Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.     

Expression of Mx protein and interferon-alpha in pigs experimentally infected with swine influenza virus

Expression of Mx protein and interferon-alpha (IFN-alpha) was examined by immunohistochemistry in pigs experimentally infected with swine influenza virus. In infected pigs euthanatized at 1 day postinoculation (dpi), the lumen of bronchioles were filled with large numbers of mononuclear cells, small numbers of neutrophils, sloughing epithelial cells, and proteinaceous fluid. Lesions at 3 and 5 dpi were similar but less severe. Alveolar spaces were filled with neutrophils. By 7 and 10 dpi, microscopic lesions were resolved. The immunohistochemical signals for Mx protein and IFN-alpha antigen were confined to cells in areas that had hybridization signal for swine influenza virus. In situ hybridization and immunohistochemistry of serial sections of lung indicated that areas containing numerous swine influenza virus RNA-positive cells also have numerous Mx and IFN-alpha antigen-positive cells. Mean immunohistochemical scores for Mx protein-positive cells were correlated with mean immunohistochemical scores for IFN-alpha antigen-positive cells (r(s) = 0.8799, P < 0.05). These results indicated that Mx protein and IFN-alpha antigen were expressed in the lung from pigs experimentally infected with swine influenza virus, but their biological functions remain to be examined.     

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.     

Expression of myeloperoxidase in swine influenza virus (SIV)-infected neutrophils in lungs from pigs experimentally infected with SIV subtype H1N2

The expression of myeloperoxidase (MPO) was examined in the swine influenza virus (SIV)-infected neutrophils in the lungs of pigs experimentally infected with swine influenza virus (SIV) subtype H1N2 by immunohistochemistry. Five pigs each from the infected and non-infected group were euthanized 1, 3, 5, 7, and 10?days post-inoculation (dpi). Immunohistochemical reactivity was mainly seen in neutrophils. The score for pulmonary histopathological lesions correlated with the score for MPO immunohistochemical reactivity (r ( s )?=?0.962, P?相似文献   

Comparison of three serological assays to determine the cross-reactivity of antibodies from eight genetically diverse U.S. Swine influenza viruses.

Swine influenza virus is an economically important pathogen to the U.S. swine industry. New influenza subtypes and isolates within subtypes with different genetic and antigenic makeup have recently emerged in U.S. swineherds. As a result of the emergence of these new viruses, diagnosticians' ability to accurately diagnose influenza infection in pigs and develop appropriate vaccine strategies has become increasingly difficult. The current study compares the ability of subtype-specific commercial enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HI), and serum neutralization (SN) assays to detect antibodies elicited by multiple isolates within different subtypes of influenza virus. Pigs were infected with genetically and antigenically different isolates of the 3 major circulating subtypes within populations of swine (H1N1, H1N2, and H3N2). Serum was collected when all pigs within a group collectively reached HI reciprocal titers >or=160 against that group's homologous challenge virus. The antibody cross-reactivity of the sera between isolates was determined using ELISA, HI, and SN assays. In addition, the correlation between the 3 assays was determined. The assays differed in their ability to detect antibodies produced by the viruses used in the study. The results provide important information to diagnostic laboratories, veterinarians, and swine producers on the ability of 3 common serological assays used in identifying infection with influenza in pigs.     

猪源流感病毒NS1基因的克隆及原核表达

对H3N2亚型猪流感病毒NS1基因进行原核表达,获得纯化表达产物,以期为检测猪流感抗体的ELISA试剂盒的研制奠定基础。采用RT-PCR方法扩增H3N2亚型猪源流感病毒的NS1基因（693bp）,并将该片段克隆至pET-28（a）构建重组表达质粒pET-NS1。将重组质粒转化E．coli BL21（DE3）感受态细胞,以终浓度1mmol／L的IPTG在不同时间进行诱导表达,SDS-PAGE检测蛋白表达情况,并经Western-blot分析表达产物的抗原性。pET—NS1重组表达质粒表达的NS1蛋白相对分子质量约为26kD,1mmol／L的IPTG诱导4h时蛋白表达量达到高峰。Western-blot印迹分析证实表达蛋白能与阳性血清发生特异性反应,而与阴性血清不反应。NS1基因的原核表达产物可用来鉴别流感感染猪群和灭活疫苗免疫猪群。     

Classical swine fever virus infection modulates serum levels of INF-α, IL-8 and TNF-α in 6-month-old pigs

Several studies have highlighted the important role of cytokines in disease development of classical swine fever virus (CSFV) infection. In the present study, we examined the kinetics of 7 porcine cytokines in serum from pigs infected with 3 different CSFV strains. Based on the clinical picture in 6-month-old Danish pigs, the strains used for inoculation were classified as being of low (Bergen), low to moderate (Eystrup) and moderate to high (Lithuania) virulence. The cytokines interferon-alpha (INF-α), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) showed increased levels after CSFV infection with more or less comparable course in the 3 groups. However, the cytokine level peaked with a 2–3 days delay in pigs infected with the low virulent strain compared to those infected with a moderately or highly virulent strain. These findings may indicate that INF-α, IL-8 and TNF-α are involved in the immune response during CSFV infection with strains of different virulence.     

Vaccination of influenza a virus decreases transmission rates in pigs

Limited information is available on the transmission and spread of influenza virus in pig populations with differing immune statuses. In this study we assessed differences in transmission patterns and quantified the spread of a triple reassortant H1N1 influenza virus in naïve and vaccinated pig populations by estimating the reproduction ratio (R) of infection (i.e. the number of secondary infections caused by an infectious individual) using a deterministic Susceptible-Infectious-Recovered (SIR) model, fitted on experimental data. One hundred and ten pigs were distributed in ten isolated rooms as follows: (i) non-vaccinated (NV), (ii) vaccinated with a heterologous vaccine (HE), and (iii) vaccinated with a homologous inactivated vaccine (HO). The study was run with multiple replicates and for each replicate, an infected non-vaccinated pig was placed with 10 contact pigs for two weeks and transmission of influenza evaluated daily by analyzing individual nasal swabs by RT-PCR. A statistically significant difference between R estimates was observed between vaccinated and non-vaccinated pigs (p < 0.05). A statistically significant reduction in transmission was observed in the vaccinated groups where R (95%CI) was 1 (0.39-2.09) and 0 for the HE and the HO groups respectively, compared to an R_o value of 10.66 (6.57-16.46) in NV pigs (p < 0.05). Transmission in the HE group was delayed and variable when compared to the NV group and transmission could not be detected in the HO group. Results from this study indicate that influenza vaccines can be used to decrease susceptibility to influenza infection and decrease influenza transmission.     

Antigenic variant of swine influenza virus causing proliferative and necrotizing pneumonia in pigs.

A new antigenic variant of swine influenza virus was isolated from the lungs of pigs experiencing respiratory problems in 7 different swine herds in Quebec. Pigs of different ages were affected, and the main clinical signs were fever, dyspnea, and abdominal respiration. Coughing was not a constant finding of the syndrome. At necropsy, macroscopic lesions included the overall appearance of pale animals, general lymphadenopathy, hepatic congestion, and consolidation of the lungs. Histopathologic findings were mainly proliferative pneumonia with a significant macrophage invasion, necrotic inflammatory cells in the alveoli and the airways, a marked proliferation of type II pneumocytes, and thickening of the alveolar septae. Fluorescent antibody examination of lungs of sick piglets did not demonstrate porcine parvovirus, transmissible gastroenteritis virus, or encephalomyocarditis virus. However, evidence of the presence of an influenza type A infection was demonstrated by indirect immunofluorescence (IIF) staining using monoclonal antibody directed to nucleocapsid protein (NP) of human type A influenza virus. The virus was isolated either by intra-allantoic inoculation of specific-pathogen-free embryonating hens' eggs or propagation in canine kidney (MDCK) cells in the presence of trypsin. By hemagglutination inhibition tests, no cross-reactivity was demonstrated with human influenza H1N1, H2N2, and H3N2 strains, and infected MDCK cells did not react by IIF with monoclonal antibodies to NP protein of type B influenza virus. The hemagglutination activity of plaque-purified isolates was only partly inhibited by hyperimmune serum produced to subtypes A/Wisconsin/76/H1N1 and A/New Jersey/76/H1N1 of swine influenza virus. Gnotobiotic piglets that were infected intranasally with egg-adapted isolates of this new antigenic variant of swine influenza virus developed the very same type of lesions observed in field cases.     

Systemic and mucosal immune responses to H1N1 influenza virus infection in pigs

Influenza is a common respiratory disease in pigs, and since swine influenza viruses are zoonotic pathogens, they also pose human health risks. Pigs infected with influenza virus mount an effective immune response and are protected from subsequent challenge, whereas the currently available, inactivated-virus vaccine does not consistently confer complete protection to challenge. To develop and evaluate new vaccination strategies, it is imperative to fully understand the immune responses that are associated with protection following natural infection. Therefore, we have evaluated the phenotype and kinetics of immune responses to primary and re-challenge infection with H1N1 swine influenza virus in the pig. Through the use of isotype-specific antibody secreting cell ELISPOT assays, interferon-gamma ELISPOT assays and isotype-specific ELISAs on serum, nasal wash and bronchoalveolar lavage samples, we defined the humoral and cellular immune responses, both locally in the respiratory tract and systemically, to this viral infection. Virus-specific serum IgG, IgA, and HI titers all peaked 2-3 weeks after primary infection and did not substantially increase after re-challenge. The predominant virus-specific isotype in serum was IgG. Pigs responded with virus-specific IgG and IgA in both the upper (nasal washes) and lower (bronchoalveolar lavages) airways; IgA was the predominant isotype in both sites. Despite the fact that the pigs were completely protected from re-challenge, the antibody titers in the nasal washes increased. Results of the antibody-secreting cell ELISPOT assays demonstrated that the numbers of both IgG and IgA secreting cells in the nasal mucosa were dramatically higher than in any other tissue examined. In contrast, IFN-gamma secreting cells were predominantly localized to the spleen and tracheobronchial lymph nodes. These data will be helpful in the future development and evaluation of novel vaccines.     

Differential interactions of virulent and non-virulent H. parasuis strains with na?ve or swine influenza virus pre-infected dendritic cells

Pigs possess a microbiota in the upper respiratory tract that includes Haemophilus parasuis. Pigs are also considered the reservoir of influenza viruses and infection with this virus commonly results in increased impact of bacterial infections, including those by H. parasuis. However, the mechanisms involved in host innate responses towards H. parasuis and their implications in a co-infection with influenza virus are unknown. Therefore, the ability of a non-virulent H. parasuis serovar 3 (SW114) and a virulent serovar 5 (Nagasaki) strains to interact with porcine bone marrow dendritic cells (poBMDC) and their modulation in a co-infection with swine influenza virus (SwIV) H3N2 was examined. At 1 hour post infection (hpi), SW114 interaction with poBMDC was higher than that of Nagasaki, while at 8 hpi both strains showed similar levels of interaction. The co-infection with H3N2 SwIV and either SW114 or Nagasaki induced higher levels of IL-1β, TNF-α, IL-6, IL-12 and IL-10 compared to mock or H3N2 SwIV infection alone. Moreover, IL-12 and IFN-α secretion differentially increased in cells co-infected with H3N2 SwIV and Nagasaki. These results pave the way for understanding the differences in the interaction of non-virulent and virulent strains of H. parasuis with the swine immune system and their modulation in a viral co-infection.     

Serological Surveillance of Influenza A Virus Infection in Swine Populations in Fujian Province,China: No Evidence of Naturally Occurring H5N1 Infection in Pigs

Several highly pathogenic H5N1 avian influenza viruses were isolated from swine populations in Fujian Province, China, since 2001. Because it is thought that H5N1 infection in pigs might result in virus adaptation to humans, we surveyed swine populations in Fujian Province in 2004 and 2007 for serological evidence of the infection. Twenty‐five pig farms covering all nine administrative districts of Fujian Province were sampled and a total of 1407 serum specimens were collected. The haemagglutination inhibition (HI) tests revealed no evidence of H5 infection and only a few cases of H9 infection. The negative results for H5 infection were further verified by micro‐neutralization tests. By contrast, H1 influenza virus infections were prevalent in swine in both surveys according to the results of enzyme‐linked immunosorbent assay (ELISA). The H3 infection rate was reduced dramatically in 2007 compared with 2004, when examined by HI and ELISA. In summary, the results imply that the swine populations in Fujian Province had not been affected greatly by the H5N1 avian influenza virus, given that there is no serological evidence that H5N1 influenza virus has infected the pig populations. The reported isolates represent only sporadic cases.     

Induction of a cross-reactive antibody response to influenza virus M2 antigen in pigs by using a Sendai virus vector

Protecting pigs from simultaneous infection with avian, swine, and human influenza viruses would be an effective strategy to prevent the emergence of reassortants with pandemic potential. M2 protein is a candidate antigen for so-called 'universal vaccines,' which confer cross-protection to different influenza viruses in a strain- and subtype-independent manner. We tested whether a recombinant F gene-deleted Sendai virus vector that contained an M2 gene derived from an H5N1 avian influenza virus (SeV/ΔF/H5N1M2) could induce a cross-reactive antibody response to the extracellular domain of M2 protein (M2e) in pigs. SeV/ΔF/H5N1M2 induced an antibody response to M2e when the vector was inoculated intramuscularly. The antibodies induced by SeV/ΔF/H5N1M2 cross-reacted with M2e derived from different avian, swine, and human influenza viruses. In mice, however, SeV/ΔF/H5N1M2 did not confer cross-protection to challenge with a heterologous H3N2 influenza virus. Our results confirm those of other groups indicating that antibodies to M2e do not mediate protection to influenza viruses in pigs.     

Pathogenicity of concurrent infection of pigs with porcine respiratory coronavirus and swine influenza virus

Combinations of porcine respiratory coronavirus (PRCV) and either of two swine influenza viruses (H1N1 or H3N2) were administered intranasally and by aerosol to six- to eight-week-old specific pathogen-free pigs. The clinical responses, gross respiratory lesions and growth performances of these pigs were studied and compared with those of single (PRCV, H1N1 or H3N2) and mock-infected animals. PRCV infection caused fever, growth retardation and lung lesions, but no respiratory symptoms. Infection with swine influenza viruses caused rather similar, mild symptoms of disease, with H1N1 infection being the least severe. Combined infections with influenza viruses and PRCV did not appear to enhance the pathogenicity of these viruses. Furthermore, viruses were isolated more frequently from tissues and nasal swabs taken from 'single' than 'dual' infected animals, suggesting a possible in vivo interference between replication of PRCV and swine influenza virus.     

Increase in chemokines CXCL10 and CCL2 in blood from pigs infected with high compared to low virulence African swine fever virus isolates

Modulation of the expression of chemokines and chemokine receptors in whole blood was compared following infection of pigs with high and low virulence isolates of African swine fever virus. Levels of mRNAs for CCL2, CCL3L1, CCL4, CXCL10, CCR1 and CCR5 were significantly increased in at least one time point following infection in two experiments and CCL5, CCR9 and CXCR4 mRNA were significantly increased in one of the experiments. The results showed that greatest fold increases in mRNAs for CXCL10 and CCL2 were observed following infection of pigs. CXCL10 mRNA was increased by up to 15 fold in infected compared to uninfected pigs. CXCL10 protein was also detected in serum from pigs infected with the high virulence Benin 97/1 isolate. Levels of CCL2 mRNA were increased in pigs infected with high virulence Benin 97/1 isolate compared to low virulence OURT88/3 isolate and this correlated with an increase of greater than 30 fold in levels of CCL2 protein detected in serum from pigs infected with this isolate. An increase in overall chemotaxis active compounds in defibrinated plasma samples from Benin 97/1 infected pigs was observed at 3 days post-infection (dpi) and a decrease by 7 dpi as measured by chemotaxis assay using normal pig leucocytes in vitro. Increased levels of CXCL10 may either contribute to the activation of lymphocyte priming toward the Th1 phenotype or induction of T lymphocyte apoptosis. Increased levels of CCL2, a chemoattractant for macrophages, may result in increased recruitment of monocytes from bone marrow thus increasing the pool of cells susceptible to infection.     

Isolation and genetic characterization of avian origin H9N2 influenza viruses from pigs in China

As pigs are susceptible to infection with both avian and human influenza A viruses, they have been proposed to be an intermediate host for the adaptation of avian influenza viruses to humans. In April 2006, a disease caused by highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) occurred in several pig farms and subsequently overwhelmed almost half of China with more than 2,000,000 cases of pig infection. Here we report a case in which four swine H9N2 influenza viruses were isolated from pigs infected by highly pathogenic PRRSVs in Guangxi province in China. All the eight gene segments of the four swine H9N2 viruses are highly homologous to A/Pigeon/Nanchang/2-0461/00 (H9N2) or A/Wild Duck/Nanchang/2-0480/00 (H9N2). Phylogenetic analyses of eight genes show that the swine H9N2 influenza viruses are of avian origin and may be the descendants of A/Duck/Hong Kong/Y280/97-like viruses. Molecular analysis of the HA gene indicates that our H9N2 isolates might have high-affinity binding to the alpha2,6-NeuAcGal receptor found in human cells. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially after the emergence of highly pathogenic PRRSVs in pigs in China.     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

猪瘟病毒感染后对机体免疫系统的影响

为初步探讨猪瘟病毒的致病机理及机体的免疫应答机制,进行了猪瘟病毒感染试验,利用流式细胞术、阻断ELISA、白细胞计数和剖检观察对猪的体液免疫、细胞免疫、白细胞数量及病理损伤进行了研究。结果表明,感染猪的CD3^＋和CD4^＋T细胞亚群的数量在感染过程中均出现了明显的降低;CD8^＋T细胞亚群在感染初期变化幅度不大,后期明显升高;猪瘟抗体于第7天开始产生,呈上升趋势。白细胞数量在感染期间呈下降趋势。病理学观察可知感染猪的肺脏、肾脏、扁桃体等出现了不同程度的损伤。猪瘟病毒感染对机体的免疫系统造成了较大的损伤,抑制了机体的免疫应答。