Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge

Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza.     

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.     

Acute phase protein response during subclinical infection of pigs with H1N1 swine influenza virus

In the present study acute phase proteins (APPs) responses in pigs after subclinical infection with H1N1 swine influenza virus (SwH1N1) were evaluated. Fourteen 5 weeks old, seronegative piglets, both sexes were used. Ten of them were infected intranasally with SwH1N1. C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA) and pig major acute phase protein (Pig-MAP) concentrations in serum were measured using commercial ELISAs. No significant clinical signs were observed in any of the infected pigs, however, all infected animals developed specific antibodies against SwH1N1 and viral shedding was observed from 2 to 5dpi. Only concentrations of Hp and SAA were significantly induced after infection, with mean maximum levels from days 1 to 2 post infection (dpi). The concentrations of CRP and Pig-MAP remained generally unchanged, however in half of infected pigs the concentration of CRP tended to increase at 1dpi (but without statistical significance). The results of our study confirmed that monitoring of APPs may be useful for detection of subclinically infected pigs. The use of SAA or Hp and Pig-MAP may be a valuable in combination [i.e. Hp (increased concentration) and Pig-MAP (unchanged concentration)] to detect subclinically SIV infected pigs, or to identify pigs actually producing a large amount of virus. Additional studies need to be done in order to confirm these findings.     

Pandemic H1N1 influenza virus infection in a Canadian cat

A cat was presented for necropsy after being found dead at home. Histologic findings suggested viral pneumonia. Polymerase chain reaction and viral typing revealed influenza A(H1N1)pdm09. This is the first report of influenza in a Canadian cat and highlights the importance of considering influenza virus in the differential diagnosis for feline respiratory distress.     

The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination

This study investigated the efficacy of a bivalent swine influenza virus (SIV) vaccine in piglets challenged with a heterologous H1N1 SIV isolate. The ability of maternally derived antibodies (MDA) to provide protection against a heterologous challenge and the impact MDA have on vaccine efficacy were also evaluated. Forty-eight MDA(+) pigs and 48 MDA(-) pigs were assigned to 8 different groups. Vaccinated pigs received two doses of a bivalent SIV vaccine at 3 and 5 weeks of age. The infected pigs were challenged at 7 weeks of age with an H1N1 SIV strain heterologous to the H1N1 vaccine strain. Clinical signs, rectal temperature, macroscopic and microscopic lesions, virus excretion, serum and local antibody responses, and influenza-specific T-cell responses were measured. The bivalent SIV vaccine induced a high serum hemagglutination-inhibition (HI) antibody titer against the vaccine virus, but antibodies cross-reacted at a lower level to the challenge virus. This study determined that low serum HI antibodies to a challenge virus induced by vaccination with a heterologous virus provided protection demonstrated by clinical protection and reduced pneumonia and viral excretion. The vaccine was able to prime the local SIV-specific antibody response in the lower respiratory tract as well as inducing a systemic SIV-specific memory T-cell response. MDA alone were capable of suppressing fever subsequent to infection, but other parameters showed reduced protection against infection compared to vaccination. The presence of MDA at vaccination negatively impacted vaccine efficacy as fever and clinical signs were prolonged, and unexpectedly, SIV-induced pneumonia was increased compared to pigs vaccinated in the absence of MDA. MDA also suppressed the serum antibody response and the induction of SIV-specific memory T-cells following vaccination. The results of this study question the effectiveness of the current practice of generating increased MDA levels through sow vaccination in protecting piglets against disease.     

Immune responses and protective efficacy of a recombinant swinepox virus co-expressing HA1 genes of H3N2 and H1N1 swine influenza virus in mice and pigs

The recombinant swine poxvirus rSPV/H3-2A-H1 co-expressing HA1 genes of H3N2 and H1N1 subtype SIV has been constructed and identified. Inoculations of rSPV/H3-2A-H1 yielded ELISA and neutralization antibodies against SIV H1N1 and H3N2, and elicited potent H1N1 and H3N2 SIV-specific INF-γ response from T-lymphocytes in mice and pigs in this study. Complete protection against SIV H1N1 or H3N2 challenge in pigs was observed.     

Effect of maternally derived antibodies on the clinical signs and immune response in pigs after primary and secondary infection with an influenza H1N1 virus

The aim of this study was to determine the role of maternally derived antibodies (MDA) against an influenza H1N1 virus in the clinical protection of piglets and especially their effect on the development of the active immunity after an infection with a homologous influenza H1N1 virus. Twenty piglets with MDA and 10 piglets without MDA were housed together and inoculated twice with influenza H1N1 virus, at 7 and 15 weeks of age. Nine piglets without MDA were added to these groups at 12 weeks of age to be inoculated at 15 weeks of age only. Clinical signs, body temperature, growth performance, virus excretion, antibody responses, and influenza-specific T-cell response were monitored. It was shown that MDA protect piglets against the clinical consequences of a primary influenza infection, but that this protection is not complete. A short but significant rise in body temperature was observed and growth seemed to be inhibited due to the infection. Piglets with MDA shed virus for a longer period after an infection than piglets without MDA. Piglets with and without MDA were protected against the clinical consequences of a secondary infection. However, both after primary and secondary infection significant differences in immune responses were observed that indicated that pigs with MDA developed a weaker immunity than pigs without MDA. Furthermore, overall growth performances from weaning to slaughter show a trend in favour of pigs without maternal antibodies, compared to pigs with maternal antibodies, mainly caused by a significant better performance in the second half of the finishing period. The results of this study provide us insight in the role of MDA in clinical protection and their influence on active immunity after an influenza virus infection of pigs. Furthermore, it leads us to the discussion about the profitability of massive sow herd vaccinations in an attempt to increase MDA levels in piglets, taking into account the overall performance of these piglets and the possible effects on antigenic drift.     

Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin.

Serpulina hyodysenteriae infection of pigs, swine dysentery, causes a mucohemorrhagic diarrhoea resulting in significant economic losses to swine producers. The pathogenesis of this disease is poorly understood. Regardless, commercial vaccines have been developed and are in use. Thus, the present study was designed to examine cellular immune responses induced by parenteral S. hyodysenteriae vaccination. Significant antigen-specific interferon-gamma (IFN-gamma) and blastogenic responses were detected from peripheral blood lymphocytes isolated from vaccinated pigs. However, poor IFN-gamma responses were detected from colonic lymph node lymphocytes from these same pigs despite significant antigen-specific blastogenic responses. In addition, peripheral blood IFN-gamma responses were diminished by either in vitro depletion of CD4 expressing cells or by in vitro treatment with porcine IL-10. Colonic lymph node IFN-gamma responses were not inhibited by treatment with porcine IL-10. Vaccination also resulted in increased percentages of both mucosal and peripheral blood CD8 single positive cells with concurrent decreases in percentages of CD4 single positive cells as compared to percentages of these same populations from non-vaccinated pigs. In conclusion, these studies show that parenteral S. hyodysenteriae vaccination results in cellular immune responses detectable both peripherally (systemic immunity) as well as at the site of infection (mucosal immunity). However, it appears that regulatory mechanisms affecting IFN-gamma production in response to S. hyodysenteriae antigen differ between peripheral and colonic compartments.     

Novel H1N2 swine influenza reassortant strain in pigs derived from the pandemic H1N1/2009 virus

Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.     

Comparison of humoral and cellular immune responses to inactivated swine influenza virus vaccine in weaned pigs

Humoral and cellular immune responses to inactivated swine influenza virus (SIV) vaccine were evaluated and compared. Fifty 3-week-old weaned pigs were randomly divided into the non-vaccinated control group and vaccinated group containing 25 pigs each. Pigs were vaccinated intramuscularly twice with adjuvanted UV-inactivated A/SW/MN/02011/08 (MN/08) H1N2 SIV vaccine at 6 and 9 weeks of age. Whole blood samples for multi-parameter flow cytometry (MP-FCM) and serum samples for hemagglutination inhibition (HI) assay were collected at 23 and 28 days after the second vaccination, respectively. A standard HI assay and MP-FCM were performed against UV-inactivated homologous MN/08 and heterologous pandemic A/CA/04/2009 (CA/09) H1N1 viruses. While the HI assay detected humoral responses only to the MN/08 virus, the MP-FCM detected strong cellular responses against the MN/08 virus and significant heterologous responses to the CA/09 virus, especially in the CD4+CD8+ T cell subset. The cellular heterologous responses to UV-inactivated virus by MP-FCM suggested that the assay was sensitive and potentially detected a wider range of antigens than what was detected by the HI assay. Overall, the adjuvanted UV-inactivated A/SW/MN/02011/08 H1N2 SIV vaccine stimulated both humoral and cellular immune responses including the CD4-CD8+ T cell subset.     

Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype

Swine influenza virus (SIV) and Mycoplasma hyopneumoniae (Mhp) are widespread in farms and are major pathogens involved in the porcine respiratory disease complex (PRDC). The aim of this experiment was to compare the pathogenicity of European avian-like swine H1N1 and European human-like reassortant swine H1N2 viruses in na?ve pigs and in pigs previously infected with Mhp. Six groups of SPF pigs were inoculated intra-tracheally with either Mhp, or H1N1, or H1N2 or Mhp+H1N1 or Mhp+H1N2, both pathogens being inoculated at 21 days intervals in these two last groups. A mock-infected group was included. Although both SIV strains induced clinical signs when singly inoculated, results indicated that the H1N2 SIV was more pathogenic than the H1N1 virus, with an earlier shedding and a greater spread in lungs. Initial infection with Mhp before SIV inoculation increased flu clinical signs and pathogenesis (hyperthermia, loss of appetite, pneumonia lesions) due to the H1N1 virus but did not modify significantly outcomes of H1N2 infection. Thus, Mhp and SIV H1N1 appeared to act synergistically, whereas Mhp and SIV H1N2 would compete, as H1N2 infection led to the elimination of Mhp in lung diaphragmatic lobes. In conclusion, SIV would be a risk factor for the severity of respiratory disorders when associated with Mhp, depending on the viral subtype involved. This experimental model of coinfection with Mhp and avian-like swine H1N1 is a relevant tool for studying the pathogenesis of SIV-associated PRDC and testing intervention strategies for the control of the disease.     

不同H5N1亚型禽流感病毒在豚鼠体内的复制和传播能力评估

本研究以豚鼠作为哺乳动物模型,评价了6株H5N1亚型禽流感病毒(AIV)的复制和水平传播能力。经滴鼻接种后,检测到6株病毒中有5株病毒在豚鼠上呼吸道和下呼吸道复制,病毒滴度为0.8LogEID50/mL～3.5LogEID50/mL(或g),豚鼠在感染后第2d至第10d可排出病毒。另外一株病毒A/duck/GX/22/02则仅能在豚鼠的下呼吸道低水平复制。在传播实验中,6株病毒中只有A/duck/GX/35/01能够在豚鼠间水平传播。上述研究结果表明,豚鼠适用于作为高致病性AIV哺乳动物水平传播的模型,而且H5N1亚型AIV在哺乳动物间的水平传播能力不同。本研究为进一步研究AIV在哺乳动物间水平传播的分子机制奠定了良好的基础。     

Descriptive clinical and epidemiological characteristics of influenza A H1N1 2009 virus infections in pigs in England

Infection of pigs with influenza A H1N1 2009 virus (A(H1N1)pdm09) was first detected in England in November 2009 following global spread of the virus in the human population. This paper describes clinical and epidemiological findings in the first English pig farms in which A(H1N1)pdm09 influenza virus was detected. These farms showed differences in disease presentation, spread and duration of infection. The factors likely to influence these features are described and relate to whether pigs were housed or outdoors, the age of the pigs, inter-current disease and the management system of the unit. Infection could be mild or clinically inapparent in breeding pigs with more typical respiratory disease being identified later in their progeny. Mortality was low where disease was uncomplicated by environmental stresses or concurrent infections. Where deaths occurred in pigs infected with A(H1N1)pdm09 influenza, they were mainly due to other infections, including streptococcal disease due to Streptococcus suis infection. This paper demonstrates the ease with which A(H1N1)pdm09 virus was transmitted horizontally and maintained in a pig population.     

辽宁省H1N1亚型猪流感病毒的分离鉴定与遗传演化分析

本研究2012年底从辽宁省某屠宰场猪鼻咽拭子样品中分离到1株流感病毒,经HA—HI试验和RT—PCR鉴定为H1N1亚型猪流感病毒株,命名为A／swine/Liaoning／01／2012（H1N1）,通过对病毒的8个基因片段克隆并测序,并利用分子生物学软件进行遗传演化分析。结果表明,分离株HA基因裂解位点附近的氨基酸序列为IPSIQSRjG,符合低致病力流感病毒的分子特征。全基因组进化树结果表明,分离株的8个基因片段与A／swine／Jiangsu／40／2011（H1N1）株核苷酸同源性最高,分离株处在类禽型H1N1亚型遗传进化分支上;由于类禽型H1N1猪流感病毒具有潜在感染人的潜力,在国外和国内均有感染人的报道,因此,辽宁省首次分离到该型猪流感病毒对全省养猪业和公共卫生安全具有重要意义,值得深入研究。     

Genetic characterization of H7N2 influenza virus isolated from pigs

Because pigs have respiratory epitheliums which express both α2-3 and α2-6 linked sialic acid as receptors to influenza A viruses, they are regarded as mixing vessel for the generation of pandemic influenza viruses through genetic reassortment. A H7N2 influenza virus (A/swine/KU/16/2001) was isolated from pig lungs collected from the slaughterhouse. All eight genes of the influenza virus were sequenced and phylogenetic analysis indicated that A/swine/KU/16/2001 originated in Hong Kong and genetic reassortment had occurred between the avian H7N2 and H5N3 influenza viruses. The first isolation of H7 influenza virus in pigs provides the opportunity for genetic reassortment of influenza viruses with pandemic potential and emphasizes the importance of surveillance for atypical swine influenza viruses.     

Evaluation of transmission of swine influenza type A subtype H1N2 virus in seropositive pigs

OBJECTIVE: To examine clinical signs, virus infection and shedding, and transmission of swine influenza virus (SIV) subtype H1N2 among seropositive pigs. ANIMALS: Eighteen 3-week-old pigs with maternal antibodies against SIV subtypes H1N1, H3N2, and H1N2. PROCEDURE: Ten pigs (principal) were inoculated intranasally with subtype H1N2 and 2 groups of contact pigs (n = 4) each were mixed with principal pigs on day 7 (group 1) or 28 (group 2). Two principal pigs each were necropsied on days 4, 14, 21, 28, and 42 days after inoculation. Four pigs in each contact group were necropsied 35 and 14 days after contact. Virus excretion was evaluated after inoculation or contact. Lung lesions and the presence of SIV in various tissues were examined. RESULTS: Mild coughing and increased rectal temperature were observed in principal pigs but not in contact pigs. Nasal virus shedding was detected in all principal pigs from day 2 for 3 to 5 days, in group 1 pigs from day 2 for 4 to 9 days after contact, and in group 2 pigs from day 4 for 2 to 6 days after contact. Trachea, lung, and lymph node specimens from infected pigs contained virus. Antibody titers against all 3 subtypes in all pigs gradually decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Protection from viral infection and shedding was not observed in pigs with maternal antibodies, but clinical disease did not develop. Vaccination programs and good management practices should be considered for control of SIV subtype H1N2 infection on swine farms.