Novel swine influenza virus subtype H3N1, United States

Influenza A virus infects various animal species and transmits among different hosts, especially between humans and swine. Swine may serve as a mixing vessel to create new reassortants that could infect humans. Thus, monitoring and characterizing influenza viruses in swine are important in preventing interspecies transmission. We report the emergence and characterization of a novel H3N1 subtype of swine influenza virus (SIV) in the United States. Phylogenetic analysis showed that the H3N1 SIVs may have acquired the hemagglutinin gene from an H3N2 turkey isolate, the neuraminidase gene from a human H1N1 isolate, and the remaining genes from currently circulating SIVs. The H3N1 SIVs were antigenically related to the turkey virus. Lung lesions and nasal shedding occurred in swine infected with the H3N1 SIVs, suggesting the potential to transmit among swine and to humans. Further surveillance will help determine whether this novel subtype will continue to circulate in swine populations.     

Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection

Previously we reported the generation of a new potential live attenuated influenza vaccine (LAIV) named SIV/606 that expresses H1 and H3 HAs. We also demonstrated intratracheal vaccination of SIV/606 conferred protection against infections with both H1 and H3 swine influenza virus subtypes in pigs. Here we vaccinated pigs with SIV/606 intranasally, which is a more suitable route for LAIV, and evaluated vaccine efficacy. Intranasal vaccination of SIV/606 induced serum IgG antibody responses against both H1N1 and H3N2 SIVs and high titer of virus neutralizing antibodies against H1N1 SIV but not against H3N2 SIV. When we challenged the pigs with H1N1 and H3N2 SIVs, we observed marked reduction of lung lesions and viral titer in lung tissue in vaccinated pigs. Our analyses also showed that vaccinated pigs had more IFN-γ secreting cells in trachea–bronchial lymph nodes. Our studies demonstrated that intranasal vaccination of SIV/606 is efficacious for H1N1 and H3N2 SIVs infections. Moreover, our results may help explaining the protection from H3N2 SIV infection despite the low viral neutralizing antibody titer.     

Swine influenza virus antibodies in humans, western Europe, 2009

Serologic studies for swine influenza viruses (SIVs) in humans with occupational exposure to swine have been reported from the Americas but not from Europe. We compared levels of neutralizing antibodies against 3 influenza viruses--pandemic (H1N1) 2009, an avian-like enzootic subtype H1N1 SIV, and a 2007-08 seasonal subtype H1N1--in 211 persons with swine contact and 224 matched controls in Luxembourg. Persons whose profession involved contact with swine had more neutralizing antibodies against SIV and pandemic (H1N1) 2009 virus than did the controls. Controls also had antibodies against these viruses although exposure to them was unlikely. Antibodies against SIV and pandemic (H1N1) 2009 virus correlated with each other but not with seasonal subtype H1N1 virus. Sequential exposure to variants of seasonal influenza (H1N1) viruses may have increased chances for serologic cross-reactivity with antigenically distinct viruses. Further studies are needed to determine the extent to which serologic responses correlate with infection.     

Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States

As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir.     

Vaccination-challenge studies with a Port Chalmers/73 (H3N2)-based swine influenza virus vaccine: Reflections on vaccine strain updates and on the vaccine potency test

The human A/Port Chalmers/1/73 (H3N2) influenza virus strain, the supposed ancestor of European H3N2 swine influenza viruses (SIVs), was used in most commercial SIV vaccines in Europe until recently. If manufacturers want to update vaccine strains, they have to perform laborious intratracheal (IT) challenge experiments and demonstrate reduced virus titres in the lungs of vaccinated pigs. We aimed to examine (a) the ability of a Port Chalmers/73-based commercial vaccine to induce cross-protection against a contemporary European H3N2 SIV and serologic cross-reaction against H3N2 SIVs from Europe and North America and (b) the validity of intranasal (IN) challenge and virus titrations of nasal swabs as alternatives for IT challenge and titrations of lung tissue in vaccine potency tests. Pigs were vaccinated with Suvaxyn Flu^® and challenged by the IT or IN route with sw/Gent/172/08. Post-vaccination sera were examined in haemagglutination-inhibition assays against vaccine and challenge strains and additional H3N2 SIVs from Europe and North America, including an H3N2 variant virus. Tissues of the respiratory tract and nasal swabs were collected 3 days post challenge (DPCh) and from 0–7 DPCh, respectively, and examined by virus titration. Two vaccinations consistently induced cross-reactive antibodies against European H3N2 SIVs from 1998–2012, but minimal or undetectable antibody titres against North American viruses. Challenge virus titres in the lungs, trachea and nasal mucosa of the vaccinated pigs were significantly reduced after both IT and IN challenge. Yet the reduction of virus titres and nasal shedding was greater after IT challenge. The Port Chalmers/73-based vaccine still offered protection against a European H3N2 SIV isolated 35 years later and with only 86.9% amino acid homology in its HA1, but it is unlikely to protect against H3N2 SIVs that are endemic in North America. We use our data to reflect on vaccine strain updates and on the vaccine potency test.     

Potential Threats to Human Health from Eurasian Avian-Like Swine Influenza A(H1N1) Virus and Its Reassortants

During 2018–2020, we isolated 32 Eurasian avian-like swine influenza A(H1N1) viruses and their reassortant viruses from pigs in China. Genomic testing identified a novel reassortant H3N1 virus, which emerged in late 2020. Derived from G4 Eurasian H1N1 and H3N2 swine influenza viruses. This virus poses a risk for zoonotic infection.     

H3N2 influenza virus transmission from swine to turkeys, United States

In 1998, a novel H3N2 reassortant virus emerged in the United States swine population. We report the interspecies transmission of this virus to turkeys in two geographically distant farms in the United States in 2003. This event is of concern, considering the reassortment capacity of this virus and the susceptibility of turkey to infection by avian influenza viruses. Two H3N2 isolates, A/turkey/NC/16108/03 and A/turkey/MN/764/03, had 98.0% to 99.9% nucleotide sequence identity to each other in all eight gene segments. All protein components of the turkey isolates had 97% to 98% sequence identity to swine H3N2 viruses, thus demonstrating interspecies transmission from pigs to turkeys. The turkey isolates were better adapted to avian hosts than were their closest swine counterparts, which suggests that the viruses had already begun to evolve in the new host. The isolation of swine-like H3N2 influenza viruses from turkeys raises new concerns for the generation of novel viruses that could affect humans.     

The repeated introduction of the H3N2 virus from human to swine during 1979–1993 in China

Limited data are available regarding the swine influenza viruses (SIVs) that circulated in Mainland China prior to the 1990s. Eleven H3N2 virus strains were isolated from swine populations from 1979 to 1992. To determine the origin and tendency of these SIVs, the phylogenetic and antigenic properties of these viruses were analyzed based on the whole genome sequenced and the HI titrations with post-infection ferret antisera against influenza A (H3N2) virus isolates of swine and human origin. The results revealed that these 11 SIVs originated from humans and were not maintained in swine populations, indicating the interspecies transmission from humans to pigs occurred frequently and independently throughout these periods. However, human H3N2 viruses might not have the ability to circulate in pig herds.     

Reassortant Pandemic (H1N1) 2009 virus in pigs, United Kingdom

Surveillance for influenza virus in pigs in the United Kingdom during spring 2010 detected a novel reassortant influenza virus. This virus had genes encoding internal proteins from pandemic (H1N1) 2009 virus and hemagglutinin and neuraminidase genes from swine influenza virus (H1N2). Our results demonstrate processes contributing to influenza virus heterogeneity.     

Efficacy of intranasal administration of a truncated NS1 modified live influenza virus vaccine in swine

In the U.S., despite available swine influenza virus (SIV) vaccines, multiple influenza subtypes as well as antigenic and genetic variants within subtypes continue to circulate in the swine population. One of the challenges to control and eliminate SIV is that the currently used inactivated influenza virus vaccines do not provide adequate cross-protection against multiple antigenic variants of SIV in the field. We previously generated a recombinant H3N2 swine influenza virus (SIV) based on the influenza A/SW/TX/4199-2/98 virus (TX98) containing an NS1 gene expressing a truncated NS1 protein of 126 amino acids, TX98-NS1Delta126 virus. This recombinant strain was demonstrated to be highly attenuated in swine and showed potential for use as a modified live-virus vaccine (MLV) after intratracheal application in pigs. However, this route of inoculation is not practical for vaccination in the field. In the present study, we first compared intramuscular and intranasal routes of application of the MLV, and found that the intranasal route was superior in priming the local (mucosal) immune response. Pigs were then vaccinated via the intranasal route and challenged with wild type homologous TX98 H3N2 virus, with a genetic and antigenic variant H3N2 SIV (influenza A/SW/CO/23619/99 virus, CO99) and a heterosubtypic H1N1 SIV (influenza A/SW/IA/00239/2004 virus, IA04). The intranasally vaccinated pigs were completely protected against homologous challenge. In addition, MLV vaccination provided nearly complete protection against the antigenic H3N2 variant CO99 virus. When challenged with the H1N1 IA04 virus, MLV vaccinated animals displayed reduced fever and virus titers despite minimal reduction in lung lesions. In vaccinated pigs, there was no serologic cross-reactivity by HI assays with the heterologous or heterosubtypic viruses. However, there appeared to be substantial cross-reactivity in antibodies at the mucosal level with the CO99 virus in MLV vaccinated pigs.     

Generation of reassortant influenza viruses within the non-industrial poultry system

We compared the genetic and biologic characteristics of 35 influenza viruses of different epidemiological backgrounds in Korea, including H3N2 canine influenza virus (CIV). Phylogenetic analysis revealed that chicken adapted H9N2 viruses (A/chicken/Korea/96006/96 [CK/Kor/96006-like]) have acquired aquatic avian gene segments through reassortment, and these reassorted H9N2 viruses were more frequently detected from minor poultry species than from industrial poultry. Conversely, gene segments from CK/Kor/96006-like viruses were also detected in most of the viruses from domestic ducks. Interestingly, domestic ducks, rather than wild aquatic birds, harbored close relatives of all eight gene segments of H3N2 CIV, which preferred binding to avian receptors. Therefore, bidirectional virus transmission events are assumed to have occurred between land-based poultry and aquatic poultry, in particular within the non-industrial poultry system. These events have contributed to the generation of a novel reassortant, H3N2 CIV. To prevent generating other reassortants capable of interspecies transmission, gene movements in the non-industrial poultry systems should be clarified and managed.     

几种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法的验证

目的比较和验证5种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法。方法首先使用美国NCBI网站的Primer—BLAST程序验证5种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法引物的理论特异性,然后使用猪流感H1N1病毒核酸、2009年新甲型H1N1流感病毒核酸、H5N1禽流感核酸、能力验证样品作为试验样品验证方法的实验特异性。结果理论验证结果显示5对来源于文献的引物均适合于猪流感的检测,前3对可用于新型甲型H1Nl流感的检测,但只限于个别来源的毒株,与预期略有差别。实验验证结果显示,方法l可正确检出甲型流感病毒;方法2可正确检出猪流感H1N1亚型病毒、新甲型H1N1流感病毒核酸;方法3检测新甲型H1N1流感核酸时未获得阳性结果;方法4可以正确检测出猪流感H1病毒核酸;方法5可以正确检测出猪流感H1N1病毒核酸,但检测新甲型H1N1流感核酸、禽流感H5N1核酸时也为阳性。结论方法1可用于猪甲型流感和新甲型H1N1流感病毒的初筛;方法2可用于猪流感H1N1亚型病毒和新甲型H1N1流感病毒核酸的初筛;方法3不适合新甲型H1N1流感病毒核酸的检测;方法4可用于猪流感H1亚型的检测;方法5不适合于猪流感H1N1亚型病毒的检测。采用SYBRGreenI荧光RT—PCR方法检测甲型H1N1流感病毒核酸,特异性普遍不十分理想,只能用于初筛检测。     

Generation and evaluation of the trivalent inactivated reassortant vaccine using human, avian, and swine influenza A viruses

Reassortant technology was used to obtain three interspecific reassortant influenza viruses using three influenza viruses of A/Puerto Rico/8/34(H1N1), A/swine/Hebei/1/2005(H3N2) and A/chicken/Guangdong/126/2002(H9N2). The high-growth reassortant strains were H9/PR8, H3/H9N2 and H1/H9N2 that contained hemagglutinin (HA) and neuraminidase (NA) genes from the inactivated parental viruses and the other 6 internal genes from the live parental viruses. The trivalent formalin-inactivated vaccine, containing H1, H3 and H9 subtype antigens from human, swine and avian influenza viruses respectively, was prepared using these reassortant viruses. Animal studies showed that the vaccine was safe and immunogenic. Two-dosing regimen of the influenza vaccine induced high titers of hemagglutination inhibiting (HI) antibodies and influenza-specific IgG antibodies without antigenic cross-interference. It protected 100% chickens from challenge of A/chicken/Guangdong/126/2002 virus and protected 100% mice against challenges with different combinations of the three infective parental viruses. These results indicated that the trivalent vaccine could offer multi-protection against multi-influenza viruses synchronously. This kind of multivalent inactivated reassortant influenza vaccine maybe enlightens the pandemic influenza preparedness as the emergency measure.     

Transmission of avian influenza virus (H3N2) to dogs

In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) isolate. The beagles shed virus through nasal excretion, seroconverted, and became ill with severe necrotizing tracheobronchitis and bronchioalveolitis with accompanying clinical signs (e.g., high fever). Consistent with histologic observation of lung lesions, large amounts of avian influenza virus binding receptor (SAalpha 2,3-gal) were identified in canine tracheal, bronchial, and bronchiolar epithelial cells, which suggests potential for direct transmission of avian influenza virus (H3N2) from poultry to dogs. Our data provide evidence that dogs may play a role in interspecies transmission and spread of influenza virus.     

Efficacy of seasonal live attenuated influenza vaccine against virus replication and transmission of a pandemic 2009 H1N1 virus in ferrets

In March 2009, a swine origin influenza A (2009 H1N1) virus was introduced into the human population and quickly spread from North America to multiple continents. Human serologic studies suggest that seasonal influenza virus vaccination or infection would provide little cross-reactive serologic immunity to the pandemic 2009 H1N1 virus. However, the efficacy of seasonal influenza infection or vaccination against 2009 H1N1 virus replication and transmission has not been adequately evaluated in vivo. Here, ferrets received one or two doses of the US licensed 2008-2009 live attenuated influenza vaccine (LAIV) intranasally. An additional group of ferrets were inoculated with the A/Brisbane/59/07 (H1N1) virus to model immunity induced by seasonal influenza virus infection. All vaccinated and infected animals possessed high titer homologous hemagglutination-inhibition (HI) and neutralizing antibodies, with no demonstrable cross-reactive antibodies against 2009 H1N1 virus. However, in comparison to non-immune controls, immunized ferrets challenged with pandemic A/Mexico/4482/09 virus displayed a significant reduction in body temperature and virus shedding. The impact of single-dose LAIV inoculation on 2009 H1N1 disease and virus transmission was also measured in vaccinated ferrets that were challenged with pandemic A/Netherlands/1132/09 virus. Although a single dose of LAIV reduced virus shedding and the frequency of transmission following homologous seasonal virus challenge, it failed to reduce respiratory droplet transmission of 2009 H1N1 virus. The results demonstrate that prior immunization with seasonal LAIV or H1N1 virus infection provides some cross-protection against the 2009 H1N1 virus, but had no significant effect on the transmission efficiency of the 2009 H1N1 virus.     

Human Case of Swine Influenza A (H1N1) Triple Reassortant Virus Infection, Wisconsin

Zoonotic infections with swine influenza A viruses are reported sporadically. Triple reassortant swine influenza viruses have been isolated from pigs in the United States since 1998. We report a human case of upper respiratory illness associated with swine influenza A (H1N1) triple reassortant virus infection that occurred during 2005 following exposure to freshly killed pigs.     

Serologic Cross-Reactivity with Pandemic (H1N1) 2009 Virus in Pigs, Europe

We tested serum samples from pigs infected or vaccinated with European swine influenza viruses (SIVs) in hemagglutination-inhibition assays against pandemic (H1N1) 2009 virus and related North American SIVs. We found more serologic cross-reaction than expected. Data suggest pigs in Europe may have partial immunity to pandemic (H1N1) 2009 virus.     

Influenza activity in China: 1998-1999

During 1989-1999, influenza A H3N2 and H1N1 subtypes and B type viruses were still co-circulating in human population in China, while influenza A (H3N2) virus was predominant strain. The two antigenically and genetically distinguishable strains of influenza B virus were also still co-circulating in men in southern China. The antigenic analysis indicated that most of the H3N2 viruses were A/Panama/2007/99 (H3N2)-like strain, the most of the H1N1 viruses were antigenically similar to A/Beijing/262/95 (H1N1) virus. However, most of the influenza B viruses were B/Beijing/184/93-like strain, but few of them were antigenically similar to B/Shandong/7/97 virus. In the summer of 1998, the influenza outbreaks caused by H3N2 subtype of influenza A virus occurred widely in southern China. Afterwards, during 1998-1999 influenza season, a severe influenza epidemic caused by H3N2 virus emerged in northern China. The morbidity was reached as high as 10% in Beijing area. It was interesting that during influenza, surveillance from 1998 to 1999, five strains of avian influenza A (H9N2) virus were isolated from outpatients with influenza-like illness in July-August of 1998, and another one was repeatedly isolated from a child suffering from influenza-like disease in November of 1999 in Guangdong province. The genetic analysis revealed that the five strains isolated in 1998 were genetically closely related to H9N2 viruses being isolated from chickens (G9 lineage virus), whereas, A/Guangzhou/333/99 (H9N2) virus was a reassortant derived from reassortment between G9 and G1 lineage of avian influenza A (H9N2) viruses due to its genes encoding the HA, NA, NP and NS proteins, closely related to G9 lineage virus, the rest of the genes encoding the M and three polymerase (PB2, PB1 and PA) were closely related to G1 lineage strain of H9N2 virus. However, no avian influenza A (H5N1) virus has so far been isolated neither from in or outpatients with influenza-like disease in mainland China. Unfortunately, where did the reassortment occur and how did the reassortant transmit to men? These questions are still unknown.     

Pandemic H1N1 influenza virus-like particles are immunogenic and provide protective immunity to pigs

The outbreak of the 2009 influenza pandemic underscored the important role of swine in influenza virus evolution and the emergence of novel viruses with pandemic potential. Vaccination is the most common practice to control swine influenza in swine industry. Influenza virus-like particle (VLP) vaccines are an alternative approach and have been demonstrated to be immunogenic and confer protection against influenza virus challenge in chickens, mice and ferrets. In this study, we generated VLPs consisting of HA, NA and M1 proteins derived from pandemic virus A/California/04/2009 in insect cells. The immunogenicity and efficacy following vaccination of VLPs were evaluated in swine. Our data showed that vaccination using VLPs elicited robust levels of serum IgG, mucosal IgA, and viral neutralizing antibodies against A/Sw/Manitoba/MAFRI32/2009 H1N1. Following challenge with pandemic H1N1 2009, vaccinated pigs were protected, displaying reduced lung lesions, virus shedding and inhibition of virus replication in the lungs compared to non-vaccinated control pigs. Thus, VLPs can serve as a promising vaccination strategy to control influenza in swine.     

Immunogenicity and protective efficacy of an elastase-dependent live attenuated swine influenza virus vaccine administered intranasally in pigs

Influenza A virus is an important respiratory pathogen of swine that causes significant morbidity and economic impact on the swine industry. Vaccination is the first choice for prevention and control of influenza infections. Live attenuated influenza vaccines (LAIV) are approved for use in humans and horses and their application provides broad protective immunity, however no LAIV against swine influenza virus (SIV) exists in the market. Previously we reported that an elastase-dependant mutant SIV A/Sw/Sk-R345V (R345V) derived from A/Sw/Saskatchewan/18789/02 (H1N1) (SIV/Sk02) is highly attenuated in pigs. Two intratracheal administrations of R345V induced strong cell-mediated and humoral immune responses and provided a high degree of protection to antigenically different SIV infection in pigs. Here we evaluated the immunogenicity and the protective efficacy of R345V against SIV infection by intranasal administration, the more practical route for vaccination of pigs in the field. Our data showed that intranasally administered R345V live vaccine is capable of inducing strong antigen-specific IFN-γ response from local tracheo-bronchial lymphocytes and antibody responses in serum and respiratory mucosa after two applications. Intranasal vaccination of R345V provided pigs with complete protection not only from parental wild type virus infection, but also from homologous antigenic variant A/Sw/Indiana/1726/88 (H1N1) infection. Moreover, intranasal administration of R345V conferred partial protection from heterologous subtypic H3N2 SIV infection in pigs. Thus, R345V elastase-dependent mutant SIV can serve as a live vaccine against antigenically different swine influenza viruses in pigs.