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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Effect of receptor specificity of A/Hong Kong/1/68 (H3N2) influenza virus variants on replication and transmission in pigs</title><author><name sortKey="Van Poucke, Sjouke" sort="Van Poucke, Sjouke" uniqKey="Van Poucke S" first="Sjouke" last="Van Poucke">Sjouke Van Poucke</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Uhlendorff, Jennifer" sort="Uhlendorff, Jennifer" uniqKey="Uhlendorff J" first="Jennifer" last="Uhlendorff">Jennifer Uhlendorff</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Wang, Zhongfang" sort="Wang, Zhongfang" uniqKey="Wang Z" first="Zhongfang" last="Wang">Zhongfang Wang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Billiau, Veerle" sort="Billiau, Veerle" uniqKey="Billiau V" first="Veerle" last="Billiau">Veerle Billiau</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Nicholls, John" sort="Nicholls, John" uniqKey="Nicholls J" first="John" last="Nicholls">John Nicholls</name><affiliation><nlm:aff id="a4"></nlm:aff></affiliation></author><author><name sortKey="Matrosovich, Mikhail" sort="Matrosovich, Mikhail" uniqKey="Matrosovich M" first="Mikhail" last="Matrosovich">Mikhail Matrosovich</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Van Reeth, Kristien" sort="Van Reeth, Kristien" uniqKey="Van Reeth K" first="Kristien" last="Van Reeth">Kristien Van Reeth</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22564359</idno><idno type="pmc">5780757</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780757</idno><idno type="RBID">PMC:5780757</idno><idno type="doi">10.1111/j.1750-2659.2012.00376.x</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000978</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000978</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Effect of receptor specificity of A/Hong Kong/1/68 (H3N2) influenza virus variants on replication and transmission in pigs</title><author><name sortKey="Van Poucke, Sjouke" sort="Van Poucke, Sjouke" uniqKey="Van Poucke S" first="Sjouke" last="Van Poucke">Sjouke Van Poucke</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Uhlendorff, Jennifer" sort="Uhlendorff, Jennifer" uniqKey="Uhlendorff J" first="Jennifer" last="Uhlendorff">Jennifer Uhlendorff</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Wang, Zhongfang" sort="Wang, Zhongfang" uniqKey="Wang Z" first="Zhongfang" last="Wang">Zhongfang Wang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Billiau, Veerle" sort="Billiau, Veerle" uniqKey="Billiau V" first="Veerle" last="Billiau">Veerle Billiau</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Nicholls, John" sort="Nicholls, John" uniqKey="Nicholls J" first="John" last="Nicholls">John Nicholls</name><affiliation><nlm:aff id="a4"></nlm:aff></affiliation></author><author><name sortKey="Matrosovich, Mikhail" sort="Matrosovich, Mikhail" uniqKey="Matrosovich M" first="Mikhail" last="Matrosovich">Mikhail Matrosovich</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Van Reeth, Kristien" sort="Van Reeth, Kristien" uniqKey="Van Reeth K" first="Kristien" last="Van Reeth">Kristien Van Reeth</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author></analytic><series><title level="j">Influenza and Other Respiratory Viruses</title><idno type="ISSN">1750-2640</idno><idno type="eISSN">1750-2659</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Please cite this paper as:</italic> Van Poucke <italic>et al.</italic> (2013) Effect of receptor specificity of A/Hong Kong/1/68 (H3N2) influenza virus variants on replication and transmission in pigs. Influenza and Other Respiratory Viruses 7(2) 151–159.</p><p><bold>Background </bold> Several arguments plead for an important role of pigs in human influenza ecology, including the similar receptor expression pattern in the respiratory tract of both species. How virus receptor binding specificity affects transmission in pigs, on the other hand, has not been studied so far.</p><p><bold>Objectives </bold> Using recombinant viruses R1‐HK, which harbored all genes from the original pandemic virus A/Hong Kong/1/68 (H3N2), and R2‐HK, which differed by L226Q and S228G mutations in the hemagglutinin and conversion to an avian‐virus‐like receptor specificity, we assessed the role of receptor specificity on (i) replication in porcine respiratory explants, (ii) pig‐to‐pig transmission, and (iii) replication and organ tropism in pigs.</p><p><bold>Results </bold> In nasal, tracheal, and bronchial explants, we noticed a 10‐ to 100‐fold lower replication of R2‐HK compared with R1‐HK. In the lung explants, the viruses replicated with comparable efficiency. These observations correlated with the known expression level of Siaα2,3‐galactose in these tissues. In the pathogenesis study, virus titers in the respiratory part of the nasal mucosa, the trachea, and the bronchus were in line with the <italic>ex vivo</italic> results. R2‐HK replicated less efficiently in the lungs of pigs than R1‐HK, which contrasted with the explants results. R2‐HK also showed a pronounced tropism for the olfactory part of the nasal mucosa. Transmissibility experiments revealed that pig‐to‐pig transmission was abrogated when the virus obtained Siaα2,3‐galactose binding preference.</p><p><bold>Conclusions </bold> Our data suggest that Siaα2,6‐galactose binding is required for efficient transmission in pigs.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type="iso-abbrev">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type="doi">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type="publisher-id">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type="ppub">1750-2640</issn><issn pub-type="epub">1750-2659</issn><publisher><publisher-name>Blackwell Publishing Ltd</publisher-name><publisher-loc>Oxford, UK</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22564359</article-id><article-id pub-id-type="pmc">5780757</article-id><article-id pub-id-type="doi">10.1111/j.1750-2659.2012.00376.x</article-id><article-id pub-id-type="publisher-id">IRV376</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Articles</subject></subj-group><subj-group subj-group-type="overline"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Effect of receptor specificity of A/Hong Kong/1/68 (H3N2) influenza virus variants on replication and transmission in pigs</article-title><alt-title alt-title-type="left-running-head">Van Poucke <italic>et al.</italic></alt-title><alt-title alt-title-type="right-running-head">Influenza binding specificity and pig transmission</alt-title></title-group><contrib-group><contrib id="cr1" contrib-type="author"><name><surname>Van Poucke</surname><given-names>Sjouke</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr2" contrib-type="author"><name><surname>Uhlendorff</surname><given-names>Jennifer</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr3" contrib-type="author"><name><surname>Wang</surname><given-names>Zhongfang</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr4" contrib-type="author"><name><surname>Billiau</surname><given-names>Veerle</given-names></name><xref ref-type="aff" rid="a3"><sup>3</sup></xref></contrib><contrib id="cr5" contrib-type="author"><name><surname>Nicholls</surname><given-names>John</given-names></name><xref ref-type="aff" rid="a4"><sup>4</sup></xref></contrib><contrib id="cr6" contrib-type="author"><name><surname>Matrosovich</surname><given-names>Mikhail</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr7" contrib-type="author"><name><surname>Van Reeth</surname><given-names>Kristien</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib></contrib-group><aff id="a1"><label><sup>1</sup></label>Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium</aff><aff id="a2"><label><sup>2</sup></label>Institute of Virology, Philipps University, Marburg, Germany</aff><aff id="a3"><label><sup>3</sup></label>Department for Biomedical Research, VIB, Zwijnaarde, Belgium</aff><aff id="a4"><label><sup>4</sup></label>Department of Pathology, University of Hong Kong, Hong Kong SAR, China.</aff><author-notes><corresp id="correspondenceTo">Kristien Van Reeth, Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B‐9820 Merelbeke, Belgium. E‐mail: <email>kristien.vanreeth@ugent.be</email></corresp></author-notes><pub-date pub-type="epub"><day>04</day><month>5</month><year>2012</year></pub-date><pub-date pub-type="ppub"><month>3</month><year>2013</year></pub-date><volume>7</volume><issue>2</issue><issue-id pub-id-type="doi">10.1111/irv.2013.7.issue-2</issue-id><fpage>151</fpage><lpage>159</lpage><history>Accepted 3 April 2012. Published Online 4 May 2012.</history><permissions><copyright-statement content-type="article-copyright">© 2012 Blackwell Publishing Ltd</copyright-statement></permissions><self-uri content-type="pdf" xlink:type="simple" xlink:href="file:IRV-7-151.pdf"></self-uri><abstract><p><italic>Please cite this paper as:</italic> Van Poucke <italic>et al.</italic> (2013) Effect of receptor specificity of A/Hong Kong/1/68 (H3N2) influenza virus variants on replication and transmission in pigs. Influenza and Other Respiratory Viruses 7(2) 151–159.</p><p><bold>Background </bold> Several arguments plead for an important role of pigs in human influenza ecology, including the similar receptor expression pattern in the respiratory tract of both species. How virus receptor binding specificity affects transmission in pigs, on the other hand, has not been studied so far.</p><p><bold>Objectives </bold> Using recombinant viruses R1‐HK, which harbored all genes from the original pandemic virus A/Hong Kong/1/68 (H3N2), and R2‐HK, which differed by L226Q and S228G mutations in the hemagglutinin and conversion to an avian‐virus‐like receptor specificity, we assessed the role of receptor specificity on (i) replication in porcine respiratory explants, (ii) pig‐to‐pig transmission, and (iii) replication and organ tropism in pigs.</p><p><bold>Results </bold> In nasal, tracheal, and bronchial explants, we noticed a 10‐ to 100‐fold lower replication of R2‐HK compared with R1‐HK. In the lung explants, the viruses replicated with comparable efficiency. These observations correlated with the known expression level of Siaα2,3‐galactose in these tissues. In the pathogenesis study, virus titers in the respiratory part of the nasal mucosa, the trachea, and the bronchus were in line with the <italic>ex vivo</italic> results. R2‐HK replicated less efficiently in the lungs of pigs than R1‐HK, which contrasted with the explants results. R2‐HK also showed a pronounced tropism for the olfactory part of the nasal mucosa. Transmissibility experiments revealed that pig‐to‐pig transmission was abrogated when the virus obtained Siaα2,3‐galactose binding preference.</p><p><bold>Conclusions </bold> Our data suggest that Siaα2,6‐galactose binding is required for efficient transmission in pigs.</p></abstract><kwd-group><kwd id="k1">A/Hong Kong/1/68</kwd><kwd id="k2">pathogenesis</kwd><kwd id="k3">pigs</kwd><kwd id="k4">receptor specificity</kwd><kwd id="k5">respiratory explants</kwd><kwd id="k6">transmission</kwd></kwd-group><counts><fig-count count="4"></fig-count><table-count count="2"></table-count><page-count count="9"></page-count></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>March 2013</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_NLMPMC version:4.6.9 mode:remove_FC converted:04.11.2015</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="ss1"><title>Introduction</title><p>Introduction of a new influenza virus to which the human population has no or little preexisting immunity and that is capable of transmitting from humans to humans poses a pandemic threat. Such a virus can arise from genetic reassortment between avian and mammalian influenza viruses or by the adaptation of a non‐human influenza virus.<xref rid="b1" ref-type="ref"><sup>1</sup></xref> Although aquatic birds are the major influenza virus reservoir, pigs are supposed to play an important role in human influenza ecology. Swine have been postulated to be either an intermediate host for the transmission of avian influenza viruses (AIVs) to humans or a mixing vessel for avian and mammalian viruses.<xref rid="b2" ref-type="ref"><sup>2</sup></xref>, <xref rid="b3" ref-type="ref"><sup>3</sup></xref>, <xref rid="b4" ref-type="ref"><sup>4</sup></xref> This is based on the observation that most, if not all, avian virus subtypes can replicate in swine.<xref rid="b5" ref-type="ref"><sup>5</sup></xref>, <xref rid="b6" ref-type="ref"><sup>6</sup></xref> Furthermore, both terminal sialic acid (Sia)α2,3‐galactose and Siaα2,6‐galactose, the preferred virus receptors of avian and human influenza viruses respectively, were shown to be present in the porcine trachea.<xref rid="b7" ref-type="ref"><sup>7</sup></xref> More recent research has confirmed the abundant expression of Siaα2,6‐galactose on the epithelial lining of the porcine respiratory tract, but Siaα2,3‐galactose was detected only sparsely in the upper respiratory tract.<xref rid="b8" ref-type="ref"><sup>8</sup></xref>, <xref rid="b9" ref-type="ref"><sup>9</sup></xref>, <xref rid="b10" ref-type="ref"><sup>10</sup></xref> Hence, the receptor distribution pattern in the respiratory tract of pigs shows extensive similarity with that of humans.<xref rid="b11" ref-type="ref"><sup>11</sup></xref>, <xref rid="b12" ref-type="ref"><sup>12</sup></xref> More detailed pathogenesis studies have shown that although pigs support replication of many AIVs, the titers reached in the respiratory tract are modest.<xref rid="b13" ref-type="ref"><sup>13</sup></xref>, <xref rid="b14" ref-type="ref"><sup>14</sup></xref>, <xref rid="b15" ref-type="ref"><sup>15</sup></xref>, <xref rid="b16" ref-type="ref"><sup>16</sup></xref> Also, low and highly pathogenic AIVs could not transmit from pig to pig, while swine‐adapted viruses could under the same experimental conditions.<xref rid="b5" ref-type="ref"><sup>5</sup></xref>, <xref rid="b13" ref-type="ref"><sup>13</sup></xref>, <xref rid="b14" ref-type="ref"><sup>14</sup></xref></p><p>A large body of data highlights the importance of the viral hemagglutinin (HA) glycoprotein for the host range restriction. Thus, the pandemic “Hong Kong” H3N2 influenza virus emerged by reassortment of the then circulating human H2N2 virus with an avian H3 influenza virus and the avian HA showed a switch in receptor binding specificity shortly after the introduction of the virus in the human population in 1968.<xref rid="b17" ref-type="ref"><sup>17</sup></xref>, <xref rid="b18" ref-type="ref"><sup>18</sup></xref></p><p>Residues 226 and 228 in the HA of H3N2 influenza virus strains have been shown to determine receptor binding specificity. When the HA harbors leucine at 226 and serine at 228, the virus binds Siaα2,6‐galactose (human‐type receptor), whereas glutamic acid at 226 and glycine at 228 preferentially recognize Siaα2,3‐galactose (avian‐type receptor).<xref rid="b19" ref-type="ref"><sup>19</sup></xref>, <xref rid="b20" ref-type="ref"><sup>20</sup></xref> In cultures of human tracheobronchial epithelial cells, recombinant viruses with these mutations displayed differences in cell tropism, similar to the ones observed with wild‐type avian and human influenza viruses.<xref rid="b21" ref-type="ref"><sup>21</sup></xref>, <xref rid="b22" ref-type="ref"><sup>22</sup></xref> Roberts and colleagues used a pair of recombinant influenza viruses with L226Q/S228G substitutions in the A/Victoria/3/75 (H3N2) backbone to examine their transmissibility in ferrets. They observed a lack of transmission of the mutant virus with avian‐virus‐like receptor specificity.<xref rid="b23" ref-type="ref"><sup>23</sup></xref></p><p>Global surveillance of influenza viruses in bird, swine, and human populations is considered a key factor in the early detection of viruses with pandemic potential. A lack of detained knowledge on molecular determinants of viral host range restriction, especially in pigs, hampers predictions of the pandemic potential of emerging influenza viruses as illustrated by the 2009 pandemic.<xref rid="b24" ref-type="ref"><sup>24</sup></xref> Therefore, we aimed to determine whether L226Q/S228G mutations in the HA of A/Hong Kong/1/68 (H3N2) would affect (i) replication potential in porcine respiratory explants, (ii) replication potential and organ tropism in pigs, and (iii) pig‐to‐pig transmission.</p></sec><sec id="ss2"><title>Materials and methods</title><sec id="ss3"><title>Animals</title><p>Six‐ to 8‐week‐old pigs were obtained from a commercial herd serologically negative for influenza. All animals were tested for influenza virus antibodies before the start of the experiment by hemagglutination inhibition (HI) assay, by a competitive anti‐influenza A nucleoprotein enzyme immunoassay (Idexx Laboratories, Hoofddorp, Netherlands), and by immunoperoxidase monolayer assay (IPMA). Swine/Belgium/1/98 (H1N1), swine/Flanders/1/98 (H3N2), and swine/Gent/7625/99 (H1N2) were used in the HI and IPMA. Pigs were housed in separate high‐efficiency particulate air‐filtered isolation units. At arrival, they were treated intramuscularly with ceftiofur (Naxcel<sup>®</sup>, Pfizer‐1 ml/20 kg body weight).</p><p>All experiments were authorized by the Ethical and Animal Welfare Committee of the Faculty of Veterinary Medicine, Ghent University.</p></sec><sec id="ss4"><title>Viruses</title><p>The recombinant viruses R1‐HK and R2‐HK have been described previously.<xref rid="b19" ref-type="ref"><sup>19</sup></xref> They were generated using the eight plasmids reverse genetic system described by Hoffmann <italic>et al.</italic><xref rid="b25" ref-type="ref"><sup>25</sup></xref> and site‐directed mutagenesis to introduce the amino acid substitutions. R1‐HK harbors all eight original genes of the pandemic human virus A/Hong Kong/1/68 (H3N2). R2‐HK differs from R1‐HK solely by the L226Q/S228G mutations in the HA, which lead to a switch in receptor specificity from preferential binding of Siaα2,6‐galactose (R1‐HK) to preferential recognition of Siaα2,3‐galactose (R2‐HK).<xref rid="b19" ref-type="ref"><sup>19</sup></xref> Virus stocks were prepared in MDCK cells, and their infectivity was determined by plaque titration in MDCK cells. The low pathogenic AIV duck/Italy/3139‐1/06 (H3N2) was provided by Dr. I. Capua. Swine/Flanders/1/98 represents a European human‐like H3N2 swine influenza virus. The HA and NA genes of the swine H3N2 virus lineage are derived from descendants of A/Hong Kong/1/68 and the six gene fragments coding for internal and non‐structural proteins from the avian‐like H1N1. The amino acids at locations 226 and 228 are identical to those of R1‐HK.</p></sec><sec id="ss5"><title>Porcine respiratory explants systems</title><p>Porcine nasal (NE), tracheal (TE), bronchial (BE), and lung (LE) explants were prepared as described elsewhere.<xref rid="b9" ref-type="ref"><sup>9</sup></xref> The replication potential of R1‐HK, R2‐HK, duck/Italy/3139‐1/06 (dk/It/06), and swine/Flanders/1/98 (sw/Fl/1/98) inoculated at three different doses [10<sup>4</sup>, 10<sup>5</sup>, and 10<sup>6</sup> plaque forming units (PFU)] was assessed by determining the virus titers in supernatant at 1, 24, and 48 hours post inoculation. Virus titrations were performed on MDCK cells in 96‐well plates followed by an immunocytochemical staining with monoclonal antibody HB‐65 against viral nucleoprotein, and virus titers were calculated by the method of Reed and Muench.<xref rid="b26" ref-type="ref"><sup>26</sup></xref></p></sec><sec id="ss6"><title>Transmission studies</title><p>For each of the recombinant viruses, we used six directly inoculated (donor) pigs and six contact pigs. The six donor pigs of each group were housed in a separate isolation unit and inoculated intranasally with 10<sup>6</sup> PFU of R1‐HK or R2‐HK on day 0, as described earlier.<xref rid="b5" ref-type="ref"><sup>5</sup></xref> Two days after primary inoculation, six contact pigs were introduced into each group. The housing allowed both direct and airborne contact between inoculated and sentinel animals. Virus shedding was monitored by collecting nasal swabs from 0 through 9 days post inoculation (pi) or post contact (pc). Nasal swabs were put into 1 ml of transport medium and used for virus titration in MDCK cells. Blood samples for serological examination were collected at 14 and 28 dpi/dpc. In the group of R2‐HK contact pigs, blood was collected at 17 instead of 28 dpc. Transmission was defined by detection of virus from the nasal swabs and/or seroconversion in contact animals.</p></sec><sec id="ss7"><title>Pathogenesis study</title><p>Two separate groups, of five pigs each, were inoculated intranasally with 10<sup>6</sup> PFU of R1‐HK or R2‐HK as described for the transmission study. From 1 until 5 dpi, one pig from each group was euthanized. The following samples were collected for virus titration: nasal mucosa respiratory part, nasal mucosa olfactory part, tonsils, trachea, bronchus (at the level of the bifurcation), bulbus olfactorius, and lung. Five different areas of the lung were collected and titrated separately: (i) apical + cardiac lobe right (ii) apical + cardiac lobe left (iii) diaphragmatic lobe right (iv) diaphragmatic lobe left, and (v) accessory lung lobe. All tissue samples for virus titration were weighed and put in phosphate‐buffered saline containing 10 IU/ml penicillin and 10 μg/ml streptomycin to prepare 20% tissue homogenates.</p></sec><sec id="ss8"><title>Serological examination</title><p>Antibody titers against the homologous viruses were determined by hemagglutination inhibition (HI), virus neutralization (VN), and IPMA. Antibodies against the viral NP were detected by a competitive anti‐influenza A nucleoprotein enzyme immunoassay (NP‐ELISA). HI and VN assays were performed as described earlier.<xref rid="b27" ref-type="ref"><sup>27</sup></xref> Sera from the R2‐HK group were tested in parallel HI assays with chicken and horse erythrocytes, while sera from R1‐HK were tested with chicken erythrocytes only, because the R1‐HK virus did not agglutinate the horse erythrocytes. In short, the sera were heat inactivated (56°C,30′), pre‐treated with receptor‐destroying enzyme, and adsorbed onto either chicken or horse erythrocytes. Sera were incubated with four hemagglutinating units of virus for 1 hour and then left to hemagglutinate 0·5% chicken erythrocytes or 1% horse erythrocytes during 1 or 2 hours at room temperature, respectively. In the VN assay, twofold serum dilutions were incubated with 10<sup>2</sup> TCID<sub>50</sub> of the homologous virus during 1 hour. Next, 100 μl of MDCK cells was added at a concentration of 300 000 cells/ml, and after 24 hours, the plates were fixed. Lack of infection was confirmed by immunocytochemical staining. For the IPMA, monolayers of MDCK cells were inoculated with 10<sup>3</sup> TCID<sub>50</sub> per well and fixed after 24 hours. They were incubated with twofold dilutions of the swine sera followed by incubation with goat anti‐swine peroxidase.<xref rid="b5" ref-type="ref"><sup>5</sup></xref> The influenza A virus antibody test kit<sup>®</sup>, purchased from IDEXX Laboratories and carried out following the manufacturer’s instructions, was a blocking ELISA. If NP antibodies are present in the serum sample to be tested, they will prevent the conjugate from binding with the nucleoprotein that is adsorbed to the ELISA plate and color development will be blocked. Starting dilutions of the sera in the serological assays were 1:2 in VN, 1:5 in IPMA, and 1:10 in HI and NP‐ELISA. All sera were tested in duplicate. Antibody titers were expressed as the reciprocal of the highest serum dilutions that completely inhibited virus replication in MDCK cells (VN assay), that stained influenza infected MDCK cells (IPMA), and that inhibited hemagglutination (HI assay). Results from the NP‐ELISA were expressed as S/N ratio. The S/N response is the ratio of the sample optical density (OD) reading to the kit negative control OD reading.</p></sec><sec id="ss9"><title>Lectin histochemistry</title><p>The pronounced tropism of R2‐HK for the olfactory part of the nasal mucosa triggered us to study the receptor expression in this tissue. Siaα2,6‐galactose distribution was examined using a digoxigenin labeled <italic>Sambucus nigra</italic> agglutinin (SNA). Two different isoforms of the <italic>Maackia amurensis</italic> agglutinin, MAA‐I and MAA‐II, were applied to identify the α2,3‐linked Sia moiety of the receptor. Staining was developed with New Fuchsin.<xref rid="b9" ref-type="ref"><sup>9</sup></xref></p></sec><sec id="ss10"><title>Data analyses</title><p>Replication differences between viruses in the explants were compared by a paired <italic>t</italic>‐test. <italic>P </italic><<italic> </italic>0·05 was considered significant. The average virus excretion in the nasal swabs from each group of pigs was determined by calculating the area under the curve (AUC). To compare the AUC between groups, the two sample Mann–Whitney test was used and <italic>P </italic><<italic> </italic>0·05 was considered significant. All analyses were performed with Prism5, version 5·0c.</p></sec></sec><sec id="ss11"><title>Results</title><sec id="ss12"><title>Replication in porcine respiratory explants</title><p>Results from replication studies in porcine respiratory explants are shown in <xref rid="f1" ref-type="fig">Figure 1</xref>. Although there was a clear dose‐dependent effect, virus replication in NE and TE was comparable, it was poor for dk/It/06, moderate for R2‐HK, high for R1‐HK, and highest for sw/Fl/1/98. In BE, R1‐HK and sw/Fl/1/98 showed equally high virus titers (<italic>P </italic>=<italic> </italic>0·92). Replication of R2‐HK and dk/It/06 in BE was also increased compared with NE and TE, but remained significantly different from R1‐HK (<italic>P </italic>=<italic> </italic>0·008 and <italic>P </italic>=<italic> </italic>0·010) and sw/Fl/98 (<italic>P </italic>=<italic> </italic>0·019 and <italic>P </italic>=<italic> </italic>0·009) respectively when 10<sup>6</sup> PFU was administrated. In LE, in contrast, virus titers did not significantly differ between the four viruses at the inoculation doses of 10<sup>6</sup> and 10<sup>5</sup> PFU.</p><fig fig-type="Figure" xml:lang="en" id="f1" orientation="portrait" position="float"><label>Figure 1</label><caption><p> Virus yield in the supernatant of porcine respiratory explants at 48 hpi. Three different inoculation doses were assessed for each virus: 10<sup>4</sup> (white bars), 10<sup>5</sup> (gray bars) and 10<sup>6</sup> plaque forming units (black bars). Bars show the mean and standard deviation of 4 repeats. Swine/Flanders/1/98 (sw/Fl/1/98) and duck/Italy/3139‐1/06 (dk/It/06) represent a swine‐adapted and non‐swine‐adapted influenza virus, respectively.</p></caption><graphic id="nlm-graphic-1" xlink:href="IRV-7-151-g001"></graphic></fig></sec><sec id="ss13"><title>Transmission studies</title><p>An overview of the nasal virus shedding in all groups is depicted in <xref rid="f2" ref-type="fig">Figure 2</xref>. After inoculation with R1‐HK, all pigs shed virus in nasal swabs until six dpi. Virus titers showed little individual variation between pigs, resulting in an average AUC of 23·1. The six contact animals in this group also excreted virus, starting at 1–3 dpc. The duration of shedding was similar to that of the inoculated pigs, but the average AUC for the contact group was only 17·4. This was because of the very low AUC values in three of six pigs.</p><fig fig-type="Figure" xml:lang="en" id="f2" orientation="portrait" position="float"><label>Figure 2</label><caption><p> Nasal virus shedding of R1‐K (left panel) and R2‐HK (right panel) influenza viruses of inoculated and sentinel pigs. The virus titers are shown as mean ± standard deviation. R2‐HK was undetectable in nasal swabs of all contact pigs. The numbers <italic>in italics</italic> represent the area under the curve for each group of pigs.</p></caption><graphic id="nlm-graphic-3" xlink:href="IRV-7-151-g002"></graphic></fig><p>All R2‐HK‐inoculated pigs were positive for virus excretion. Their excretion profile was similar to that of R1‐HK‐inoculated pigs in terms of start and duration of viral shedding, but the average AUC was only 15·7 (significantly lower than in the R1‐HK‐inoculated group). Four of six pigs showed a remarkable drop in virus excretion at 2 and 3 dpi. By 4 dpi, the nasal shedding in all six pigs had increased again to levels between 3·8 and 5·3 log<sub>10</sub> TCID<sub>50</sub>/ml. In contrast, none of the contact animals in the R2‐HK group had detectable virus titers in nasal swabs.</p></sec><sec id="ss14"><title>Pathogenesis study</title><p>Of 11 tissues analyzed for virus titers, only one was negative during five subsequent days for R1‐HK compared with four negative tissues for R2‐HK. The bulbus olfactorius tested negative for both R1‐HK and R2‐HK, while the tonsil, the right diaphragmatic, and the left diaphragmatic lung lobes were also negative for R2‐HK. With the exception of the olfactory part of the nasal mucosa, the average virus titers obtained for R2‐HK were at least 20‐fold lower than for R1‐HK. The virus titers obtained for R2‐HK were particularly low in bronchus and lungs, with the exception of those in the accessory lung lobe (<xref rid="f3" ref-type="fig">Figure 3</xref>).</p><fig fig-type="Figure" xml:lang="en" id="f3" orientation="portrait" position="float"><label>Figure 3</label><caption><p> Replication of R1‐HK (gray bars) and R2‐HK (black bars) influenza virus in various parts of the respiratory tract and in the bulbus olfactorius of pigs during five subsequent days post inoculation. Each bar represents the virus titer of an individual pig. The thin dashed line represents the detection limit.</p></caption><graphic id="nlm-graphic-5" xlink:href="IRV-7-151-g003"></graphic></fig></sec><sec id="ss15"><title>Serological examination</title><p>The results of the serological assays in the R1‐HK and R2‐HK groups are shown in <xref rid="t1" ref-type="table-wrap">1</xref>, <xref rid="t2" ref-type="table-wrap">2</xref>, respectively. In the HI test performed with 0·5% chicken red blood cells (RBC), all R1‐HK‐inoculated and contact pigs had seroconverted by 14 dpi/dpc (titers 10–40). None of the 12 pigs of the R2‐HK group reacted in this assay. In contrast, in the HI test with 1% horse RBC, all R2‐HK‐inoculated pigs had antibody titers (10–40) by 14 dpi but none of the contact animals showed seroconversion. The VN test largely confirmed the results of the HI test, but antibody titers for the R2‐HK group were significantly lower than for the R1‐HK group, even by 28 dpi. One of the six contact pigs in the R2‐HK group showed seroconversion in both the NP‐ELISA and IPMA. The IPMA gave the highest number of seropositive animals in both R1‐HK and R2‐HK groups by 14 dpi.</p><table-wrap id="t1" xml:lang="en" orientation="portrait" position="float"><label>Table 1</label><caption><p> Antibody responses in pigs directly inoculated with R1‐HK virus and in contact pigs</p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="3" valign="bottom" align="left" colspan="1">Assay</th><th colspan="6" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Number of serologically positive pigs and range of antibody titers (in parentheses)</th></tr><tr style="border-bottom:solid 1px #000000"><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Directly inoculated pigs
Days post inoculation</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Contact pigs
Days post contact</th></tr><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1">0</th><th align="left" valign="bottom" rowspan="1" colspan="1">14</th><th align="left" valign="bottom" rowspan="1" colspan="1">28</th><th align="left" valign="bottom" rowspan="1" colspan="1">0</th><th align="left" valign="bottom" rowspan="1" colspan="1">14</th><th align="left" valign="bottom" rowspan="1" colspan="1">28</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">HI (chicken RBC)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (10–40)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (10–40)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (10–40)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (10–40)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">VN</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<2)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (24–64)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (12–64)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<2)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (16–96)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (32–64)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">NP‐ELISA</td><td align="left" valign="top" rowspan="1" colspan="1">0 (≥0·6)</td><td align="left" valign="top" rowspan="1" colspan="1">2 (0·565–0·508)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (0·584–0·445)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (≥0·6)</td><td align="left" valign="top" rowspan="1" colspan="1">4 (0·529–0·427)</td><td align="left" valign="top" rowspan="1" colspan="1">5 (0·524–0·377)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">IPMA</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<5)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (1280–5120)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (1280–10240)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<5)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (640–5120)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (1280–5120)</td></tr></tbody></table><table-wrap-foot><fn id="t1_note38"><p>RBC, red blood cells.</p></fn><fn id="t1_note39"><p>The number of positive pigs among the total of six pigs is shown. Range of antibody titers in positive pigs are expressed as the reciprocal of the serum dilutions for virus neutralization, immunoperoxidase monolayer assay, and HI assays and as S/N ratio for the NP‐ELISA. S/N ratios ≥0·6 are considered negative.</p></fn></table-wrap-foot></table-wrap><table-wrap id="t2" xml:lang="en" orientation="portrait" position="float"><label>Table 2</label><caption><p> Antibody responses in pigs directly inoculated with R2‐HK virus and in contact pigs</p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="3" valign="bottom" align="left" colspan="1">Assay</th><th colspan="6" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Number of serologically positive pigs and range of antibody titers (in parentheses)</th></tr><tr style="border-bottom:solid 1px #000000"><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Directly inoculated pigs
Days post inoculation</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Contact pigs
Days post contact</th></tr><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1">0</th><th align="left" valign="bottom" rowspan="1" colspan="1">14</th><th align="left" valign="bottom" rowspan="1" colspan="1">28</th><th align="left" valign="bottom" rowspan="1" colspan="1">0</th><th align="left" valign="bottom" rowspan="1" colspan="1">14</th><th align="left" valign="bottom" rowspan="1" colspan="1">17</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">HI (chicken RBC)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">HI (horse RBC)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (10–20)</td><td align="left" valign="top" rowspan="1" colspan="1">5 (20–40)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<10)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">VN</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<2)</td><td align="left" valign="top" rowspan="1" colspan="1">4 (2–6)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (3–12)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<2)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<2)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<2)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">NP‐ELISA</td><td align="left" valign="top" rowspan="1" colspan="1">0 (≥0·6)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (0·581–0·250)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (0·538–0·343)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (≥0·6)</td><td align="left" valign="top" rowspan="1" colspan="1">1 (0·458)</td><td align="left" valign="top" rowspan="1" colspan="1">1 (0·565)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">IPMA</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<5)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (640–2560)</td><td align="left" valign="top" rowspan="1" colspan="1">6 (1280–5120)</td><td align="left" valign="top" rowspan="1" colspan="1">0 (<5)</td><td align="left" valign="top" rowspan="1" colspan="1">1 (320)</td><td align="left" valign="top" rowspan="1" colspan="1">1 (640)</td></tr></tbody></table><table-wrap-foot><fn id="t2_note45"><p>RBC, red blood cells.</p></fn><fn id="t2_note46"><p>The number of positive pigs among the total of six pigs is shown. Range of antibody titers in positive pigs are expressed as the reciprocal of the serum dilutions for virus neutralization, immunoperoxidase monolayer assay, and HI assays and as S/N ratio for the NP‐ELISA. S/N ratios ≥0·6 are considered negative.</p></fn></table-wrap-foot></table-wrap></sec><sec id="ss16"><title>Lectin histochemistry</title><p>There was strong binding of both MAA‐I and MAA‐II to the epithelial lining of the olfactory part of the nasal mucosa (indicated by the arrows in <xref rid="f4" ref-type="fig">Figure 4A,B</xref>), indicating the presence of both N and O‐linked Siaα2,3‐linked glycans. Siaα2,6‐galactose was not detected in any of the epithelial cell layers as there was no binding by SNA (<xref rid="f4" ref-type="fig">Figure 4C</xref>).</p><fig fig-type="Figure" xml:lang="en" id="f4" orientation="portrait" position="float"><label>Figure 4</label><caption><p> Tissue binding of <italic>Maackia amurensis</italic> agglutinin I (A), <italic>Maackia amurensis</italic> agglutinin II (B) and <italic>Sambucus nigra</italic> agglutinin (C) in the porcine olfactory part of the nasal mucosa. Siaα2,3‐galactoseβ1,4‐glucosamine (A) was abundantly expressed on the epithelial lining, Siaα2,3‐galactoseβ1,3‐glucosamine (B) moderately (indicated by the arrows). Sia α2,6‐galactose (C) was not detected in any of the epithelial cell layers.</p></caption><graphic id="nlm-graphic-7" xlink:href="IRV-7-151-g004"></graphic></fig></sec></sec><sec id="ss17"><title>Discussion</title><p>Techniques such as reverse genetics, enabling the generation of large numbers of mutant or reassortant influenza viruses, have emphasized the need for reliable <italic>ex vivo</italic> tools to study these viruses and to predict their behavior <italic>in vivo</italic>. Because pigs play an important role in influenza ecology and zoonotic transmission, they are the species of choice to investigate the molecular determinants, both at viral and host levels, of inter‐ and intraspecies transmission. We have previously shown that replication efficiency of influenza viruses isolated from porcine, human, and avian hosts differs significantly in NE, TE, and BE but not in LE.<xref rid="b9" ref-type="ref"><sup>9</sup></xref> The restricted replication of AIVs in tissues of the upper respiratory tract was in line with the limited expression of Siaα2,3‐galactose in these tissues.<xref rid="b8" ref-type="ref"><sup>8</sup></xref>, <xref rid="b10" ref-type="ref"><sup>10</sup></xref> Using R1‐HK and R2‐HK viruses that differ solely by two amino acids in the receptor binding site, we have examined the usefulness of this <italic>ex vivo</italic> model of the porcine respiratory tract to assess replication differences between viruses with genetic modifications. The avian‐like R2‐HK replicated less efficiently than the human‐like R1‐HK in NE, TE, and BE. On the other hand, virus titers of R2‐HK were consistently higher than those of dk/It/06. This finding suggests that the two viruses could still differ in receptor binding properties or that the low replication level of the entirely AIV was not only attributed to a non‐optimal receptor binding tropism. A similar observation was the lower replication of R1‐HK compared with sw/Fl/1/98 in NE and TE, despite the human‐like tropism of both viruses. A reduction in the inoculation dose from 10<sup>6</sup> to 10<sup>4</sup> PFU resulted in a stronger drop of virus replication of R2‐HK than of R1‐HK, suggesting a lower minimal infectious dose of the latter virus. Roberts <italic>et al.</italic><xref rid="b23" ref-type="ref"><sup>23</sup></xref> indeed showed that a similar A/Victoria/3/75 mutant virus with avian‐like receptor specificity required a 40‐fold higher infectious dose in ferrets than the wild‐type virus. To assess whether the replication differences observed in these explants were biologically relevant <italic>in vivo</italic>, we used the recombinant viruses for transmission and pathogenesis studies in pigs.</p><p>Although seroconversion was observed in one of six contact pigs of the R2‐HK group, our results indicate that a switch in the receptor preference of H3 from Siaα2,6‐galactose to Siaα2,3‐galactose was sufficient to block efficient transmission in pigs, as this virus was not isolated in any of the nasal swabs from contact animals. In previous studies in ferrets and guinea pigs, intraspecies transmission was also shown to be influenced by receptor binding preferences of viruses.<xref rid="b28" ref-type="ref"><sup>28</sup></xref>, <xref rid="b29" ref-type="ref"><sup>29</sup></xref>, <xref rid="b30" ref-type="ref"><sup>30</sup></xref> Tumpey <italic>et al.</italic><xref rid="b28" ref-type="ref"><sup>28</sup></xref> showed that a switch toward an avian‐virus‐like receptor preference of the 1918 H1N1 pandemic virus abolished airborne transmission between ferrets. Pappas <italic>et al.</italic><xref rid="b29" ref-type="ref"><sup>29</sup></xref> have also shown that an early isolate of the 1957 H2N2 pandemic with avian‐virus‐like receptor specificity did not efficiently spread between ferrets. A Q226L mutant with human‐virus‐like receptor specificity, which emerged during the course of their experiment, was transmissible even through respiratory droplets. When compared to R1‐HK, the nasal virus shedding of R2‐HK‐inoculated pigs was characterized by overall lower virus titers, a temporary drop at 2 and 3 dpi, and an identical duration. The lower AUC of R2‐HK, representing the overall viral load built up in the stable, could at least in part explain for the lack of transmission. To examine this possibility, we increased the inoculation dose of R2‐HK to 10<sup>8</sup> PFU in a preliminary experiment. As the AUC of R2‐HK was not significantly increased (AUC = 18) under these conditions, the hypothesis could not be confirmed. In literature, data on the role of the virus excretion level on transmission efficiency are contradicting. Mubareka <italic>et al.</italic><xref rid="b31" ref-type="ref"><sup>31</sup></xref> showed that reduced virus shedding of a human H1N1 compared with a human H3N2 influenza virus was associated with less efficient transmission in guinea pigs. In another transmission study in ferrets, a human H3N2 and an avian H5N1 virus achieved similar peak mean titers in nasal washes but only the human virus transmitted efficiently.<xref rid="b32" ref-type="ref"><sup>32</sup></xref></p><p>The pathogenesis study confirmed the hampered replication potential of R2‐HK in porcine tissues, except for one particular region, the olfactory part of the nasal mucosa. The lower replication of R2‐HK could be the result of an imbalance between the receptor preference of HA and substrate specificity of NA. However, Scull <italic>et al.</italic><xref rid="b21" ref-type="ref"><sup>21</sup></xref> showed that A/Victoria/3/75 with L226Q and S228G mutations in the HA was not significantly less fit than its wild‐type counterpart as both viruses infected similar numbers of human ciliated airway epithelium cells and replicated to comparable peak titers when incubated at 37°C. In the LE, we observed similar peak virus titers for R2‐HK and R1‐HK, indicating that the virus itself is replication‐competent. Another explanation for the limited R2‐HK replication could be a restricted binding of this virus to Sia receptors on target cells. This hypothesis is in agreement with our observations in the explants. R2‐HK preferably binds Siaα2,3‐galactose, and the expression of this linkage is absent or limited in NE, TE, and BE.<xref rid="b9" ref-type="ref"><sup>9</sup></xref> Still, the expected enhanced tropism of R2‐HK for the lungs, based on replication in LE and the Siaα2,3‐galactose expression in this organ, was not observed <italic>in vivo.</italic> This may result from a difference in exposure of the tissues to the inoculum. In the LE, virus is directly brought onto the tissue, while it must overcome several barriers to reach the LRT after intranasal inoculation. Furthermore, we demonstrated a difference in predilection for the olfactory part of the nasal mucosa between R2‐HK and R1‐HK. The preference of AIVs for this particular tissue was noticed before in pathogenesis studies in our laboratory,<xref rid="b5" ref-type="ref"><sup>5</sup></xref> unpublished data (De Vleeschauwer, Kyriakis). Here, we have shown a correlation with the absence of Siaα2,6‐galactose and the abundant expression of Siaα2,3‐galactose on the epithelial lining. Despite a lack of Siaα2,6‐galactose, swine/Belgium/1/98 (H1N1) could successfully infect this olfactory part of the nasal mucosa,<xref rid="b15" ref-type="ref"><sup>15</sup></xref> indicating that the SNA lectin may not stain all α2,6 variants or that another receptor mediates the endocytotic uptake of the virus.</p><p>In conclusion, our data suggest that Siaα2,6‐galactose binding is a prerequisite for efficient transmission of influenza viruses in pigs. 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