Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

Elastase-Dependent Live Attenuated Swine Influenza A Viruses Are Immunogenic and Confer Protection against Swine Influenza A Virus Infection in Pigs

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.Swine influenza virus (SIV) is the causative pathogen of swine influenza, a highly contagious, acute respiratory viral disease of swine. The mortality of SIV-infected pigs is usually low, although morbidity may approach 100%. Swine influenza is characterized by sudden onset, coughing, respiratory distress, weight loss, fever, nasal discharge, and rapid recovery (38). SIV is a member of the influenza virus A genus in the Orthomyxoviridae family, and the virus has a genome consisting of eight segments of negative-sense single-stranded RNA (29). Epithelial cells in the swine respiratory tract have receptors for both avian and mammalian influenza viruses (13); thus, pigs could potentially serve as “mixing vessels” for the generation of new reassortant strains of influenza A virus that have pandemic capacity. There are a number of reports in which the direct transmission of influenza viruses from pigs to humans has been documented (6, 12, 52), and several of these cases have resulted in human fatalities (19, 35, 40, 53). Consequently, effective control of SIV would be beneficial to both humans and animals.Until 1998, classical H1N1 SIVs were the predominant isolates from pigs in the United States and Canada (5, 28). In 1997 to 1998, a dramatic change in the epidemiologic pattern of SIV began. Serological studies conducted by Olsen and colleagues in 1997 to 1998 detected a significant increase in H3-seropositive individuals, and H3N2 SIVs were isolated from pigs in both the United States and Canada (17, 54). Furthermore, reassortment between H3N2 viruses and classical H1N1 SIV resulted in the appearance of H1N2 reassortant viruses (14, 15). In addition to the isolation of H4N6 viruses, which are of duck origin, in pigs in Canada (16), wholly avian viruses of the H3N3 and H1N1 subtypes have also been isolated from Canadian pigs (18). In general, three major SIV subtypes exist, i.e., H1N1, H1N2, and H3N2, each of which has multiple genetic and antigenic variants circulating in North American swine populations (18, 28). The increased incidence of avian-like or human-like SIV reassortants raises concerns for public health and requires research devoted to the development of cross-protective SIV vaccines.Currently available swine influenza vaccines are based on inactivated whole virus of the H1N1 and H3N2 subtypes. Application of these vaccines reduces the severity of disease but does not provide consistent protection from infection (3, 22). In contrast to killed vaccines that are administered intramuscularly, intranasally administered live attenuated influenza vaccines (LAIV) induce an immune response at the site of natural infection. Therefore, an LAIV has the potential to induce broad humoral and cellular immune responses that could provide protection against antigenically different influenza viruses. LAIV based on attenuation of the virus by cold adaptation are available for humans (2) and horses (41). However, to date, no SIV LAIV are commercially available for use in swine in North America. Recent studies by Solorzano et al. showed that a mutant SIV with a truncated NS1 protein was highly attenuated in pigs (36). In addition, this SIV/NS1 LAIV was capable of stimulating a protective immune response against homologous SIVs and a partial protection against heterologous subtypic wild-type (WT) SIVs (31, 50). Stech and colleagues demonstrated that the conversion of a conserved cleavage site in the influenza virus hemagglutinin (HA) protein from a trypsin-sensitive site to an elastase-sensitive site results in in vivo attenuation of the influenza virus in mouse models (9, 37). Furthermore, these elastase-dependent LAIV were able to induce protective systemic and mucosal immune responses. Recently, we showed that two elastase-dependent SIVs derived from A/Sw/Saskatchewan/18789/02 (SIV/Sk02), R345V and R345A, are attenuated in their natural host, pigs (23). In the current study, we addressed the immunogenic and cross-protective abilities of these mutants.     

Designing Inhibitors of M2 Proton Channel against H1N1 Swine Influenza Virus

Background

M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591–595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.

Methodology

Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.

Conclusions

1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a “barrel hoop”, holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.     

A Nine-Segment Influenza A Virus Carrying Subtype H1 and H3 Hemagglutinins

Influenza virus genomic RNAs possess segment-specific packaging signals that include both noncoding regions (NCRs) and adjacent terminal coding region sequences. Using reverse genetics, an A/Puerto Rico/8/34 (A/PR/8/34) virus was rescued that contained a modified PB1 gene such that the PB1 packaging sequences were exchanged for those of the neuraminidase (NA) gene segment. To accomplish this, the PB1 open reading frame, in which the terminal packaging signals were inactivated by serial synonymous mutations, was flanked by the NA segment-specific packaging sequences including the NCRs and the coding region packaging signals. Next, the ATGs located on the 3′ end of the NA packaging sequences of the resulting PB1 chimeric segment were mutated to allow for correct translation of the full-length PB1 protein. The virus containing this chimeric PB1 segment was viable and able to stably carry a ninth, green fluorescent protein (GFP), segment flanked by PB1 packaging signals. Utilizing this method, we successfully generated an influenza virus that contained the genes coding for both the H1 hemagglutinin (HA) from A/PR/8/34 and the H3 HA from A/Hong Kong/1/68 (A/HK/1/68); both subtypes of HA protein were also incorporated into the viral envelope. Immunization of mice with this recombinant virus conferred complete protection from lethal challenge with recombinant A/PR/8/34 virus and with X31 virus that expresses the A/HK/1/68 HA and NA. Using the described methodology, we show that a ninth segment can also be incorporated by manipulation of the PB2 or PA segment-specific packaging signals. This approach offers a means of generating a bivalent influenza virus vaccine.Influenza viruses possess segmented, negative-sense RNA genomes and belong to the family of Orthomyxoviridae. Three types of influenza viruses have been identified: A, B, and C (24). Based on the two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), type A viruses are further divided into different subtypes; there are now 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) of influenza A viruses (24). Current influenza A viruses circulating in humans include the H1N1 and H3N2 subtypes.The genomes of influenza A and B viruses consist of eight RNAs, while C viruses have only seven segments. Influenza virus genomic RNAs associate with nucleoprotein (NP) and three viral polymerase subunits (PB2, PB1, and PA), to form the ribonucleoprotein (RNP) complexes within virions (24). Previous data indicated that each segment of the influenza A/WSN/33 (H1N1) virus possesses segment-specific RNA packaging signals that include both the 3′ and 5′ noncoding regions (NCRs), as well as coding sequences at the two ends of each open reading frame (ORF) (4, 5, 10, 11, 13, 15, 22, 23, 28; and see Fig. 47.23 in reference 24). In addition, an electron microscopy study showed that the wild-type influenza A virus contains exactly eight RNPs within the virions, with seven RNPs surrounding a central one (19). These results suggest that influenza virus genome packaging is a specific process, with each particle containing eight unique RNA segments. Additional evidence supporting a specific packaging theory came from studies of defective interfering (DI) RNAs which contain internal deletions in the coding sequences. These short RNAs can be incorporated into the virus particles despite the fact that they do not encode full-length functional proteins. The finding that incorporation of DI RNAs interferes with the parent full-length RNAs in a segment-specific manner (1, 16, 17) also suggests that influenza virus genome packaging is a specific process.However, there are also data arguing that influenza virus RNA packaging can be nonspecific. First, studies showed that the two different RNA segments of influenza virus can be engineered to share the same set of 3′ and 5′ NCRs, which are important components of the influenza virus RNA packaging signals (18, 31). In addition, under specific circumstances, influenza virus is able to contain nine RNA segments, in which two of them share identical NCRs and partially identical coding region sequences (2, 29). Titrations of the nine-segment virus revealed a linear relationship between dilutions and plaque numbers, suggesting an influenza virus virion can incorporate more than eight segments (2).Herein, we describe a novel approach for the generation of nine-segment influenza viruses based on the manipulation of the segment-specific packaging signals. When the packaging sequences of the PB1 (or PB2 or PA) segment were replaced by those of the NA segment, influenza A/PR/8/34 virus was able to stably incorporate a ninth segment flanked by the PB1 (or PB2 or PA) packaging signals. Using this property, we successfully generated influenza viruses encoding two full-length HA glycoproteins: a subtype H1 A/PR/8/34 HA and a subtype H3 A/HK/1/68 HA. Immunization of mice with the virus carrying two HAs protected them from the lethal challenge with either A/PR/8/34 or X31 virus, the latter of which carries the HA and NA genes of A/HK/1/68. This approach can be used to construct live attenuated influenza vaccine viruses targeting two heterologous strains.     

Efficacy in Pigs of Inactivated and Live Attenuated Influenza Virus Vaccines against Infection and Transmission of an Emerging H3N2 Similar to the 2011-2012 H3N2v

Vaccines provide a primary means to limit disease but may not be effective at blocking infection and pathogen transmission. The objective of the present study was to evaluate the efficacy of commercial inactivated swine influenza A virus (IAV) vaccines and experimental live attenuated influenza virus (LAIV) vaccines against infection with H3N2 virus and subsequent indirect transmission to naive pigs. The H3N2 virus evaluated was similar to the H3N2v detected in humans during 2011-2012, which was associated with swine contact at agricultural fairs. One commercial vaccine provided partial protection measured by reduced nasal shedding; however, indirect contacts became infected, indicating that the reduction in nasal shedding did not prevent aerosol transmission. One LAIV vaccine provided complete protection, and none of the indirect-contact pigs became infected. Clinical disease was not observed in any group, including nonvaccinated animals, a consistent observation in pigs infected with contemporary reassortant H3N2 swine viruses. Serum hemagglutination inhibition antibody titers against the challenge virus were not predictive of efficacy; titers following vaccination with a LAIV that provided sterilizing immunity were below the level considered protective, yet titers in a commercial vaccine group that was not protected were above that level. While vaccination with currently approved commercial inactivated products did not fully prevent transmission, certain vaccines may provide a benefit by limitating shedding, transmission, and zoonotic spillover of antigenically similar H3N2 viruses at agriculture fairs when administered appropriately and used in conjunction with additional control measures.     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。     

Investigation of Pathogenesis of H1N1 Influenza Virus and Swine Streptococcus suis Serotype 2 Co-Infection in Pigs by Microarray Analysis

Swine influenza virus and Streptococcus suis are two important contributors to the porcine respiratory disease complex, and both have significant economic impacts. Clinically, influenza virus and Streptococcus suis co-infections in pigs are very common, which often contribute to severe pneumonia and can increase the mortality. However, the co-infection pathogenesis in pigs is unclear. In the present study, co-infection experiments were performed using swine H1N1 influenza virus and Streptococcus suis serotype 2 (SS2). The H1N1-SS2 co-infected pigs exhibited more severe clinical symptoms, serious pathological changes, and robust apoptosis of lungs at 6 days post-infection compared with separate H1N1 and SS2 infections. A comprehensive gene expression profiling using a microarray approach was performed to investigate the global host responses of swine lungs against the swine H1N1 infection, SS2 infection, co-infection, and phosphate-buffered saline control. Results showed 457, 411, and 844 differentially expressed genes in the H1N1, SS2, and H1N1-SS2 groups, respectively, compared with the control. Noticeably, genes associated with the immune, inflammatory, and apoptosis responses were highly overexpressed in the co-infected group. Pathway analysis indicated that the cytokine–cytokine receptor interactions, MAPK, toll-like receptor, complement and coagulation cascades, antigen processing and presentation, and apoptosis pathway were significantly regulated in the co-infected group. However, the genes related to these were less regulated in the separate H1N1 and SS2 infection groups. This observation suggested that a certain level of synergy was induced by H1N1 and SS2 co-infection with significantly stronger inflammatory and apoptosis responses, which may lead to more serious respiratory disease syndrome and pulmonary pathological lesion.     

A VLP Vaccine Induces Broad-Spectrum Cross-Protective Antibody Immunity against H5N1 and H1N1 Subtypes of Influenza A Virus

The recent threats of influenza epidemics and pandemics have prioritized the development of a universal vaccine that offers protection against a wider variety of influenza infections. Here, we demonstrate a genetically modified virus-like particle (VLP) vaccine, referred to as H5M2eN1-VLP, that increased the antigenic content of NA and induced rapid recall of antibody against HA(2) after viral infection. As a result, H5M2eN1-VLP vaccination elicited a broad humoral immune response against multiple viral proteins and caused significant protection against homologous RG-14 (H5N1) and heterologous A/California/07/2009 H1N1 (CA/07) and A/PR/8/34 H1N1 (PR8) viral lethal challenges. Moreover, the N1-VLP (lacking HA) induced production of a strong NA antibody that also conferred significant cross protection against H5N1 and heterologous CA/07 but not PR8, suggesting the protection against N1-serotyped viruses can be extended from avian-origin to CA/07 strain isolated in humans, but not to evolutionally distant strains of human-derived. By comparative vaccine study of an HA-based VLP (H5N1-VLP) and NA-based VLPs, we found that H5N1-VLP vaccination induced specific and strong protective antibodies against the HA(1) subunit of H5, thus restricting the breadth of cross-protection. In summary, we present a feasible example of direction of VLP vaccine immunity toward NA and HA(2), which resulted in cross protection against both seasonal and pandemic influenza strains, that could form the basis for future design of a better universal vaccine.     

Tmprss2 Is Essential for Influenza H1N1 Virus Pathogenesis in Mice

Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

Prediction of Mutations Initiated by Internal Power in H3N2 Hemagglutinins of Influenza A Virus from North America

Probably the best way to predict mutations is to find the cause for mutations, by which the cause–mutation relationship can be built. However, many causes which have resulted in mutations in the past might not leave any trace due to the changes in environments. As well, the current proteins may not be sensitive to the causes, which led to mutations in the past, because of evolution. Thus we might have recorded many mutations, but few of their corresponding causes, and it would be difficult to establish the one-to-one cause–mutation relationship. However, the internal power engineering mutations within a protein would exist, of which randomness should play an important role. Since 1999, we have developed three methods to quantify the randomness within a protein by which we can build a cause–mutation relationship because we can classify the occurrence and non-occurrence of mutation as unity and zero, and transfer this relationship into the classification problem, which can be solved using logistic regression. Recently, we used the logistic regression to predict the mutation positions in H5N1 hemagglutinins from influenza A virus, and applied the amino-acid mutating probability to predict the would-be-mutated amino acids at predicted positions as the concept-initiated study. However, we still need to conduct many proof-of-concept studies to test whether this cause–mutation relationship is independent of protein subtypes, whether the logistic regression is powerful enough, etc. In this study, we attempted to use the logistic regression to predict the mutation positions in H3N2 hemagglutinins of influenza A virus from North America to answer the questions in the proof-of-concept stage.     

Swine Influenza H1N1 Virus Induces Acute Inflammatory Immune Responses in Pig Lungs: a Potential Animal Model for Human H1N1 Influenza Virus

Pigs are capable of generating reassortant influenza viruses of pandemic potential, as both the avian and mammalian influenza viruses can infect pig epithelial cells in the respiratory tract. The source of the current influenza pandemic is H1N1 influenza A virus, possibly of swine origin. This study was conducted to understand better the pathogenesis of H1N1 influenza virus and associated host mucosal immune responses during acute infection in humans. Therefore, we chose a H1N1 swine influenza virus, Sw/OH/24366/07 (SwIV), which has a history of transmission to humans. Clinically, inoculated pigs had nasal discharge and fever and shed virus through nasal secretions. Like pandemic H1N1, SwIV also replicated extensively in both the upper and lower respiratory tracts, and lung lesions were typical of H1N1 infection. We detected innate, proinflammatory, Th1, Th2, and Th3 cytokines, as well as SwIV-specific IgA antibody in lungs of the virus-inoculated pigs. Production of IFN-γ by lymphocytes of the tracheobronchial lymph nodes was also detected. Higher frequencies of cytotoxic T lymphocytes, γδ T cells, dendritic cells, activated T cells, and CD4⁺ and CD8⁺ T cells were detected in SwIV-infected pig lungs. Concomitantly, higher frequencies of the immunosuppressive T regulatory cells were also detected in the virus-infected pig lungs. The findings of this study have relevance to pathogenesis of the pandemic H1N1 influenza virus in humans; thus, pigs may serve as a useful animal model to design and test effective mucosal vaccines and therapeutics against influenza virus.Swine influenza is a highly contagious, acute respiratory viral disease of swine. The causative agent, swine influenza virus (SwIV), is a strain of influenza virus A in the Orthomyxoviridae family. Clinical disease in pigs is characterized by sudden onset of anorexia, weight loss, dyspnea, pyrexia, cough, fever, and nasal discharge (21). Porcine respiratory tract epithelial cells express sialic acid receptors utilized by both avian (α-2,3 SA-galactose) and mammalian (α-2,6 SA-galactose) influenza viruses. Thus, pigs can serve as “mixing vessels” for the generation of new reassortant strains of influenza A virus that may contain RNA elements of both mammalian and avian viruses. These “newly generated” and reassorted viruses may have the potential to cause pandemics in humans and enzootics in animals (52).Occasional transmission of SwIV to humans has been reported (34, 43, 52), and a few of these cases resulted in human deaths. In April 2009, a previously undescribed H1N1 influenza virus was isolated from humans in Mexico. This virus has spread efficiently among humans and resulted in the current human influenza pandemic. Pandemic H1N1 virus is a triple reassortant (TR) virus of swine origin that contains gene segments from swine, human, and avian influenza viruses. Considering the pandemic potential of swine H1N1 viruses, it is important to understand the pathogenesis and mucosal immune responses of these viruses in their natural host. Swine can serve as an excellent animal model for the influenza virus pathogenesis studies. The clinical manifestations and pathogenesis of influenza in pigs closely resemble those observed in humans. Like humans, pigs are also outbred species, and they are physiologically, anatomically, and immunologically similar to humans (9, 23, 39, 40). In contrast to the mouse lung, the porcine lung has marked similarities to its human counterpart in terms of its tracheobronchial tree structure, lung physiology, airway morphology, abundance of airway submucosal glands, and patterns of glycoprotein synthesis (8, 10, 17). Furthermore, the cytokine responses in bronchoalveolar lavage (BAL) fluid from SwIV-infected pigs are also identical to those observed for nasal lavage fluids of experimentally infected humans (20). These observations support the idea that the pig can serve as an excellent animal model to study the pathogenesis of influenza virus.Swine influenza virus causes an acute respiratory tract infection. Virus replicates extensively in epithelial cells of the bronchi and alveoli for 5 to 6 days followed by clearance of viremia by 1 week postinfection (48). During the acute phase of the disease, cytokines such as alpha interferon (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-12, and gamma interferon (IFN-γ) are produced. These immune responses mediate both the clinical signs and pulmonary lesions (2). In acute SwIV-infected pigs, a positive correlation between cytokines in BAL fluid, lung viral titers, inflammatory cell infiltrates, and clinical signs has been detected (2, 48).Infection of pigs with SwIV of one subtype may confer complete protection from subsequent infections by homologous viruses and also partial protection against heterologous subtypes, but the nature of the immune responses generated in the swine are not fully delineated. Importantly, knowledge related to host mucosal immune responses in the SwIV-infected pigs is limited. So far only the protective virus-specific IgA and IgG responses in nasal washes and BAL fluid, as well as IgA, IgG, and IgM responses in the sera of infected pigs, have been reported (28). Pigs infected with H3N2 and H1N1 viruses have an increased frequency of neutrophils, NK cells, and CD4 and CD8 T cells in the BAL fluid (21). Pigs infected with the pandemic H1N1 virus showed activated CD4 and CD8 T cells in the peripheral blood on postinfection day (PID) 6 (27). Proliferating lymphocytes in BAL fluid and blood and virus-specific IFN-γ-secreting cells in the tracheobronchial lymph nodes (TBLN) and spleen were detected in SwIV-infected pigs (7). Limited information is available on the mucosal immune responses in pig lungs infected with SwIV, which has a history of transmission to humans.In this study, we examined the acute infection of SwIV (strain SwIV OH07) in pigs with respect to viral replication, pathology, and innate and adaptive immune responses in the respiratory tract of these pigs. This virus was isolated from pigs which suffered from respiratory disease in Ohio, and the same virus was also transmitted to humans and caused clinical disease (43, 55). Interestingly, like pandemic H1N1 influenza virus, SwIV also infects the lower respiratory tract of pigs. Delineation of detailed mucosal immune responses generated in pig lungs during acute SwIV OH07 infection may provide new insights for the development of therapeutic strategies for better control of virus-induced inflammation and for the design and testing of effective vaccines.     

DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice

In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine’s protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.