPs to elicit antigen-specific immunity was evaluated. Serum antibody titers and production of cytokines were measured in BALB/C mice immunized intramuscularly three times with different doses (0.5, 1.0, 2.0, and 4.0 μg) of the VLP vaccine. A commercial vaccine consisting of inactivated PRRSV and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. IgG titers to GP5 were significantly higher in all groups of mice vaccinated with the VLPs than in control mice. Neutralizing antibodies were only detected in mice vaccinated with 2.0 and 4.0 μg of the VLPs. Cytokine levels were determined in cell culture supernatants after in vitro stimulation of splenocytes with the VLPs for 3 days. Mice immunized with 4.0 μg of the VLPs produced a significantly higher amount of interferon-gamma (IFN-γ) than mice immunized with the commercial inactivated PRRSV vaccine and PBS. In contrast, immunization with the commercial vaccine induced higher production of IL-4 and IL-10 in mice than mice vaccinated with VLPs. These data together demonstrate the capacity of VLPs to induce both neutralizing antibodies and IFN-γ in immunized mice. The VLP vaccine developed in this study could serve as a platform for the generation of improved VLP vaccines to control PRRSV.

Arsenic (As) is highly toxic and carcinogenic to humans. It is a frequently found contaminant in contaminated sites due to its many industrial applications such as tanning, wood preservation, pesticide and herbicide. We developed a combined process of dispersion and magnetic separation for the remediation of As contaminated soil. We collected the As contaminated silt loam soil from a smelting site. We tested the soil dispersion rate at various pH and concentrations of Na-orthophosphate, Na-pyrophosphate and Na-hexametaphosphate and determined an optimum dispersion condition. We also tested the change in As concentration of the soil after and before passing its suspension through a magnetic field. The dispersion rate increased with increasing phosphate concentration and showed the following order at the same concentration: hexametaphosphate > pyrophosphate > orthophosphate. The dispersion rate also sharply increased at the solution pH 11. We concluded that the optimum dispersion condition was pH 11 10mM Na-hexametaphosphate. The soil dispersed at the optimum conditions was separated into magnetic and nonmagnetic particles with a high gradient magnetic separator (HGMS) at 12,000 gauss. The bulk soil with 37.7 mg kg -1 of As was separated into 20% of magnetic fraction and 80% of nonmagnetic fraction. The magnetic fraction had a higher As concentration, 48.0 mg kg -1 but the nonmagnetic fraction had a lower As concentration, 20.0 mg kg -1 , comparing with the bulk soil. The result

Background Interferon gamma (IFN-γ), an immunoregulatory cytokine, is known to control many microbial infections. In a previous study, chicken interferon gamma (chIFN-γ) was found to be up-regulated following avian influenza virus (AIV) infection in specific pathogen-free chickens. We aimed to investigate whether the pre-immune state induced by chIFN-γ could generate an antiviral response against influenza virus. Methods We generated a chIFN-γ-expressing plasmid and transfected it into chicken embryo fibroblasts (CEFs) and then infected the cells with human origin H1N1 or avian origin H9N2 influenza viruses. Viral titers of culture medium were evaluated in MDCK cell and the viral RNA and IFN-stimulated genes (ISGs) were then quantified by real-time reverse transcriptase polymerase. To further evaluate the role of the antiviral effect of chIFN-γ by using a backward approach, synthetic small interfering RNAs (siRNA) targeting chIFN-γ were used to suppress chIFN-γ. Results The chIFN-γ-stimulated CEFs inhibited the replication of viral RNA (vRNA) and showed a mild decrease in the infectious virus load released in the culture medium. Compared to the mock-transfected control, the messenger RNA (mRNA) levels of type I IFNs and IFN-stimulated genes were up-regulated in the cells expressing chIFN-γ. After treatment with the siRNA, we detected a higher expression of viral genes than that observed in the mock-transfected control. Conclusions Our results suggest that apart from the important role played by chIFN-γ in the antiviral state generated against influenza virus infection, the pre-immune state induced by chIFN-γ can be helpful in mitigating the propagation of influenza virus. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0527-1) contains supplementary material, which is available to authorized users.

The classical swine fever (CSF) vaccine, which is derived from the LOM strain of the CSF virus (CSFV), induces protective immunity against CSFV infection. However, several factors influence vaccine efficacy. Evidence suggests that infection by porcine reproductive and respiratory syndrome virus (PRRSV) and/or porcine circovirus 2 (PCV2) reduces the efficacy of several vaccines. Here, we examined the effect of PRRSV or PCV2 alone or co-infection by PRRSV/PCV2 on the potency of the LOM vaccine in pigs. Neither CSFV antibody levels nor the period during which CSFV antigens were detectable in LOM-vaccinated pigs were negatively affected by infection by PRRSV or PCV2. However, co-infection with PRRSV/PCV2 may affect the replication or activity of the CSF vaccine virus in pigs vaccinated with the LOM strain, although CSFV antibody levels were not negatively affected. Nevertheless, the LOM vaccine afforded complete protection against a virulent strain of CSFV.

Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4 kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level.

Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND) are considered as the most devastating poultry infections, owing to their worldwide distribution and economical threat. Vaccines have been widely used to control these diseases in the poultry industry in endemic countries. However, vaccination policy without differentiating infected animals from vaccinated animals (DIVA) makes the virus surveillance difficult. In this study, we developed a bivalent virus-like particle (VLP) vaccine that is composed of the hemagglutinin (HA) and matrix 1 (M1) proteins of the H5N1 HPAI virus (HPAIV) and a chimeric protein containing the ectodomain of the ND virus (NDV) fusion (F) protein fused with the cytoplasmic and transmembrane domains of the HPAIV HA protein. A single immunization of chickens with the chimeric VLP vaccine induced high levels of hemagglutination inhibition (HI) antibody titers against H5N1 HPAI virus and anti-NDV antibody detected in ELISA and protected chickens against subsequent lethal HPAIV and NDV infections. Furthermore, we could easily perform DIVA test using the commercial NP-cELISA tests against HPAIV and HI assay against NDV. These results strongly suggest that utilization of chimeric VLP vaccine in poultry species would be a promising strategy for the better control of HPAI and ND simultaneously.

CEFs were transfected with 50nM and 10nM concentration of siRNA targeting chIFN-γ. The cells were harvested at 12 and 24 h post transfection and analyzed by rRT-PCR. Relative expression levels of chIFN-γ mRNA was normalized and calculated using the comparative 2-2∆∆Ct method. Error bars are standard deviation of the average. Asterisk represent significance when compared to control siRNA transfection (P

Genes, GenBank Accession numbers, and Primers used in Real-time RT-PCR. (DOCX 14 kb)

Objective: Change in thoracic fluid content (TFC) derived via a bioreactance technique with a noninvasive cardiac output monitoring device (NICOM) reportedly shows a good correlation with the amount of fluid removed. The present study prospectively evaluated the utility and clinical application of TFC in the intraoperative fluid management of paediatric patients with congenital heart disease, undergoing cardiac surgery with bioreactance-based noninvasive monitoring. Methods: Haemodynamic parameters, patient body weight and parameters derived from the NICOM device (including cardiac output, cardiac index, TFC, percentage change in TFC compared with baseline [TFCd0%] and stroke volume variation) were recorded after anaesthesia induction but before surgical incision, and just before departure from the operating room to the intensive care unit. Results: In the 80 paediatric patients included in this study, linear regression analyses demonstrated good correlations between body weight gain and TFCd0%, between body weight gain % and TFCd0%, and between intra -operative fluid balance and TFCd0%. Conclusion: TFCd0% may be a useful indicator for intraoperative fluid management in paediatric patients with congenital heart disease, undergoing cardiac surgery.

Interferons (IFNs) are commonly grouped into type I and type II IFN. Type I IFNs are known as antiviral IFNs including IFN-α, IFN-β, and IFN-ω whereas type II IFN is referred to immune IFN and IFN-γ is only member of the type II IFN. Type I IFNs are induced by virus invading however type II IFN is produced by mitogenic or antigenic stimuli. IFN-τ was first identified in ruminant ungulates as a pregnancy recognition hormone, trophoblastin. IFN-τ constitutes a new class of type I IFN, which possesses the common features of type I IFN, such as the ability to prevent viral infection and to limit cell proliferation. In addition, IFN-τ is unique in that it is induced by pregnancy unlike other type I IFNs. We cloned Bos taurus ( B. T. ) Coreanae IFN-τ from peripheral blood mononuclear cells. The amino acid sequence of B. T. Coreanae IFN-τ shares only 90.3% identity with that of Holstein dairy cow. Recombinant B. T. Coreanae and Holstein IFN-τ proteins were expressed in Escherichia coli and the antiviral activity of IFN-τ proteins were examined. Both recombinant proteins were active and protected human WISH and bovine MDBK cells from the cytopathic effect of vesicular stomatitis virus. The recombinant IFN-τ protein of B. T. Coreanae and Holstein properly induced the expression of antiviral genes including 2′,5′-oligoadenylate synthetase (OAS) and Mx GTPase 1 (Mx-1).

Purpose In spite of an extensive vaccination program, parvoviral infections still pose a major threat to the health of dogs. Materials and Methods We isolated a novel canine parvovirus (CPV) strain from a dog with enteritis. Nucleotide and amino acid sequence analysis of the isolate showed that it is a novel type 2b CPV with asparagine at the 426th position and valine at the 555th position in VP2. To develop a vaccine against CPV infection, we passaged the isolate 4 times in A72 cells. Results The attenuated isolate conferred complete protection against lethal homologous CPV infection in dogs such that they did not develop any clinical symptoms, and their antibody titers against CPV were significantly high at 7-11 days post infection. Conclusion These results suggest that the virus isolate obtained after passaging can be developed as a novel vaccine against paroviral infection.

Background Currently, Asian lineage highly pathogenic avian influenza (HPAI) H5N1 has become widespread across continents. These viruses are persistently circulating among poultry populations in endemic regions, causing huge economic losses, and raising concerns about an H5N1 pandemic. To control HPAI H5N1, effective vaccines for poultry are urgently needed. Objective In this study, we developed HPAI virus-like particle (VLP) vaccine as a candidate poultry vaccine and evaluated its protective efficacy and possible application for differentiating infected from vaccinated animals (DIVA). Methods Specific pathogen-free chickens received a single injection of HPAI H5N1 VLP vaccine generated using baculovirus expression vector system. Immunogenicity of VLP vaccines was determined using hemagglutination inhibition (HI), neuraminidase inhibition (NI), and ELISA test. Challenge study was performed to evaluate efficacy of VLP vaccines. Results and Conclusions A single immunization with HPAI H5N1 VLP vaccine induced high levels of HI and NI antibodies and protected chickens from a lethal challenge of wild-type HPAI H5N1 virus. Viral excretion from the vaccinated and challenged group was strongly reduced compared with a mock-vaccinated control group. Furthermore, we were able to differentiate VLP-vaccinated chickens from vaccinated and then infected chickens with a commercial ELISA test kit, which offers a promising strategy for the application of DIVA concept.

The detection of West Nile virus (WNV) in areas endemic for Japanese encephalitis virus (JEV) is complicated by the extensive serological cross-reactivity between the two viruses. A testing algorithm was developed and employed for the detection of anti-WNV antibody in areas endemic for JEV. Using this differentiation algorithm, a serological survey of poultry (2004 through 2009) and horses (2007 through 2009) was performed. Among 2681 poultry sera, 125 samples were interpreted as being positive for antibodies against JEV, and 14 were suspected to be positive for antibodies against undetermined flaviviruses other than WNV and JEV. Of the 2601 horse sera tested, a total of 1914 (73.6%) were positive to the initial screening test. Of these positive sera, 132 sera (5.1%) had been collected from horses that had been imported from the United States, where WNV is endemic. These horses had WNV vaccination records, and no significant pattern of increasing titer was observed in paired sera tests. Of the remaining 1782 positive sera 1468 sera (56.4%) were also found to contain anti-JEV antibodies, and were interpreted to be JEV-specific antibodies by the differentiation algorithm developed in this study. The remaining 314 horses (12.1%) for which a fourfold difference in neutralizing antibody titer could not be demonstrated, were determined to contain an antibody against an unknown (unidentified or undetermined) flavivirus. No evidence of WNV infections were found during the period of this study.

Suppression of interleukin (IL)-6 production has beneficial effects against various inflammatory diseases. Through a rapid screening system, we found that nitazoxanide, or 2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide, which is a well-known antiparasitic agent, suppressed lipopolysaccharide (LPS)-induced production of IL-6 from RAW 264.7 cells and mouse peritoneal macrophages, with 50% inhibitory concentrations (IC(50)s) of 1.54 mM and 0.17 mM, respectively. Nitazoxanide also inhibited the LPS-induced expression of IL-6 mRNA in RAW 264.7 cells. To investigate the effects of nitazoxanide in vivo, we orally administered nitazoxanide at a dose of 100mg/kg to mice 2h before a 1-mL intraperitoneal injection of 4% thioglycollate (TG). Six hours after TG injection, plasma IL-6 levels were markedly lower (by 90%) than the levels in vehicle-treated mice. These data suggest that nitazoxanide could be a promising lead compound for agents against various diseases associated with overproduction of IL-6.

Newcastle disease virus (NDV) has a worldwide distribution and is often carried by wild ducks, which may represent one of the natural reservoirs. However, the epidemiological relatedness of NDV between wild ducks and domestic poultry is unclear. A total of 14 isolates were obtained from 8439 samples from live bird markets (LBMs) and wild bird populations in Korea during from 2007 to 2010. These isolates were characterized genetically and phylogenetic analysis was conducted to investigate the relatedness between isolates from wild birds, LBM and poultry farms. In phylogenetic analysis, all 14 isolates belonged to genotype I virus within class II. Of these, nine isolates from wild birds were most closely related to the Aomori-like cluster. The five LBM isolates were most closely related to the V4-like cluster. All isolates in this study were closely related to isolates from domestic duck farms in Korea and Chinese LBM isolates. The results indicate that NDV exchange occurs between wild birds, poultry farms, LBMs and neighboring countries. Enhanced NDV surveillance is required to monitor the introduction of variant NDV in consequence of evolution in LBMs and to investigate NDV epidemiology in various species of putative hosts.

An outbreak of avian influenza, caused by an H9N2 low-pathogenic avian influenza virus (AIV), occurred in a chicken farm and caused severe economic losses due to mortality and diarrhea. AIV was isolated and identified in a sample from an affected native Korean chicken. Genetic analysis of the isolate revealed a high sequence similarity to genes of novel reassortant H9N2 viruses isolated from slaughterhouses and live bird markets in Korea in 2008 and 2009. Animal challenge studies demonstrated that the replication kinetics and pathogenicity of the isolate were considerably altered due to adaptation in chickens. Vaccine protection studies indicated that commercial vaccine was not able to prevent virus shedding and clinical disease when chickens were challenged with the isolate. These results suggest that the novel H9N2 virus possesses the capacity to replicate efficiently in the respiratory system against vaccination and to cause severe disease in domestic chickens. The results also highlight the importance of appropriate updating of vaccine strains, based on continuous surveillance data, to prevent the possibility of a new H9N2 epidemic in Korea.

The highly pathogenic avian influenza (HPAI) subtype H5N1 virus has caused continuous outbreaks in the poultry industry with devastating economic losses and is a severe threat to public health. In the present study, we developed a low-density microarray for the rapid detection and identification of avian influenza virus hemagglutinin subtypes H5, H7, and H9 and neuraminidase subtypes N1, N2, and N3. We evaluated this diagnostic microarray using 6 HPAI H5N1 clade 2.3.2 viruses that caused the South Korean outbreaks during the 2010–2011 winter season. Cy3-labeled DNA targets were generated by reverse transcription polymerase chain reaction (RT-PCR) using Cy3-labeled universal primers. The Cy3-labeled RTPCR products were then hybridized to the microarray. All subtypes and pathotypes were correctly determined and were found to be identical to the nucleotide sequencing results. This diagnostic microarray has enormous potential for the rapid subtyping and pathotyping of avian influenza viruses.

Since 2007, 55 adenovirus strains have been isolated from commercial chicken flocks in Korea and have been identified and the pathogenicity of these isolates was confirmed in specific-pathogen-free chickens of different age. Based on sequencing analysis of the hexon gene, 55 FAdV isolates were genetically related to the IBH-2A strain of FAdV3 (4 isolates, 99.2% to 100%), the KR5 strain of FAdV4 (22 isolates, 97.9% to 99.2%), the 764 strain of FAdV9 (11 isolates, 99.1% to 99.3%), and the 1047 strain of FAdV11 (18 isolates,99%). Experimental infections with four serotypes of FAdV resulted in high mortality of 18-day-old chicken embryos and 1-day-old chicks with marked liver necrosis similar to those observed in the natural outbreaks. Notably, specific hydropericardium was observed in chicks challenged with the K531 strain (serotype 4). However, 3-wk-old chickens challenged with FAdVs, regardless of serotype, did not show any clinical signs or mortality except histologic lesions of focal hepatocytic necrosis with mild lymphocytic infiltration. The results indicate that four FAdV serotypes (3, 4, 9, and 11) are the dominant serotypes of FAdVs in the Korea and are pathogenic enough to cause clinical disease in young chicks. The present investigation provides important information on the epidemiology and pathogenesis of FAdVs and highlights the importance of control strategies against FAdV infection in Korea.

Highly pathogenic avian influenza (HPAI) H5N1 virus circulates among a variety of free-ranging wild birds and continually poses a threat to animal and human health. During the winter of 2010-2011, we surveyed Korean wild bird habitats. From 728 fresh fecal samples, 14 HPAI H5N1 viruses were identified. The isolates phylogenetically clustered with other recently isolated clade 2.3.2 HPAI H5N1 viruses isolated from wild birds in Mongolia. All HPAI-positive fecal samples were analyzed by DNA barcoding for host-species identification. Twelve of the 14 HPAI-positive samples were typed as Mandarin Duck (Aix galericulata). The high incidence of HPAI subtype H5N1 viruses in wild Mandarin Duck droppings is a novel finding and underscores the need for enhanced avian influenza virus surveillance in wild Mandarin Ducks. Further investigation of the susceptibility of Mandarin Ducks to HPAI H5N1 clade 2.3.2 virus would aid the understanding of HPAI ecology and epidemiology in wild birds.

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions.

The infectious bronchitis virus (IBV) is continuously evolving through point mutation and recombination of their genome, subsequently the emergence of IBV variants complicates disease control. The objective of this study was to investigate genetic characterization of new IBV variants isolated from commercial chicken flocks in Korea collected between 2005 and 2010. Phylogenetic analysis revealed that all new IBV isolates belonged to Korean group II (K-II), which included the nephropathogenic IBV strains. However, the isolates formed a new gene cluster that was distinguished from the two distinct K-II subgroups (KM91-like and QX-like). Recombination events were identified in the S1 gene, with their putative parental strains being the KM91-like or QX-like subgroup. In addition, two crossover sites were observed in the S1 gene of IBV isolates. These results suggest that natural genetic recombination between heterologous strains classified into different genetic groups has occurred and may have caused the emergence of new IBV strains. This finding provides important information on IBV evolution and is essential for the effective control of IB in Korea.

It is well known that Norovirus (NoV) and Sapovirus (SaV) identified in humans and pigs have heterogeneous genome sequences. In this study, a total of three strains of NoV and 37 strains of SaV were detected in 567 porcine fecal samples by RT-PCR, corresponding detection rates of 0.5 and 6.5%, respectively. Phylogenetic analyses were conducted using amino acid sequences of the partial RNA-dependent RNA polymerase (RdRp) and complete capsid proteins of both viruses to determine their genogroups. Analysis with the RdRp sequences indicated that all three NoV strains HW41, DG32, and DO35 detected in this study were classified into genogroup II (GII). A further analysis with the complete capsid sequence demonstrated that the DO35 strain belonged to subgenotype b in GII-21 (GII-21b) along with the SW918 strain. A total of 26 strains out of 27 strains that were selected from the 37 porcine SaVs were classified into genogroup III when they were analyzed with the RdRp sequences. The remaining strain (DO19) was not clustered with any of the previously classified SaV strains, thereby suggesting the advent of a new genogroup virus. Additional analyses with the amino acid sequence of the capsid and the nucleotide sequence of the RdRp and capsid junction region supported the notion that the DO19 strain belonged to a novel genogroup of SaV. To the best of our knowledge, this is the first report to describe a novel porcine SaV belonging to an unknown genogroup in Korea.

Avian influenza viruses (AIV) can be genetically distinguished by geographical origin. The present study found evidence of intercontinental transfer of North American lineage AIV into Asia via migratory bird populations. The North American lineage genes were detected in live animal markets during avian influenza surveillance, seemed to have reassorted with Eurasian AIV in wild bird habitats, and had transmitted to live animal markets. Enhanced AIV surveillance is required to understand the influence of newly transferred North American lineage AIV genes on AIV evolution in Asia and to investigate AIV ecology in various transcontinental migrant species.

Interleukin-32 (IL-32) is a new cytokine produced mainly by T-cells, natural killer cells, and epithelial cells after stimulation with IL-2, IL-12 plus IL-18, and IFNγ, respectively. IL-32 induces various proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8 in monocytes and macrophages. In this study, we cloned bovine IL-32 cDNA from peripheral blood mononuclear cells (PBMC) of a Holstein ( Bos Taurus ). The bovine IL-32 cDNA has an entire open reading frame, encoding 171 amino acid residues and the deduced amino acid sequence has 27.5% identity with human IL-32 beta isoform. Recombinant bovine IL-32 protein was produced in E. coli and its molecular weight was approximately 26 kDa. The biological activity of the bovine IL-32 was examined for cytokine induction using human monocytic THP-1 cells and bovine PBMC. The THP-1 cells responded to stimulation by recombinant bovine IL-32 and produced IL-8. Stimulation by recombinant bovine IL-32 induced mRNA for TNFα and IL-6 in bovine PBMC suggesting conservation of the biological activity and function in cattle.

Infection of intestinal crypt epithelial cells by the obligate intracellular bacterium Lawsonia intracellularis is directly linked to marked proliferation of the infected enterocytes within 3-5days post-infection. The virulence factor for this unique host cell-proliferative response is not known, but is considered to involve altered crypt cell cycle or differentiation events. McCoy mouse fibroblast cells were infected with L. intracellularis, and then harvested for expressed mRNA at daily time points, with matching non-infected control cell cultures. Mouse DNA microarray (>44,000 transcript targets) analysis of cDNA derived from matching mRNA samples showed over 40 identifiable genes with at least 4-fold changes between days 0 and 3 after infection with L. intracellularis. These included altered transcription of typical host cell 'alarm' response genes, such as interferon-related response genes Isgf3g and Igtp, known to be associated with invading microbial agents. Altered transcription of several genes in these cells known to be active in regulation of the cell cycle or cell differentiation genes, including usp18, Hr, Elavl2 and Slfn2, were also detected. The altered transcription of several of these genes via RT-PCR analysis was confirmed. The microarray-detected altered transcription of cell cycle and cell differentiation genes is of possible interest for links to Lawsonia-related disturbances in epithelial cell differentiation within the intestinal crypt, but this would need to be confirmed in intestinal epithelial cell studies.

Avian influenza virus (AIV) circulates among free-ranging, wild birds. We optimized and validated a DNA barcoding technique for AIV isolation and host-species identification using fecal samples from wild birds. DNA barcoding was optimized using tissue and fecal samples from known bird species, and the method was shown to distinguish 26 bird species. Subsequently, fecal samples (n=743) collected from wild waterfowl habitats confirmed the findings from the laboratory tests. All identified AIV-positive hosts (n=35) were members of the order Anseriformes. We successfully applied the DNA barcoding technique to AIV surveillance and examined AIV epidemiology and host ecology in these wild waterfowl populations. This methodology may be useful in the design of AIV surveillance strategies.

Avian metapneumovirus (aMPV) causes upper respiratory tract infections in chickens and turkeys. Although the swollen head syndrome (SHS) associated with aMPV in chickens has been reported in Korea since 1992, this is the study isolating aMPV from chickens in this country. We examined 780 oropharyngeal swab or nasal turbinate samples collected from 130 chicken flocks to investigate the prevalence of aMPV and to isolate aMPV from chickens from 2004-2008. Twelve aMPV subtype A and 13 subtype B strains were detected from clinical samples by the aMPV subtype A and B multiplex real-time reverse transcription polymerase chain reaction (RRT-PCR). Partial sequence analysis of the G glycoprotein gene confirmed that the detected aMPVs belonged to subtypes A and B. Two aMPVs subtype A out of the 25 detected aMPVs were isolated by Vero cell passage. In animal experiments with an aMPV isolate, viral RNA was detected in nasal discharge, although no clinical signs of SHS were observed in chickens. In contrast to chickens, turkeys showed severe nasal discharge and a relatively higher titer of viral excretion than chickens. Here, we reveal the co-circulation of aMPV subtypes A and B, and isolate aMPVs from chicken flocks in Korea.

In a previous study, we optimized DNA barcoding techniques for avian influenza virus (AIV) isolation and host identification, using fecal samples from wild birds, for high-throughput surveillance of migratory waterfowls. In the present study, we surveyed AIV in Mongolia during the breeding season and, subsequently, in Korea in winter, to compare prevalent AIV subtypes and hosts using DNA barcoding. In Korea, H4 and H5 subtypes were the most abundantly detected HA subtypes, and most AIVs were isolated from the major population (mallards, Anas platyrhynchos) of wild bird habitats. On the other hand, in Mongolia, H3 and H4 subtypes were the most abundantly detected HA subtypes, and most AIVs were isolated from a small population of wild bird habitats that were not visible at the sampling site. In conclusion, AIV isolation using fecal samples, accompanied with DNA barcoding techniques as a host bird species identification tool, could be useful for monitoring major and minor populations of wild bird habitats. Further, continuous, and large-scale surveillance could be helpful for understanding the AIV epidemiology, evolution, and ecology in wild waterfowl.

Zoonotic transmission of hepatitis E virus (HEV) is mostly mediated by HEV-3 and HEV-4 genotypes, and domestic pigs are an important reservoir of these genotypes. A survey of 14 pig farms in Korea revealed HEV RNA in 30 of 148 (20.3%) fecal samples. HEV-3a and HEV-4c subtypes were identified in five pig farms (35.7%) and two pig farms (14.3%), respectively. Phylogenetic analysis indicated that the isolated HEV strains were closely related to previously reported zoonotic strains in Korea. The results of the genetic analysis partially explain the possible source of the zoonotic transmission of HEV to humans in Korea.

본 연구는 한우의 모색발현에 정확히 어떤 유전자가 어떤 유전기작에 의해 관여하고 있는가를 규명하고자 황갈색의 모색을 지닌 한우와 검정모색을 지닌 홀스타인과의 교배를 통해 만든 F2집단의 DNA를 이용하여, MC1R, ASIP 및 TYRP1 유전자형과 한우모색 발현양상을 연관분석 하였으며, 또 한우 집단내에서의 이들 유전자형 빈도를 조사하여 황갈색 한우모색 다양성과 후보유전자 변이의 연관성연구에 필요한 정보를 제공하고자 하였다. MC1R 유전자의 경우 황갈색을 지닌 3두의 유전자형은 모두 e/e 형으로 밝혀졌으며, 검정모색을 지니고 태어난 나머지 3두의 두 좌위에서의 유전자형은 ED/e임을 확인하였는데 황갈색과 검정모색의 비율이 1:1로 나온다는 것은 MC1R 단일유전자가 한우의 모색에 중요한 영향을 미치는 것으로 사료된다. MC1R 이외의 모색발현에 영향을 줄 수 있는 ASIP와 TYRP1 유전자들은 F2 집단에서 염기서열을 분석한 결과 이들 유전자들이 한우 황갈색모색에 주된 영향을 미치지 않는 것으로 나타났다. 하지만 TYRP1 유전자에서 발견된 329번 (Glu329Lys) 아미노산 변이는 TYRP1 단백질의 구조와 화학적 성질에 영향을 줄 수 있는 것으로 사료되어 한우집단에서 황갈색바탕의 모색변이에 영향을 줄 수 있는지에 대한 추가적인 연구가 이루어져야 할 것이다. 【Most cattle breeds have a coat color pattern that is characteristic for the breed. Korean cattle(Hanwoo) has a coat color ranging from yellowish brown to dark brown including a red coat color. Variation in the Hanwoo coat color is likely to be the effects of modified genes segregating within the Hanwoo breed. MC1R encoded by the Extension(E) locus was almost fixed with recessive red e allele in the Hanwoo, but other gene(s) might be affecting the variation of the Hanwoo coat color into yellowish to red brown. We have analyzed a segregation of coat color in the F2 families generated from two Hanwoo bulls(yellowish brown) mated to six F1 dams(black) derived from Hanwoo and Holstein crosses. Segregation of coat color in the offspring found a ratio of 1(yellowish brown) : 1(black) and this ratio indicates that a single gene may play a major role for the Hanwoo coat color. We further investigated SNPs in MC1R, ASIP and TYRP1 loci to determine genetic cause of the Hanwoo coat color. Several polymorphisms within ASIP intron 2 and TYRP1 exons were found but not conserved within the Hanwoo population. However, the segregation of the MC1R e allele was completely associated with the Hanwoo coat color. Based on this information, it is clear that the MC1R e allele is mainly responsible for the yellowish red Hanwoo coat color. Further study is warrant to identify possible genetic interaction between MC1R e allele and other coat color related gene(s) for the variation of Hanwoo coat color from yellowish brown to dark brown. (Key words : Hanwoo, Coat color, SNP, MC1R, ASIP, TYRP1)】

This study was designed to investigate the seasonal variation in semen characteristics and the change of motility during semen frozen/thawed, and conception rates were observed following AI at the different times after estrus synchronization.Semen collected from March to May showed significantly lower semen quality than the other months (P

Hepatitis A virus (HAV) is the leading cause of acute viral hepatitis worldwide, with HAV infection being restricted to humans and nonhuman primates. In this study, HAV infection status was serologically determined in domestic pigs and experimental infections of HAV were attempted to verify HAV infectivity in pigs. Antibodies specific to HAV or HAV-like agents were detected in 3.5% of serum samples collected from pigs in swine farms. When the pigs were infected intravenously with 2 × 10(5) 50% tissue culture infectious dose (TCID50 ) of HAV, shedding of the virus in feces, viremia, and seroconversion were detected. In pigs orally infected with the same quantity of HAV, viral shedding was detected only in feces. HAV genomic RNA was detected in the liver and bile of intravenously infected pigs, but only in the bile of orally infected pigs. In further experiments, pigs were intravenously infected with 6 × 10(5) TCID50 of HAV. Shedding of HAV in feces, along with viremia and seroconversion, were confirmed in infected pigs but not in sentinel pigs. HAV genomic RNA was detected in the liver, bile, spleen, lymph node, and kidney of the infected pigs. HAV antigenomic RNA was detected in the spleen of one HAV-infected pig, suggesting HAV replication in splenic cells. Infiltration of inflammatory cells was observed in the livers of infected pigs but not in controls. This is the first experimental evidence to demonstrate that human HAV strains can infect pigs.

Immunocastration is an alternative method used to replace surgical castration commonly performed in swine farms. In boars, the main effects of immunocastration are reduction of gonadotropin-releasing hormone (GnRH) and the resulting inhibition of testicular function. The aim of this study was to evaluate immunocastration efficacy in pre-pubertal boars vaccinated with a recombinant GnRH protein conjugated with Salmonella Typhimurium flagellin fljB (STF2). A total of 35 boars were assigned to three groups: the untreated group (n = 5), the surgically castrated group (n = 5), and the immunocastrated group (n = 25). Pigs in the immunocastration group were immunized with the GnRH-STF2 vaccine at pre-pubertal ages 4 and 8 weeks. All experimental pigs were kept for 26 weeks before slaughter. Anti-GnRH antibody levels of immunocastrated pigs were significantly higher than those of untreated pigs (P0.001). In contrast, testosterone levels of immunocastrated pigs were significantly lower than those of untreated pigs (P0.001). Statistical significances were not found in the body weights and backfat thicknesses of untreated vs. immunocastrated pigs. Weights of the testes and epididymides of immunocastrated pigs were significantly lower than those of untreated pigs (P0.001). Testicular tissues of immunocastrated pigs were severely suppressed compared with those of untreated pigs. In conclusion, immunization with the STF2-GnRH vaccine effectively induced immunocastration in pre-pubertal boars.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant diseases in the swine industry. The PRRS virus (PRRSV) has genetically diverse populations, like other RNA viruses, and various field strains continue to be reported worldwide. The molecular epidemiological study of PRRSV can provide important data for use in controlling the disease. In this study, 50 oral fluid samples from conventional farms in Korea were taken to analyze nucleotide sequences of the open reading frame 5 of PRRSV. The viruses present in more than 80% of oral fluid samples genetically originated from the type 2 PRRSV, which is North American (NA) lineage. In addition 8.9% of samples contained both of the type 1 PRRSV, which is European (EU) lineage and the type 2 PRRSV. About 60% of farms involved in this study had more than two strains of PRRSV. In phylogenetic analysis, the Korean field strains of PRRSV detected from the oral fluid samples were divided into several subgroups: four subgroups of Korean field strains clustered with the type 1 PRRSV, and other five subgroups of Korean field strains clustered with the type 2. These results suggest that the type 2 PRRSV is more prevalent than the type 1 in Korea and heterologous strains of PRRSV can simultaneously infect a single pig farm.

Xenotransplantation offers a solution to the shortage of available organs for transplantation, and the pig represents an ideal source of such organs. However, porcine endogenous retrovirus (PERV), whose genome is integrated in pigs, has been suggested to pose a potential risk of xenotransmission. Expression of PERVs in different organs of pigs was carefully measured at DNA, mRNA, and protein levels, providing information valuable for the application of pig organs in xenotransplantation. An analysis of PERV DNA showed that a very similar number of PERV copies was present in the genome of all organs, whereas mRNA and protein levels of PERV varied depending on the organ, with kidney, liver, and spleen expressing high levels of both mRNA and protein. In contrast, mRNA and protein levels were dissimilar in the lung and brain, where mRNA levels were low but protein levels were high. This discrepancy indicates that mRNA levels are not always reflected in protein expression. In addition, the difference between mRNA and protein highlights the importance of choosing the proper analysis method for diagnosing viral infection. In summary, this study provides insight into the distribution of PERV in various organs at the DNA, mRNA, and protein levels, and also informs the proper selection of tissues or organs for future clinical xenotransplantation.

The oral microbiome, which is closely associated with many diseases, and the resident pathogenic oral bacteria, which can be transferred by close physical contact, are important public health considerations. Although the dog is the most common companion animal, the composition of the canine oral microbiome, which may include human pathogenic bacteria, and its relationship with that of their owners are unclear. In this study, 16S rDNA pyrosequencing was used to compare the oral microbiomes of 10 dogs and their owners and to identify zoonotic pathogens. Pyrosequencing revealed 246 operational taxonomic units in the 10 samples, representing 57 genera from eight bacterial phyla. Firmicutes (57.6%), Proteobacteria (21.6%), Bacteroidetes (9.8%), Actinobacteria (7.1%), and Fusobacteria (3.9%) were the predominant phyla in the human oral samples, whereas Proteobacteria (25.7%), Actinobacteria (21%), Bacteroidetes (19.7%), Firmicutes (19.3%), and Fusobacteria (12.3%) were predominant in the canine oral samples. The predominant genera in the human samples were Streptococcus (43.9%), Neisseria (10.3%), Haemophilus (9.6%), Prevotella (8.4%), and Veillonella (8.1%), whereas the predominant genera in the canine samples were Actinomyces (17.2%), Unknown (16.8), Porphyromonas (14.8), Fusobacterium (11.8), and Neisseria (7.2%). The oral microbiomes of dogs and their owners were appreciably different, and similarity in the microbiomes of canines and their owners was not correlated with residing in the same household. Oral-to-oral transfer of Neisseria shayeganii, Porphyromonas canigingivalis, Tannerella forsythia, and Streptococcus minor from dogs to humans was suspected. The finding of potentially zoonotic and periodontopathic bacteria in the canine oral microbiome may be a public health concern.

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 (1x10(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.