Host cell-mediated variation in H3N2 influenza viruses

The influence of the host cell on the selection of antigenic variants of influenza A H3N2 viruses and the relevance of host cell selection to the induction of immunity by these viruses have been investigated. Influenza viruses were isolated from human clinical samples during a single epidemic, were passaged in mammalian Madin-Darby Canine Kidney (MDCK) cells or in embryonated hens eggs, and were tested for antigenic variability in the hemagglutinin (HA) molecule with a panel of monoclonal antibodies. In many cases, the HA of virus cultivated in eggs was antigenically distinct from the HA of virus from the same individual grown in mammalian cells. Viruses recovered from different individuals were antigenically similar to each other when grown in mammalian cell lines yet were antigenically heterogeneous when cultivated in eggs. The HA genes of viruses isolated from different individuals during the epidemic were shown, by sequence analysis, to differ from each other by five or six amino acid residues. Sequence analyses of the HA genes of MDCK cell-grown and egg-grown virus obtained from the same individual demonstrated that the molecular changes between antigenically distinct HAs of MDCK cell- and egg-grown A/Mem/12/85 virus involved a single amino acid substitution at residue 156 in HA1, which lies at the tip of the HA molecule and immediately adjacent to the receptor-binding site. However, the amino acid sequences of HAs from MDCK-grown and egg-grown viruses (A/Mem/2/85) isolated from a second individual were identical although these viruses exhibited antigenic differences when examined with anti-HA monoclonal antibodies. Therefore, single amino acid changes in the HA molecule may not be the sole cause of antigenic changes in the HA observed between pairs of MDCK cell-grown and egg-grown viruses and genes other than that encoding the HA may contribute to the host cell-mediated antigenic variation of these viruses. Nevertheless, antigenic differences between viruses grown in eggs and MDCK cells did not influence their ability to protect, since ferrets infected with either live egg-grown or MDCK-grown virus were protected equally well from challenge with virus grown in either host cell type.     

Serological profiles after consecutive experimental infections of pigs with European H1N1, H3N2, and H1N2 swine influenza viruses

Swine influenza viruses (SIVs) of H1N1, H3N2, and H1N2 subtypes, with antigenically different hemagglutinins, are currently cocirculating in pigs in Europe. This study aimed to determine whether the hemagglutination inhibition (HI) test, which is the primary serological test for SIV, is sufficiently specific to discriminate between infections with the three subtypes. In experiment 1, pigs were consecutively inoculated with European H1N1, H3N2, and H1N2 SIVs by the intranasal route, or with the respective subtypes only. In a second experiment, a commercial, inactivated H1N1- and H3N2- based SIV vaccine was administered once to pigs previously infected with one to three SIV subtypes or to influenza-naive pigs. Sequential serum samples were examined in HI and virus-neutralizing (VN) tests to the three strains used for pig inoculations. Of the 160 sera collected after infection with one or two SIV subtypes, only 8 showed cross-reactive antibodies to the remaining subtype(s) in the HI test, and 11 in the VN test. Consecutive inoculations with H1N1 and H1N2 or vice versa were followed by a significant rise in preexisting antibody titers to the first subtype after the second inoculation. When dually infection-immune pigs were inoculated with the third, remaining SIV subtype, nasal virus excretion was undetectable or reduced and the serological response was absent to moderate. A single vaccination of infection-immune pigs resulted in a dramatic rise in HI and VN antibody titers to any of the previously encountered subtypes, whereas SIV-naive pigs barely seroconverted. Most important, pigs previously infected with H1N1 but not with H1N2 developed crossreactive antibodies to H1N2 after the vaccination. In conclusion, the HI test remains adequate for the differential diagnosis of infections with H1N1, H3N2, and H1N2 in European swine populations if it is properly used and if the SIV vaccination status is taken into account.     

Genetic characterization of H1N1, H1N2 and H3N2 swine influenza virus in Thailand

Swine have been known to be a suitable host for influenza A virus. In Thailand, phylogenetic analysis on swine influenza virus (SIV) has as yet not been attempted. The present report presents molecular and phylogenetic analysis performed on SIV in Thailand. In this study, 12 SIV isolates from the central and eastern part of Thailand were subtyped and the molecular genetics of hemagglutinin and neuraminidase were elucidated. Three subtypes, H1N1, H1N2 and H3N2, are described. Phylogenetic analysis of the SIV hemagglutinin and neuraminidase genes shows individual clusters with swine, human or avian influenza virus at various global locations. Furthermore, amino acid substitutions were detected either at the receptor binding site or the antigenic sites of the hemagglutinin gene.     

Isolation of two H1N2 influenza viruses from swine in France

Summary Samples collected in 1987 and 1988 in Brittany from influenzainfected swine made it possible to isolate and antigenically characterize two H1N2 recombinant viruses (Sw/France/5027/87 and Sw/France/5550/88). The former virus was cloned and reinoculated to swine to allow reproduction of the disease and reisolation of a strain similar to the original one. The serodiagnostic tests carried out on both the original sera and those from the experimentally infected animals confirmed that the virus was actually type Sw/H1N2.     

Antigenic and genetic variation in the hemagglutinins of H1N1 and H3N2 human influenza a viruses in the Shanghai area from 2005 to 2008

Continued rapid evolution of the influenza A virus is responsible for annual epidemics and occasional pandemics in the Shanghai area. In the present study, the representative strains of A/H1N1 and A/H3N2 influenza viruses isolated in the Shanghai area from 2005 to 2008 were antigenically and genetically characterized. The antigenic cartography method was carried out to visualize the hemagglutination-inhibition data. Antigenic differences were detected between circulating A/H1N1 strains isolated from 2005 to 2006 and the epidemic A/H1N1 strains isolated in 2008, which were found to be associated with the amino acid substitution K140E in HA1. The present vaccine strain A/Brisbane/59/2007 is considered to be capable of providing sufficient immunity against most of the circulating A/H1N1 viruses isolated in 2008 from the Shanghai population. The study showed that there were significant antigenic differences between the epidemic A/H3N2 strains isolated in 2007 and 2008, suggesting that antigenic drift had occurred in the A/H3N2 strains isolated in 2008. The P194L mutation was thought to be responsible for the antigenic evolution of influenza A/H3N2 viruses isolated from Shanghai in 2008. Evidence of antigenic drift suggests that the influenza A/H3N2 vaccine component needs to be updated.     

Pathogenesis and inflammatory responses of swine H1N2 influenza viruses in pigs

Swine influenza viruses are an important pathogen in pig industry. In this study, we wanted to know whether swine H1N2 influenza viruses circulating in Korean pigs would cause clinical signs in pigs when experimentally infected. When pigs were infected with swine H1N2 viruses isolated from Korean pigs, pigs suffered from severe clinical signs of coughing, nasal discharge, labored breathing, facial edema, anorexia, and diarrhea. When the level of cytokine induction was measured using lung tissues, pro-inflammatory cytokines such as TNF-alpha, IL-1, and IL-8 were induced higher in lungs of infected pigs than in lungs of uninfected pigs. However, no increased induction of the anti-inflammatory cytokines such as IL-4 and IL-10 was observed in lungs of infected pigs. These results suggest that the pathogenesis induced in pigs by H1N2 influenza viruses may be induced by pro-inflammatory cytokines instead of anti-inflammatory cytokines.     

Antigenic composition of the hemagglutinins of influenza viruses H2N2

A comparative immunological analysis of the antigenic structure of hemagglutinins of influenza H2N2 viruses isolated in 1957-1967 was carried out by the method of specific adsorption of highly active antiviral rabbit sera. The hemagglutinins of the viruses under study were shown to contain at least three antigenic determinants changing in the course of evolution, one of them shared by all the viruses of the H2N2 subtype remaining antigenically stable since 1957 until 1967.     

Isolation and characterization of H6N1 and H9N2 avian influenza viruses from Ducks in Hanoi, Vietnam

We report the genetic characterization of low pathogenic avian influenza (LPAI) viruses isolated from domestic ducks in northern Vietnam in 2009. In total, 22 influenza A viruses consisting of 21 H6N1 subtypes and one H9N2 subtype were isolated from 1488 ducks collected in February, March, and April 2009, accounting the overall virus isolation rate for 1.5%. No H5N1 strain was isolated in this study. Phylogenetic analysis indicated that all the eight genes of the H6N1 and H9N2 subtypes analyzed in this study were similar to those isolated in Korea, southeast China and northern Japan, and wild birds which migrate along the coastal East Asian Flyway are estimated to transmit these viruses. There was no evidence that the H6N1 and H9N2 subtypes share the gene segments with H5N1 subtypes. However, it is important to monitor the prevalence and genetical backgrounds of LPAI viruses among poultry in an area where several different influenza A subtypes are in circulation.     

Antigenic structure of the hemagglutinin of H9N2 influenza viruses

The hemagglutinins (HAs) of H9 influenza viruses isolated from birds and mammals of different species were antigenically and genetically analyzed. Antigenic variants were selected from A/swine/Hong Kong/10/98 (H9N2) and A/duck/Hokkaido/13/00 (H9N2) in the presence of monoclonal antibodies (MAbs). Based on the reactivity patterns of these mutants with a panel of MAbs, at least five non-overlapping antigenic sites were defined using eight MAbs which recognized seven distinct epitopes on the H9 HA molecule. Based on the reactivity patterns with the panel of monoclonal antibodies, 21 H9N2 virus strains isolated from birds and mammals were divided into 7 antigenically distinct groups. The present findings indicate that it is important to monitor the antigenic variation in H9 influenza viruses. The panel of MAbs in the present study, thus, should be useful for detailed antigenic analysis of the H9 HAs for epidemiological studies, the selection of vaccine strains, and diagnosis.     

Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2

In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies.     

Continued circulation of reassortant H1N2 influenza viruses in pigs in Japan

Summary.  In 1991 and 1992, H1N2 influenza A viruses were isolated from the lungs of pigs with overt signs of respiratory disease at farms in the Chiba and Kanagawa prefectures of Japan. To determine the genetic origin of these isolates, we phylogenetically analyzed partial nucleotide sequences of their genes. The results indicate that influenza viruses possessing the N2 of human influenza virus and seven other gene segments of classical H1N1 swine influenza virus, which were first isolated in 1980, have become established in Japanese pigs. Received April 15, 1998 Accepted May 6, 1998     

Genetic analysis of influenza A/H3N2 and A/H1N1 viruses circulating in Vietnam from 2001 to 2006

Influenza A virus has the ability to overcome immunity from previous infections through the acquisition of genetic changes. Thus, understanding the evolution of the viruses in humans is important for the surveillance and the selection of vaccine strains. A total of 30 influenza A/H3N2 viruses and 35 influenza A/H1N1 viruses that were collected in Vietnam from 2001 to 2006 were used to analyze the evolution of the hemagglutinin (HA), neuraminidase (NA), and matrix protein (M) genes. Phylogenetic analysis of individual gene segments revealed that the HA and the NA genes of the influenza A viruses evolved in a sequential way. However, the evolutionary pattern of the M gene proved to be nonlinear and was not linked with that of the HA and NA genes. Genetic drift in HA1 segments, especially in the antigenic sites of A/H3N2 viruses, occurred more frequently in A/H3N2 viruses than it did in A/H1N1 viruses. Two reassortants, one influenza A/H3N2 strain and one A/H1N1 strain, were found on the basis of the phylogenetic analysis of the three genes. While both genetic mutation and reassortment contributed to their evolution, the frequency of genetic changes and reassortment events differs between the two subtypes. As influenza viruses circulate throughout the year, we emphasize the importance of surveillance in tropical and subtropical zones, where the emergence of new strains may be detected earlier than it is in temperate zones.     

Two genotypes of H1N2 swine influenza viruses appeared among pigs in China

BackgroundH1N2 is one of the main subtypes of influenza, which circulates in swine all over the world.ObjectivesTo investigate the prevalence and genetic of H1N2 in swine of China.Study designTwo H1N2 swine influenza viruses were isolated from Tianjin and Guangdong province of China in 2004 and 2006, respectively. The molecular evolution of eight gene segments was analyzed.ResultA/Swine/Tianjin/1/2004 has low identity with A/Swine/Guangdong/2006; in the phylogenetic tree of PA gene, A/Swine/Guangdong/1/2006 and A/Swine/Guangxi/1/2006 along with the H1N2 swine isolates of North America formed a cluster; and A/Swine/Tianjin/2004 and A/Swine/Zhejiang/2004, along with the classical H1N1 swine isolates formed another cluster; except that NA gene of A/Swine/Tianjin/1/2004 fell into the cluster of the H3N2 human influenza virus, indicating the reassortment between H3N2 human and H1N1 swine influenza viruses.ConclusionTwo different genotypes of H1N2 appeared among pigs in China. A/swine/Guangdong/1/06 was probably from H1N2 swine influenza viruses of North America; while A/swine/Tianjin/1/04 maybe come from reassortments of classical H1N1 swine and H3N2 human viruses prevalent in North America.     

Emergence of novel reassortant H3N2 swine influenza viruses with the 2009 pandemic H1N1 genes in the United States

Reassortant H1 swine influenza viruses (SIVs) carrying 2009 pandemic H1N1 virus (pH1N1) genes have been isolated from pigs worldwide. Seven novel reassortant H3N2 SIVs were identified from diseased pigs in the USA from winter 2010 to spring 2011. These novel viruses contain three or five internal genes from pH1N1 and continue to circulate in swine herds. The emergence of novel reassortant H3N2 SIVs demonstrates reassortment between pH1N1 and endemic SIVs in pigs and justifies continuous surveillance.     

Antigenic analysis of recent H1N1 influenza viruses with monoclonal antibodies.

Antigenic analysis of recently isolated H1 influenza viruses was performed using haemagglutination inhibition (HI) assay with monoclonal antibodies to the haemagglutinin (HA) subunit. Tests using monoclonal antibodies against the HA of the A/England/333/80 (H1N1) and A/Yamagata/120/86 (H1N1) viruses revealed that the major antigenic drift occurred in 1985 or 1986 and A/Dunedin/6/83-like virus became a major strain after 1986.     

Antigenic and genetic characterization of swine influenza A (H1N1) viruses isolated from pneumonia patients in The Netherlands

It is generally believed that pigs can serve as an intermediate host for the transmission of avian influenza viruses to humans or as mixing vessels for the generation of avian-human reassortant viruses. Here we describe the antigenic and genetic characterization of two influenza A (H1N1) viruses, which were isolated in The Netherlands from two patients who suffered from pneumonia. Both viruses proved to be antigenically and genetically similar to avian-like swine influenza A (H1N1) viruses which currently circulate in European pigs. It is concluded that European swine H1N1 viruses can infect humans directly, causing serious disease without the need for any reassortment event.     

Antigenic and biochemical analysis of influenza “A” H3N2 viruses isolated from pigs

Summary Four influenza A-H3N2 viruses isolated in pigs from different herds in Central Italy in the period 1981/82 have been antigenically and biochemically analysed. Three of them A/Sw/Italy/2/81, A/Sw/Italy/7/81, A/Sw/Italy/8/82 were found to be serologically related to A/Bangkok/1/79 (H3N2). These three viruses were shown to have an identical electrophoretic pattern, as regards virus induced polypeptides and were clearly distinguishable from the virus A/Sw/Italy/6/81 which was antigenically related to A/England/42/72 (H3N2) and A/Sw/Taiwan/7310/70 as shown by specific monoclonal and polyclonal antisera.The observed biochemical variations underline the importance of the changes occurring by genetic reassortments or mutations in human influenza viruses, during their maintenance in pigs.With 3 Figures     

Immunogenic properties of epidemic and recombinant strains of influenza viruses H1N1 and H3N2

The study of the immunogenic properties of epidemic influenza viruses H1N1 and H3N2 isolated from patients in 1979-1982 revealed a high immunogenic activity of H3N2 viruses. Recombinant strains of both subtypes (H1N1 and H3N2) had a higher immunogenicity than the original viruses. The intensity of immunity determined for an antigenically close pathogenic strain was considerably higher than for an antigenically remote pathogenic virus which is important for obtaining accurate information on the immunogenic activity of influenza viruses. No correlation between the immunity intensity and the antibody titres was observed.     

Genetic characterization of H1N1 swine influenza A viruses isolated in eastern China

Three influenza H1N1 viruses were isolated in 2005 from pigs with respiratory disease on a farm in eastern China. The three isolates were characterized to determine their probable origin. Each of the eight genes of the isolates was most closely related to the corresponding gene from the classical swine H1N1 virus. Also, phylogenetic analysis further confirmed that each of the eight genes of the isolates was closely related to the classical swine H1N1 viruses, especially those isolated in China. The HA1 proteins of the three isolates were identical to that of A/Swine/Guangdong/1/01, a virus isolated in 2001 in China, even though three nucleotide differences were observed. These results further support the concept that swine can serve as a reservoir of genetically stable influenza viruses.