Novel Swine Influenza Virus Reassortants in Pigs, China

During swine influenza virus surveillance in pigs in China during 2006–2009, we isolated subtypes H1N1, H1N2, and H3N2 and found novel reassortment between contemporary swine and avian panzootic viruses. These reassortment events raise concern about generation of novel viruses in pigs, which could have pandemic potential.     

Serological studies of influenza viruses in pigs in Great Britain 1991-2.

Samples from a sow serum bank representative of the pig population of Great Britain collected during 1991-2, were examined for antibodies to influenza A, B and C viruses, using viruses which had been isolated from a variety of hosts. For influenza A viruses there was evidence of the continued circulation of classical swine H1N1 virus (26%) seroprevalence), and human H3N2 viruses (39%) which are antigenically most closely-related to A/Port Chalmers/1/73 virus. In addition antibodies were detected to A/swine/England/201635/92 (8%), a strain of H3N2 virus which appears to have arisen by antigenic drift from conventional H3N2 swine strains. Specific antibodies (2%) were detected to an H1N1 virus (A/swine/England/195852/92) related most closely to avian H1N1 strains. In tests with human H1N1 and H3N2 viruses, excluding isolates from pigs, the highest seroprevalence was detected to the prevailing strains from the human population. Serological tests with avian H4 and H10, human H2, equine 1 and 2 influenza A viruses were all negative. Seven pigs seropositive by haemagglutination-inhibition, virus neutralization and immunoblotting assays for antibody to influenza B virus, were randomly distributed geographically suggesting that influenza B viruses may be transmitted to pigs but fail to spread. The seroprevalence to influenza C viruses was 9.9% indicating that these viruses are widespread in pigs. These results provide further evidence that the pig can be infected by a number of influenza viruses, some of which may have significance in the epidemiology of human influenza.     

Spread of Influenza Virus A (H5N1) Clade 2.3.2.1 to Bulgaria in Common Buzzards

On March 15, 2010, a highly pathogenic avian influenza virus was isolated from the carcass of a common buzzard (Buteo buteo) in Bulgaria. Phylogenetic analyses of the virus showed a close genetic relationship with influenza virus A (H5N1) clade 2.3.2.1 viruses isolated from wild birds in the Tyva Republic and Mongolia during 2009–2010. Designated A/common buzzard/Bulgaria/38WB/2010, this strain was highly pathogenic in chickens but had low pathogenicity in mice and ferrets and no molecular markers of increased pathogenicity in mammals. The establishment of clade 2.3.2.1 highly pathogenic avian influenza viruses of the H5N1 subtype in wild birds in Europe would increase the likelihood of health threats to humans and poultry in the region.     

Efficacy of an AS03A-adjuvanted split H5N1 influenza vaccine against an antigenically distinct low pathogenic H5N1 virus in pigs

We used the pig model of influenza to examine the efficacy of an AS03(A)-adjuvanted split H5N1 (A/Indonesia/05/2005) vaccine against challenge with a low pathogenic (LP) H5N1 avian influenza (AI) virus (duck/Minnesota/1525/1981) with only 85% amino acid homology in its HA1. Influenza seronegative pigs were vaccinated twice intramuscularly with adjuvanted vaccine at 3 antigen doses, unadjuvanted vaccine or placebo. All pigs were challenged 4 weeks after the second vaccination and euthanized 2 days later. After 2 vaccinations, all pigs in the adjuvanted vaccine groups had high hemagglutination inhibiting (HI) antibody titers to the vaccine strain (160-640), and lower antibody titers to the A/Vietnam/1194/04 H5N1 strain and to 2 LP H5 viruses with 90-91% amino acid homology to the vaccine strain (20-160). Eight out of 12 pigs had HI titers (10-20) to the challenge virus immediately before challenge. Neuraminidase inhibiting antibodies to the challenge virus were detected in most pigs (7/12) and virus neutralizing antibodies in all pigs. There was no antigen-dose dependent effect on the antibody response among the pigs immunized with adjuvanted H5N1 vaccines. After challenge, these pigs showed a complete clinical protection, reduced lung lesions and a significant protection against virus replication in the respiratory tract. Though the challenge virus showed only moderate replication efficiency in pigs, our study suggests that AS03(A)-adjuvanted H5N1 vaccine may confer a broader protection than generally assumed. The pros and cons of the pig as an H5N1 challenge model are also discussed.     

Pandemic (H1N1) 2009 Virus on Commercial Swine Farm, Thailand

A swine influenza outbreak occurred on a commercial pig farm in Thailand. Outbreak investigation indicated that pigs were co-infected with pandemic (H1N1) 2009 virus and seasonal influenza (H1N1) viruses. No evidence of gene reassortment or pig-to-human transmission of pandemic (H1N1) 2009 virus was found during the outbreak.     

Influenza in Refugees on the Thailand�CMyanmar Border, May�COctober 2009

We describe the epidemiology of influenza virus infections in refugees in a camp in rural Southeast Asia during May–October 2009, the first 6 months after identification of pandemic (H1N1) 2009 in Thailand. Influenza A viruses were detected in 20% of patients who had influenza-like illness and in 23% of those who had clinical pneumonia. Seasonal influenza A (H1N1) was the predominant virus circulating during weeks 26–33 (June 25–August 29) and was subsequently replaced by the pandemic strain. A review of passive surveillance for acute respiratory infection did not show an increase in acute respiratory tract infection incidence associated with the arrival of pandemic (H1N1) 2009 in the camp.     

Genetic and biological characterization of two novel reassortant H5N6 swine influenza viruses in mice and chickens

Novel H5N6 influenza A viruses have infected birds and human beings and caused four human clinical cases in China since 2014. The pig, as a mixing vessel, plays an important role for influenza virus reassortment and transmission. Towards this, routine surveillance for swine influenza in Guangdong province was conducted in 2014. In this study, we reported the biological characterization of two H5N6 influenza viruses isolated from healthy pigs in Guangdong province. Genetic analysis indicates that the two viruses are reassortants of 2.3.4.4 H5N1 and H6N6 avian influenza viruses with a high similarity to duck and human H5N6 influenza viruses isolated from Guangdong province. The data from chicken and mouse experiments show that the viruses are highly pathogenic in chickens and result in a systemic infection, and replicate in the mouse lung accompanying with a clinical inflammatory pathology. The results of the study demonstrate that the two H5N6 influenza viruses isolated from swine are the avian-originated viruses and have not adapted to swine population yet. However, they might keep evolving and pose a potential risk to public health and the continued surveillance of swine influenza should be strengthened.     

Genomic Signatures of Influenza A Pandemic (H1N1) 2009 Virus

Adaptive mutations that have contributed to the emergence of influenza A pandemic (H1N1) 2009 virus, which can replicate and transmit among humans, remain unknown. We conducted a large-scale scanning of influenza protein sequences and identified amino acid–conserving positions that are specific to host species, called signatures. Of 47 signatures that separate avian viruses from human viruses by their nonglycoproteins, 8 were human-like in the pandemic (H1N1) 2009 virus. Close examination of their amino acid residues in the recent ancestral swine viruses of pandemic (H1N1) 2009 virus showed that 7 had already transitioned to human-like residues and only PA 356 retained an avian-like K; in pandemic (H1N1) 2009 virus, this residue changed into a human-like R. Signatures that separate swine viruses from human viruses were also present. Continuous monitoring of these signatures in nonhuman species will help with influenza surveillance and with evaluation of the likelihood of further adaptation to humans.     

Co-circulation of pandemic 2009 H1N1, classical swine H1N1 and avian-like swine H1N1 influenza viruses in pigs in China

The pandemic A/H1N1 influenza viruses emerged in both Mexico and the United States in March 2009, and were transmitted efficiently in the human population. They were transmitted occasionally from humans to other mammals including pigs, dogs and cats. In this study, we report the isolation and genetic analysis of novel viruses in pigs in China. These viruses were related phylogenetically to the pandemic 2009 H1N1 influenza viruses isolated from humans and pigs, which indicates that the pandemic virus is currently circulating in swine populations, and this hypothesis was further supported by serological surveillance of pig sera collected within the same period. Furthermore, we isolated another two H1N1 viruses belonging to the lineages of classical swine H1N1 virus and avian-like swine H1N1 virus, respectively. Multiple genetic lineages of H1N1 viruses are co-circulating in the swine population, which highlights the importance of intensive surveillance for swine influenza in China.     

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1).     

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.     

Swine-origin influenza A (H3N2) virus infection in two children--Indiana and Pennsylvania, July-August 2011

Influenza A viruses are endemic in many animal species, including humans, swine, and wild birds, and sporadic cases of transmission of influenza A viruses between humans and animals do occur, including human infections with avian-origin influenza A viruses (i.e., H5N1 and H7N7) and swine-origin influenza A viruses (i.e., H1N1, H1N2, and H3N2). Genetic analysis can distinguish animal origin influenza viruses from the seasonal human influenza viruses that circulate widely and cause annual epidemics. This report describes two cases of febrile respiratory illness caused by swine-origin influenza A (H3N2) viruses identified on August 19 and August 26, 2011, and the current investigations. No epidemiologic link between the two cases has been identified, and although investigations are ongoing, no additional confirmed human infections with this virus have been detected. These viruses are similar to eight other swine-origin influenza A (H3N2) viruses identified from previous human infections over the past 2 years, but are unique in that one of the eight gene segments (matrix [M] gene) is from the 2009 influenza A (H1N1) virus. The acquisition of the M gene in these two swine-origin influenza A (H3N2) viruses indicates that they are "reassortants" because they contain genes of the swine-origin influenza A (H3N2) virus circulating in North American pigs since 1998 and the 2009 influenza A (H1N1) virus that might have been transmitted to pigs from humans during the 2009 H1N1 pandemic. However, reassortments of the 2009 influenza A (H1N1) virus with other swine influenza A viruses have been reported previously in swine. Clinicians who suspect influenza virus infection in humans with recent exposure to swine should obtain a nasopharyngeal swab from the patient for timely diagnosis at a state public health laboratory and consider empiric neuraminidase inhibitor antiviral treatment to quickly limit potential human transmission.     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

Novel Reassortant Influenza A(H5N8) Viruses among Inoculated Domestic and Wild Ducks,South Korea, 2014

An outbreak of highly pathogenic avian influenza, caused by a novel reassortant influenza A (H5N8) virus, occurred among poultry and wild birds in South Korea in 2014. The aim of this study was to evaluate the pathogenesis in and mode of transmission of this virus among domestic and wild ducks. Three of the viruses had similar pathogenicity among infected domestic ducks: the H5N8 viruses were moderately pathogenic (0%–20% mortality rate); in wild mallard ducks, the H5N8 and H5N1 viruses did not cause severe illness or death; viral replication and shedding were greater in H5N8-infected mallards than in H5N1-infected mallards. Identification of H5N8 viruses in birds exposed to infected domestic ducks and mallards indicated that the viruses could spread by contact. We propose active surveillance to support prevention of the spread of this virus among wild birds and poultry, especially domestic ducks.     

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.     

Study of influenza A virus in wild boars living in a major duck wintering site

Wild birds, which are reservoirs of influenza viruses, are believed to be the original source of new influenza viruses-including highly pathogenic ones-that can be transmitted to domestic animals as well as humans and represent a potential epizootic and/or pandemic threat. Despite increasing knowledge on influenza A virus dynamics in wild birds, the viral circulation in wild boars remains largely unknown. This is of particular interest since pigs can be infected with both human and avian viruses; upon co-infection, they can act as a mixing vessel through reassortment, a mechanism that resulted in the emergence of the pandemic H1N1 virus in 2009. The Camargue (Southern France) appears as an ideal study area to investigate inter-species transmission of influenza A viruses from wild birds and possibly humans to wild boars. Indeed, the important local wild boar population shares wetland use with humans and the largest concentration of wintering ducks in France, that are both susceptible to infection by influenza A viruses. Additionally, wild boars occasionally prey on ducks. We conducted a virological and serological survey on wild boars in the Camargue (Southern France) between September 2009 and November 2010. No influenza A virus was detected in the collected nasal swabs (n=315) and no influenza specific antibodies were observed in the serological samples (n=20). As the study was mainly focused on viral excretion, which is limited in time, we cannot exclude that low or occasional influenza A virus circulation took place during the study period. Although, wild boars did not seem to be a key element in the dynamics of influenza A virus circulation in the Camargue, wild boar influenza A virus infections should be more widely studied to determine if the pattern observed here represents the normal situation or an exceptional one.     

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.     

Das pandemische H1N1-Influenzavirus/2009

The generation of pandemic influenza A viruses of the previous century as well as that of the current influenza A/H1N1/2009 pandemic appear to be governed and preceded by reassortment events in other mammalian species. So far, it could not be shown that transmission of avian influenza viruses to humans will directly cause a pandemic. Zoonotic transmissions of avian and also of porcine influenza viruses of diverse subtypes have been repeatedly described. However, these events did not lead to further spread and establishment of these viruses. This is in contrast to the current A/H1N1/2009 viruses which already have started to outcompete seasonal human influenza viruses. The actual molecular key factors required for a successful exchange of genome segments between different influenza virus strains and which factors foster the consecutive spread of certain reassortant viruses in the human population remain to be pinpointed. It has been elucidated so far that newly introduced genome segments need to be compatible with both the remaining original segments and the human hosts.     

Recent zoonoses caused by influenza A viruses

Influenza is a highly contagious, acute illness which has afflicted humans and animals since ancient times. Influenza viruses are part of the Orthomyxoviridae family and are grouped into types A, B and C according to antigenic characteristics of the core proteins. Influenza A viruses infect a large variety of animal species, including humans, pigs, horses, sea mammals and birds, occasionally producing devastating pandemics in humans, such as in 1918, when over twenty million deaths occurred world-wide. The two surface glycoproteins of the virus, haemagglutinin (HA) and neuraminidase (NA), are the most important antigens for inducing protective immunity in the host and therefore show the greatest variation. For influenza A viruses, fifteen antigenically distinct HA subtypes and nine NA subtypes are recognised at present; a virus possesses one HA and one NA subtype, apparently in any combination. Although viruses of relatively few subtype combinations have been isolated from mammalian species, all subtypes, in most combinations, have been isolated from birds. In the 20th Century, the sudden emergence of antigenically different strains in humans, termed antigenic shift, has occurred on four occasions, as follows, in 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each resulting in a pandemic. Frequent epidemics have occurred between the pandemics as a result of gradual antigenic change in the prevalent virus, termed antigenic drift. Currently, epidemics occur throughout the world in the human population due to infection with influenza A viruses of subtypes H1N1 and H3N2 or with influenza B virus. The impact of these epidemics is most effectively measured by monitoring excess mortality due to pneumonia and influenza. Phylogenetic studies suggest that aquatic birds could be the source of all influenza A viruses in other species. Human pandemic strains are thought to have emerged through one of the following three mechanisms: genetic reassortment (occurring as a result of the segmented genome of the virus) of avian and human influenza A viruses infecting the same host direct transfer of whole virus from another species the re-emergence of a virus which may have caused an epidemic many years earlier. Since 1996, the viruses H7N7, H5N1 and H9N2 have been transmitted from birds to humans but have apparently failed to spread in the human population. Such incidents are rare, but transmission between humans and other animals has also been demonstrated. This has led to the suggestion that the proposed reassortment of human and avian viruses occurs in an intermediate animal with subsequent transference to the human population. Pigs have been considered the leading contender for the role of intermediary because these animals may serve as hosts for productive infections of both avian and human viruses and, in addition, the evidence strongly suggests that pigs have been involved in interspecies transmission of influenza viruses, particularly the spread of H1N1 viruses to humans. Global surveillance of influenza is maintained by a network of laboratories sponsored by the World Health Organization. The main control measure for influenza in human populations is immunoprophylaxis, aimed at the epidemics occurring between pandemics.