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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Development of a new candidate H5N1 avian influenza virus for pre‐pandemic vaccine production</title><author><name sortKey="Dong, Jie" sort="Dong, Jie" uniqKey="Dong J" first="Jie" last="Dong">Jie Dong</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Matsuoka, Yumiko" sort="Matsuoka, Yumiko" uniqKey="Matsuoka Y" first="Yumiko" last="Matsuoka">Yumiko Matsuoka</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Maines, Taronna R" sort="Maines, Taronna R" uniqKey="Maines T" first="Taronna R." last="Maines">Taronna R. Maines</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Swayne, David E" sort="Swayne, David E" uniqKey="Swayne D" first="David E." last="Swayne">David E. Swayne</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="O Eill, Eduardo" sort="O Eill, Eduardo" uniqKey="O Eill E" first="Eduardo" last="O Eill">Eduardo O Eill</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Davis, C Todd" sort="Davis, C Todd" uniqKey="Davis C" first="C. Todd" last="Davis">C. Todd Davis</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Van Oven, Neal" sort="Van Oven, Neal" uniqKey="Van Oven N" first="Neal" last="Van-Hoven">Neal Van-Hoven</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Balish, Amanda" sort="Balish, Amanda" uniqKey="Balish A" first="Amanda" last="Balish">Amanda Balish</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Yu, Hong Ie" sort="Yu, Hong Ie" uniqKey="Yu H" first="Hong-Jie" last="Yu">Hong-Jie Yu</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Katz, Jacqueline M" sort="Katz, Jacqueline M" uniqKey="Katz J" first="Jacqueline M." last="Katz">Jacqueline M. Katz</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Klimov, Alexander" sort="Klimov, Alexander" uniqKey="Klimov A" first="Alexander" last="Klimov">Alexander Klimov</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Cox, Nancy" sort="Cox, Nancy" uniqKey="Cox N" first="Nancy" last="Cox">Nancy Cox</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Li, De In" sort="Li, De In" uniqKey="Li D" first="De-Xin" last="Li">De-Xin Li</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Wang, Yu" sort="Wang, Yu" uniqKey="Wang Y" first="Yu" last="Wang">Yu Wang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Guo, Yuan I" sort="Guo, Yuan I" uniqKey="Guo Y" first="Yuan-Ji" last="Guo">Yuan-Ji Guo</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Yang, Wei Hong" sort="Yang, Wei Hong" uniqKey="Yang W" first="Wei-Zhong" last="Yang">Wei-Zhong Yang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Donis, Ruben O" sort="Donis, Ruben O" uniqKey="Donis R" first="Ruben O." last="Donis">Ruben O. Donis</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Shu, Yue Ong" sort="Shu, Yue Ong" uniqKey="Shu Y" first="Yue-Long" last="Shu">Yue-Long Shu</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19903211</idno><idno type="pmc">4941393</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941393</idno><idno type="RBID">PMC:4941393</idno><idno type="doi">10.1111/j.1750-2659.2009.00104.x</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">000067</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000067</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Development of a new candidate H5N1 avian influenza virus for pre‐pandemic vaccine production</title><author><name sortKey="Dong, Jie" sort="Dong, Jie" uniqKey="Dong J" first="Jie" last="Dong">Jie Dong</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Matsuoka, Yumiko" sort="Matsuoka, Yumiko" uniqKey="Matsuoka Y" first="Yumiko" last="Matsuoka">Yumiko Matsuoka</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Maines, Taronna R" sort="Maines, Taronna R" uniqKey="Maines T" first="Taronna R." last="Maines">Taronna R. Maines</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Swayne, David E" sort="Swayne, David E" uniqKey="Swayne D" first="David E." last="Swayne">David E. Swayne</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="O Eill, Eduardo" sort="O Eill, Eduardo" uniqKey="O Eill E" first="Eduardo" last="O Eill">Eduardo O Eill</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Davis, C Todd" sort="Davis, C Todd" uniqKey="Davis C" first="C. Todd" last="Davis">C. Todd Davis</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Van Oven, Neal" sort="Van Oven, Neal" uniqKey="Van Oven N" first="Neal" last="Van-Hoven">Neal Van-Hoven</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Balish, Amanda" sort="Balish, Amanda" uniqKey="Balish A" first="Amanda" last="Balish">Amanda Balish</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Yu, Hong Ie" sort="Yu, Hong Ie" uniqKey="Yu H" first="Hong-Jie" last="Yu">Hong-Jie Yu</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Katz, Jacqueline M" sort="Katz, Jacqueline M" uniqKey="Katz J" first="Jacqueline M." last="Katz">Jacqueline M. Katz</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Klimov, Alexander" sort="Klimov, Alexander" uniqKey="Klimov A" first="Alexander" last="Klimov">Alexander Klimov</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Cox, Nancy" sort="Cox, Nancy" uniqKey="Cox N" first="Nancy" last="Cox">Nancy Cox</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Li, De In" sort="Li, De In" uniqKey="Li D" first="De-Xin" last="Li">De-Xin Li</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Wang, Yu" sort="Wang, Yu" uniqKey="Wang Y" first="Yu" last="Wang">Yu Wang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Guo, Yuan I" sort="Guo, Yuan I" uniqKey="Guo Y" first="Yuan-Ji" last="Guo">Yuan-Ji Guo</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Yang, Wei Hong" sort="Yang, Wei Hong" uniqKey="Yang W" first="Wei-Zhong" last="Yang">Wei-Zhong Yang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Donis, Ruben O" sort="Donis, Ruben O" uniqKey="Donis R" first="Ruben O." last="Donis">Ruben O. Donis</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Shu, Yue Ong" sort="Shu, Yue Ong" uniqKey="Shu Y" first="Yue-Long" last="Shu">Yue-Long Shu</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="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><bold>Background </bold> Highly pathogenic H5N1 avian influenza viruses currently circulating in birds have caused hundreds of human infections, and pose a significant pandemic threat. Vaccines are a major component of the public health preparedness for this likely event. The rapid evolution of H5N1 viruses has resulted in the emergence of multiple clades with distinct antigenic characteristics that require clade‐specific vaccines. A variant H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused multiple fatal infections. Vaccine candidates that match the antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain‐specific protection.</p><p><bold>Objective </bold> To address the need for a suitable seed for manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 pre‐pandemic candidate vaccine by reverse genetics and evaluated its safety and replication <italic>in vitro</italic> and <italic>in vivo</italic>.</p><p><bold>Methods </bold> A reassortant virus termed, Anhui/PR8, was produced by reverse genetics in compliance with WHO pandemic vaccine development guidelines and contains six genes from A/Puerto Rico/8/34 as well as the neuraminidase and hemagglutinin (HA) genomic segments from the A/Anhui/01/2005 virus. The multi‐basic cleavage site of HA was removed to reduce virulence.</p><p><bold>Results </bold> The reassortant Anhui/PR8 grows well in eggs and is avirulent to chicken and ferrets but retains the antigenicity of the parental A/Anhui/01/2005 virus.</p><p><bold>Conclusion </bold> These results indicate that the Anhui/PR8 reassortant lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, Thailand, and Laos.</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><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">19903211</article-id><article-id pub-id-type="pmc">4941393</article-id><article-id pub-id-type="doi">10.1111/j.1750-2659.2009.00104.x</article-id><article-id pub-id-type="publisher-id">IRV104</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Development of a new candidate H5N1 avian influenza virus for pre‐pandemic vaccine production</article-title><alt-title alt-title-type="left-running-head">Dong <italic>et al.</italic></alt-title><alt-title alt-title-type="right-running-head">Anhui/PR8 candidate vaccine virus derivation</alt-title></title-group><contrib-group><contrib id="cr1" contrib-type="author"><name><surname>Dong</surname><given-names>Jie</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr2" contrib-type="author"><name><surname>Matsuoka</surname><given-names>Yumiko</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr3" contrib-type="author"><name><surname>Maines</surname><given-names>Taronna R.</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr4" contrib-type="author"><name><surname>Swayne</surname><given-names>David E.</given-names></name><xref ref-type="aff" rid="a3"><sup>3</sup></xref></contrib><contrib id="cr5" contrib-type="author"><name><surname>O’Neill</surname><given-names>Eduardo</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr6" contrib-type="author"><name><surname>Davis</surname><given-names>C. Todd</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr7" contrib-type="author"><name><surname>Van‐Hoven</surname><given-names>Neal</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr8" contrib-type="author"><name><surname>Balish</surname><given-names>Amanda</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr9" contrib-type="author"><name><surname>Yu</surname><given-names>Hong‐jie</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr10" contrib-type="author"><name><surname>Katz</surname><given-names>Jacqueline M.</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr11" contrib-type="author"><name><surname>Klimov</surname><given-names>Alexander</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr12" contrib-type="author"><name><surname>Cox</surname><given-names>Nancy</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr13" contrib-type="author"><name><surname>Li</surname><given-names>De‐xin</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr14" contrib-type="author"><name><surname>Wang</surname><given-names>Yu</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr15" contrib-type="author"><name><surname>Guo</surname><given-names>Yuan‐ji</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr16" contrib-type="author"><name><surname>Yang</surname><given-names>Wei‐zhong</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr17" contrib-type="author"><name><surname>Donis</surname><given-names>Ruben O.</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr18" contrib-type="author"><name><surname>Shu</surname><given-names>Yue‐long</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib></contrib-group><aff id="a1"><label><sup>1</sup></label>Chinese National Influenza Center, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, China CDC, Xuanwu District Beijing, China</aff><aff id="a2"><label><sup>2</sup></label>Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA</aff><aff id="a3"><label><sup>3</sup></label>Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Athens, GA, USA</aff><author-notes><corresp id="correspondenceTo">Dr. Ruben O. Donis, Influenza Division, NCIRD, CCID, Centers for Disease Control and Prevention, 1600 Clifton Road ‐ Mail Stop G‐16 Atlanta, GA 30333, USA. E‐mail: <email>rdonis@cdc.gov</email></corresp></author-notes><pub-date pub-type="epub"><day>22</day><month>10</month><year>2009</year></pub-date><pub-date pub-type="ppub"><month>11</month><year>2009</year></pub-date><volume>3</volume><issue>6</issue><issue-id pub-id-type="doi">10.1111/irv.2009.3.issue-6</issue-id><fpage>287</fpage><lpage>295</lpage><history>Accepted 19 August 2009. Published Online 9 October 2009.</history><permissions><copyright-statement content-type="article-copyright">© 2009 Blackwell Publishing Ltd</copyright-statement></permissions><self-uri content-type="pdf" xlink:type="simple" xlink:href="file:IRV-3-287.pdf"></self-uri><abstract><p><bold>Background </bold> Highly pathogenic H5N1 avian influenza viruses currently circulating in birds have caused hundreds of human infections, and pose a significant pandemic threat. Vaccines are a major component of the public health preparedness for this likely event. The rapid evolution of H5N1 viruses has resulted in the emergence of multiple clades with distinct antigenic characteristics that require clade‐specific vaccines. A variant H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused multiple fatal infections. Vaccine candidates that match the antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain‐specific protection.</p><p><bold>Objective </bold> To address the need for a suitable seed for manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 pre‐pandemic candidate vaccine by reverse genetics and evaluated its safety and replication <italic>in vitro</italic> and <italic>in vivo</italic>.</p><p><bold>Methods </bold> A reassortant virus termed, Anhui/PR8, was produced by reverse genetics in compliance with WHO pandemic vaccine development guidelines and contains six genes from A/Puerto Rico/8/34 as well as the neuraminidase and hemagglutinin (HA) genomic segments from the A/Anhui/01/2005 virus. The multi‐basic cleavage site of HA was removed to reduce virulence.</p><p><bold>Results </bold> The reassortant Anhui/PR8 grows well in eggs and is avirulent to chicken and ferrets but retains the antigenicity of the parental A/Anhui/01/2005 virus.</p><p><bold>Conclusion </bold> These results indicate that the Anhui/PR8 reassortant lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, Thailand, and Laos.</p></abstract><kwd-group><kwd id="k1">Pandemic influenza</kwd><kwd id="k2">H5N1 vaccine</kwd><kwd id="k3">seed virus</kwd></kwd-group><counts><fig-count count="1"></fig-count><table-count count="6"></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>November 2009</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>Influenza viruses cause seasonal epidemics of acute respiratory disease with most severe consequences in pediatric and geriatric populations. Sporadically, influenza A viruses with a novel hemagglutinin (HA) subtype cause pandemics with serious public health consequences. Pandemics are caused by influenza viruses containing surface protein(s) for which the human population has little or no immunity, and endowed with the ability to spread in the human population. The three influenza pandemics of the 20th century involved the transmission of influenza viruses from animals to humans.<xref rid="b1" ref-type="ref"><sup>1</sup></xref> The most severe pandemic of the previous century was the ‘Spanish Influenza’ of 1918–1919, which spread globally in just 1 year and caused at least 40 million deaths.<xref rid="b1" ref-type="ref"><sup>1</sup></xref> The pandemic of 1957 was caused by an H2N2 reassortant virus containing avian HA, neuraminidase (NA), and polymerase basic protein 1(PB1) genes in the background of genes belonging to a circulating human H1N1 virus and rapidly displaced the circulating H1N1 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> In 1968, an H3N2 reassortant virus containing avian HA and PB1 genes with the remaining genes from the contemporary H2N2 virus emerged to cause a pandemic.<xref rid="b2" ref-type="ref"><sup>2</sup></xref>, <xref rid="b4" ref-type="ref"><sup>4</sup></xref>, <xref rid="b5" ref-type="ref"><sup>5</sup></xref> Another influenza pandemic could occur at any time and effective public health interventions based on appropriate preparedness are needed to diminish morbidity and mortality.</p><p>Numerous different subtypes of the influenza A virus exist in nature, resulting from combinations of 16 serotypes of HA (H1–H16) and nine serotypes of NA (N1–N9).<xref rid="b6" ref-type="ref"><sup>6</sup></xref> The human population has immunity to only a few of the known serotypes. To date, viruses with HA subtypes (H1, H2, H3, H5, H6, H7, and H9) and four NAs (N1, N2, N3, and N7) have been isolated (or antibodies against them have been detected) from humans.<xref rid="b4" ref-type="ref"><sup>4</sup></xref>, <xref rid="b7" ref-type="ref"><sup>7</sup></xref> Among these, subtype H5, H6, H7, and H9 viruses of avian origin have infected humans and are considered potential pandemic threats.</p><p>Highly pathogenic avian influenza (HPAI) H5N1 viruses currently circulating in birds have resulted in more than 400 instances of H5N1 transmission to humans since 2004, suggesting that this virus could initiate the next pandemic.<xref rid="b8" ref-type="ref"><sup>8</sup></xref> The HA sequences of avian H5N1 viruses have diverged into ten distinct phylogenetic clades (genetic groups).<xref rid="b9" ref-type="ref"><sup>9</sup></xref> Genetic divergence into clades is also correlated with antigenic drift and often antibodies generated against one clade do not efficiently inhibit hemagglutination or neutralize a virus from another clade.<xref rid="b10" ref-type="ref"><sup>10</sup></xref>, <xref rid="b11" ref-type="ref"><sup>11</sup></xref> As an additional complication, some clades have evolved further into second and third order clades (subclades). Most currently circulating H5N1 strains that have infected humans belong to four serologically distinct antigenic groups (clades 1, 2.1, 2.2, and 2.3.4).<xref rid="b9" ref-type="ref"><sup>9</sup></xref></p><p>The impact of viral evolution on the antigenic structure of HA becomes evident upon comparing the HA amino acid sequences of the major H5N1 clades. A large proportion of the amino acids that constitute putative antigenic sites in the HA are polymorphic suggesting that the antigenicity of the four major clades has drifted.<xref rid="b12" ref-type="ref"><sup>12</sup></xref>, <xref rid="b13" ref-type="ref"><sup>13</sup></xref> The consequence of such structural evolution is reflected in the reactivity of these viruses with panels of ferret antisera which are used to assess antigenic differences which may trigger the need to prepare a more closely matched vaccine candidate virus.<xref rid="b10" ref-type="ref"><sup>10</sup></xref>, <xref rid="b14" ref-type="ref"><sup>14</sup></xref></p><p>In this study, we analyzed the genetic and antigenic properties of a clade 2.3.4, subtype H5N1 virus which was then utilized for the derivation of a vaccine candidate by reassortment with the A/Puerto Rico/8/1934 (PR8) strain, which provided the polymerase, NP, M, and NS genes. This virus was expected to be antigenically representative of the entire clade 2.3.4 and to provide some cross‐reactive immunity outside this clade. In addition, since it has the internal genes from PR8 which should confer high growth in chicken embryo and partial attenuation in humans, it was expected to grow to reasonably high viral titers and to lack virulence.<xref rid="b15" ref-type="ref"><sup>15</sup></xref> These properties were evaluated to determine the suitability of this reassortant to develop a seed virus for the preparation of an inactivated subunit or split vaccine, the only type of H5N1 vaccine currently approved by the FDA, for use in the event of a pandemic.</p></sec><sec id="ss2"><title>Materials and methods</title><sec id="ss3"><title>Cells and viruses</title><p>Madin–Darby canine kidney and 293T human embryonic kidney cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS). Vero cells from a working cell bank qualified for vaccine production were described previously.<xref rid="b16" ref-type="ref"><sup>16</sup></xref> Vero cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) with 10% irradiated FBS (Cambrex, East Rutherford, NJ, USA) and 50 μg/ml Neomycin (Sigma, St. Louis, MO, USA). Primary chicken embryo fibroblast (CEF) and primary chicken kidney (PCK) cells were obtained from Charles River Laboratories, Wilmington, MA, USA. Influenza virus A/Anhui/1/2005 (H5N1) is a HPAI virus isolated from a fatal case of a pregnant woman in the Anhui province, China during 2005.<xref rid="b17" ref-type="ref"><sup>17</sup></xref> The virus was isolated from a tracheal aspirate, propagated in 10‐day‐old embryonated chicken eggs and its viral RNA extracted in a biosafety level (BSL)‐3‐enhanced laboratory. Harvested virus was quantified with a standard HA assay using a 0·5% turkey erythrocyte cell suspension<xref rid="b18" ref-type="ref"><sup>18</sup></xref> and also in eggs and CEF cells.</p></sec><sec id="ss4"><title>Sequence analysis of HA and NA genes, PCR amplification, mutagenesis, and cloning</title><p>Influenza viral genomes were sequenced as previously described.<xref rid="b19" ref-type="ref"><sup>19</sup></xref> Briefly, RNA was extracted with the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA), and used for RT‐PCR (OneStep RT/PCR Kit; Qiagen). The sequencing reaction was performed using BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA). Automated sequencing was performed using an ABI 3100 Genetic Analyzer. To clone the HA and at the same time remove the multi‐basic cleavage site, overlapping PCR was performed using two sets of primers; one set corresponding to HA1 (5′‐GATCGCTCTTCAGGGAGCAAAAGCAGGGG‐3′ and 5′‐CTAGGCTCTTCTTCGTTCTCGTAGAGGACTATTTCTGAG‐3′) and other corresponding to HA2 (5′‐AGTCGCTCTTCGCGAGGACTATTTGGAGCTATAGCAGG‐3′ and 5′‐ACTGGCTCTTCTATTAGTAGAAACAAGGGTGTTTTTAACTAC‐3′). For the amplification of the NA gene, we used the following universal primers which were modified from Hoffmann <italic>et al.</italic><xref rid="b20" ref-type="ref"><sup>20</sup></xref> 5′‐GATCGCTCTTCAGGGAGCAAAAGCAGGAGT‐3′ and 5′‐ACTGGCTCTTCTATTAGTAGAAACAAGGAGTTTTTT‐3′. The amplified PCR products for HA and NA genes were digested with restriction enzyme <italic>Sap</italic>I and ligated into the bidirectional transcription plasmid pCIPolISapIT (modified from pPolISapIT).<xref rid="b19" ref-type="ref"><sup>19</sup></xref></p></sec><sec id="ss5"><title>Plasmid DNA</title><p>The plasmids containing the PR8 internal genes were modified from the pPolISapIT‐derived PR8 plasmids described previously<xref rid="b21" ref-type="ref"><sup>21</sup></xref> by transferring regulatory elements including the human polymerase I promoter, mouse terminator, and PR8 genome sequences into the pCDNA3.1<sup>+</sup> mammalian expression vector (Invitrogen).</p></sec><sec id="ss6"><title>Virus recovery by DNA transfection</title><p>Plasmids containing HA and NA genes of A/Anhui/1/2005 (H5N1) together with plasmids carrying the six remaining genes, PB2, PB1, PA, NP, M, NS, of PR8 virus origin were used to transfect certified Vero cells using TransIT (Mirus, Madison, WI). Twenty four hours after transfection, PCK cells (Charles River Laboratories) were added to the Vero cells to support amplification of any released virus. The transfected cells and supernatants were harvested 48 hours later and 0·2 ml inoculated into the allantoic cavity of 10‐day‐old embryonated eggs. Eggs were incubated at 35°C for 2 days and allantoic fluid was analyzed by HA assay for the presence of virus. After one additional egg passage (E2), the infectivity and virion content was determined by titration in chicken embryos and HA assays, respectively. The former was expressed as EID<sub>50</sub> and was calculated using the Reed–Muench method from endpoint dilution.<xref rid="b22" ref-type="ref"><sup>22</sup></xref> The HA titer was determined as described previously using turkey erythrocytes.<xref rid="b23" ref-type="ref"><sup>23</sup></xref> All experiments described in this manuscript were performed with a virus stock derived in Vero cells and passaged twice in eggs (V1E2).</p></sec><sec id="ss7"><title>Plaque formation on chicken embryo fibroblast cells with or without trypsin</title><p>The viral plaque characteristics and titer were determined on CEF (Charles River Laboratories) cells as described previously.<xref rid="b24" ref-type="ref"><sup>24</sup></xref> CEF cells were grown on six‐well culture plates in DMEM supplemented with 10% FBS and antibiotics (100 μg/ml penicillin and 100 μg/ml streptomycin). After inoculation with serial dilutions of the virus stock, cell monolayers were overlaid with 0·8% agarose in DMEM without serum, with or without 0·5 μg/ml L‐1‐tosylamido‐2‐phenylethyl chloromethyl ketone (TPCK)‐treated trypsin. Plates were incubated for 48 hours and cells were stained with 0·1% crystal violet to visualize plaques.</p></sec><sec id="ss8"><title>Chicken embryo lethality test</title><p>Ten‐day‐old chicken embryos were inoculated in the allantoic sac with 0·1 ml of log<sub>10</sub> dilutions of each virus preparation with known infectious titers. The embryo viability was recorded at 48 hours post‐inoculation. The virus dose that caused death in 50% of embryos was calculated by the method of Reed and Muench<xref rid="b22" ref-type="ref"><sup>22</sup></xref> and reported as the Median Chicken Embryo Lethal Dose (CELD<sub>50</sub>).</p></sec><sec id="ss9"><title>Pathogenicity for chickens</title><p>Groups of eight 4‐week‐old specific pathogen‐free Plymouth White Rock chickens were inoculated with the reassortant virus, Anhui/PR8, or wild type A/Anhui/01/2005 virus, at a standard dose (10<sup>8·8</sup> EID<sub>50</sub>; 0·2 ml of a 1:10 dilution of stock virus) by the intravenous (i.v.) route and observed for 14 days.<xref rid="b25" ref-type="ref"><sup>25</sup></xref> Groups of eight chickens were also inoculated intranasally (i.n.) with 10<sup>6</sup> EID<sub>50</sub> in 0·1 ml of each virus and observed for 14 days.</p></sec><sec id="ss10"><title>Ferret inoculation studies</title><p>Six male ferrets (8‐month old) that were serologically negative for currently circulating influenza A viruses were used to evaluate the outcome of infection with Anhui/PR8 virus or the reverse genetics derived A/Puerto Rico/8/34 (PR8‐RG). Each ferret was i.n. inoculated with 10<sup>6</sup> or 10<sup>6·3</sup> EID<sub>50</sub> of Anhui/PR8 virus or the PR8‐RG virus, respectively. Three ferrets were killed at 3 days post‐inoculation (d.p.i.) and their spleen, lungs, whole blood, nasal turbinates, and brain were harvested for virus titration. The remaining three ferrets were monitored for clinical signs for 14 days and nasal washes were collected at 1, 3, 5, and 7 d.p.i. for virus titration. Infectious virus titers were determined by inoculation of embryonated eggs.</p></sec><sec id="ss11"><title>Antigenic characterization by HI assay</title><p>Antigenic characterization of relevant H5N1 viruses was performed using reference ferret antisera in a hemagglutination inhibition (HI) assay with turkey erythrocytes as previously described.<xref rid="b26" ref-type="ref"><sup>26</sup></xref></p></sec></sec><sec id="ss12"><title>Results</title><sec id="ss13"><title>Emergence and antigenic characteristics of clade 2.3.4 H5N1 viruses in Asia</title><p>The rapid evolution of HPAI H5N1 viruses circulating in birds has led to the emergence of multiple clades.<xref rid="b9" ref-type="ref"><sup>9</sup></xref> To identify an appropriate virus representative of clade 2.3.4 for vaccine development, a group of related clade 2 viruses were subjected to phylogenetic and antigenic analysis. A/Anhui/01/2005 virus had close phylogenetic relationship to other viruses within clade 2.3.4 (<xref rid="f1" ref-type="fig">Figure 1</xref>), suggesting that it is a suitable representative of the HA genetic diversity within in this clade.</p><fig fig-type="Figure" xml:lang="en" id="f1" orientation="portrait" position="float"><label>Figure 1</label><caption><p> Phylogenetic relationships of the A/Anhui/1/2005 hemagglutinin gene to other recent Eurasian H5N1 viruses. The phylogeny was generated by neighbor‐joining analysis using the Kimura 2‐parameter in MEGA 4 (<ext-link ext-link-type="uri" xlink:href="http://www.megasoftware.net/mega.html">http://www.megasoftware.net/mega.html</ext-link>). Bootstrap values at each node represent 1000 replicates. Scale bar represents a nucleotide distance of 0·005. Viruses included in the antigenic characterization (<xref rid="t1" ref-type="table-wrap">Table 1</xref>) are underlined; A/Anhui/1/2005 and the clade designation are highlighted in yellow.</p></caption><graphic id="nlm-graphic-1" xlink:href="IRV-3-287-g001"></graphic></fig><p>Post‐infection reference ferret antisera was generated against A/Anhui/01/2005 virus and was used in HI assays to determine the antigenic relatedness of A/Anhui/01/2005 virus compared with other viruses in the clade and <italic>vice versa</italic>. Antisera to A/Anhui/01/2005 showed strong inhibition of hemagglutination by the homologous antigen, as well as A/Indonesia/5/2005 virus (clade 2.1), but was minimally inhibitory for viruses of clade 1 and 2.2, as well as some 2.1 viruses (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). Most importantly, A/Anhui/01/2005 virus antisera yielded high HI titers with all the clade 2.3.4 viruses tested, indicating that this strain served as a representative antigen for the generation of a pre‐pandemic vaccine candidate reassortant virus.</p><table-wrap id="t1" xml:lang="en" orientation="portrait" position="float"><label>Table 1</label><caption><p> Hemagglutination inhibition assay of H5N1 viruses</p></caption><graphic id="nlm-graphic-3" xlink:href="IRV-3-287-g002"></graphic></table-wrap></sec><sec id="ss14"><title>Reassortant virus rescue and growth in embryonated eggs</title><p>To generate a PR8 reassortant virus displaying the A/Anhui/01/2005 virus surface proteins, we amplified both the HA and NA genes and cloned them into a reverse genetics plasmid. Four basic amino acid residues were removed from the HA cleavage site by directed mutagenesis. After plasmid transfection, virus recovered from Vero cell cultures, termed Anhui/PR8 was then passaged twice in embryonated chicken eggs.</p><p>To determine whether the rescued reassortant Anhui/PR8 virus retained the amino acid sequence from the parental virus surface glycoproteins, viral nucleic acid from the second egg passage was sequenced. The HA and NA proteins of the Anhui/PR8 virus were identical to that of A/Anhui/01/2005 virus except for the modified HA cleavage site which displayed the desired four amino acid deletion (R‐R‐K‐R) in the multibasic cleavage motif (<xref rid="t2" ref-type="table-wrap">Table 2</xref>). The newly created cleavage motif resembles those found in low pathogenic H5N1 influenza viruses (e.g., A/chicken/Italy/22A/98; <xref rid="t2" ref-type="table-wrap">Table 2</xref>) and complies with the WHO guidance for the development of pre‐pandemic candidate vaccine viruses.<xref rid="b10" ref-type="ref"><sup>10</sup></xref></p><table-wrap id="t2" xml:lang="en" orientation="portrait" position="float"><label>Table 2</label><caption><p> Deletion of the virulence‐inducing cleavage motif in HA of A/Anhui/01/2005</p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1"></th><th align="left" valign="bottom" rowspan="1" colspan="1">Amino acid sequence at cleavage site</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1">↓</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">A/Anhui/01/2005</td><td align="left" valign="top" rowspan="1" colspan="1">G L R N S P L R E R R R K R G L F</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Anhui/PR8</td><td align="left" valign="top" rowspan="1" colspan="1">G L R N S P L R E R ‐ ‐ ‐ ‐ G L F</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">A/CK/Italy/22A/1998</td><td align="left" valign="top" rowspan="1" colspan="1">G P R N V P Q K E T R ‐ ‐ ‐ G L F</td></tr></tbody></table><table-wrap-foot><fn id="t2_note10"><p>↓Denotes proteolytic cleavage site.</p></fn></table-wrap-foot></table-wrap><p>To determine the ability of the second egg passage Anhui/PR8 reassortant virus to replicate to acceptable yields for vaccine production, its yield in embryonated eggs was determined by different methods. As shown in <xref rid="t3" ref-type="table-wrap">Table 3</xref>, Anhui/PR8 virus demonstrated a high yield of viral infectivity (EID<sub>50</sub> and plaque forming unit (PFU)) and antigen (HA) from propagation in eggs. These results suggest that the reassortant virus replicated to levels that are likely to be scalable for industrial manufacturing of an influenza vaccine in embryonated eggs. The A/Indonesia/5/2005‐(H5N1)‐PR8 (Indo5/PR8) reassortant virus, which was successfully manufactured in eggs as split vaccine (<xref rid="t3" ref-type="table-wrap">Table 3</xref>) displayed similar replication characteristics as the Anhui/PR8 virus (M. Perdue, DHHS, personal communication).</p><table-wrap id="t3" xml:lang="en" orientation="portrait" position="float"><label>Table 3</label><caption><p> Hemagglutination and infectivity titers of reassortant viruses</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><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="2" valign="bottom" align="left" colspan="1">Virus</th><th rowspan="2" valign="bottom" align="left" colspan="1">Passage history*</th><th rowspan="2" valign="bottom" align="left" colspan="1">HA titer<sup>†</sup></th><th colspan="2" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Infectivity titer</th></tr><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1">Eggs<sup>‡</sup></th><th align="left" valign="bottom" rowspan="1" colspan="1">MDCK<sup>‡</sup></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">Anhui/PR8</td><td align="left" valign="top" rowspan="1" colspan="1">V1E2</td><td align="left" valign="top" rowspan="1" colspan="1">1:640</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>8·5</sup> EID<sub>50</sub>/ml</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>9</sup> PFU/ml</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Indo5/PR8</td><td align="left" valign="top" rowspan="1" colspan="1">V1E2</td><td align="left" valign="top" rowspan="1" colspan="1">1:1024</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>9·5</sup> EID<sub>50</sub>/ml</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>8</sup> PFU/ml</td></tr></tbody></table><table-wrap-foot><fn id="t3_note16"><p>*V1E2: produced by transfection in Vero cells and two passages in eggs.</p></fn><fn id="t3_note17"><p><sup>†</sup>With turkey erythrocytes.</p></fn><fn id="t3_note18"><p><sup>‡</sup>Host system used for titration.</p></fn><fn id="t3_note19"><p>MDCK, Madin–Darby canine kidney.</p></fn></table-wrap-foot></table-wrap></sec><sec id="ss15"><title>Trypsin‐dependent plaque formation on CEF cells</title><p>Highly pathogenic viruses can undergo multicycle <italic>in vitro</italic> replication in the absence of trypsin due to the presence of a multibasic cleavage motif in HA that is readily cleaved by furin‐like proteases expressed by all vertebrate cells.<xref rid="b1" ref-type="ref"><sup>1</sup></xref>, <xref rid="b27" ref-type="ref"><sup>27</sup></xref> The genetic changes introduced into the newly developed pre‐pandemic candidate vaccine viruses are expected to impart dependence on trypsin for growth in chicken fibroblasts, which is a property displayed by low pathogenicity viruses. Indeed, Anhui/PR8 virus failed to form plaques on CEF cell cultures lacking trypsin (<xref rid="t4" ref-type="table-wrap">Table 4</xref>). In the presence of trypsin, the reassortant virus was capable of forming plaques on CEF cells suggesting that the absence of plaques in cultures devoid of trypsin was not due to a defect inherent to the reverse genetics derivation (<xref rid="t4" ref-type="table-wrap">Table 4</xref>). These results indicate that Anhui/PR8 virus lacks an essential virulence determinant of HPAI as evidenced by a strict trypsin dependence for plaque formation on CEF cells.<xref rid="b1" ref-type="ref"><sup>1</sup></xref>, <xref rid="b27" ref-type="ref"><sup>27</sup></xref></p><table-wrap id="t4" xml:lang="en" orientation="portrait" position="float"><label>Table 4</label><caption><p> Trypsin‐dependent plaque formation on CEF cells</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><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="3" valign="bottom" align="left" colspan="1">Virus</th><th colspan="4" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Plaque formation on CEF cells</th></tr><tr style="border-bottom:solid 1px #000000"><th colspan="2" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">With trypsin</th><th colspan="2" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Without trypsin</th></tr><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1"> PFU/ml</th><th align="left" valign="bottom" rowspan="1" colspan="1">Diameter (mm)</th><th align="left" valign="bottom" rowspan="1" colspan="1"> PFU/ml</th><th align="left" valign="bottom" rowspan="1" colspan="1">Diameter (mm)</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">Anhui/PR8</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>8·3</sup></td><td align="left" valign="top" rowspan="1" colspan="1">3</td><td align="left" valign="top" rowspan="1" colspan="1">≤1</td><td align="left" valign="top" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">A/Anhui/01/2005</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>8·54</sup></td><td align="left" valign="top" rowspan="1" colspan="1">2·5–3</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>8·6</sup></td><td align="left" valign="top" rowspan="1" colspan="1">2–2·5</td></tr></tbody></table><table-wrap-foot><fn id="t4_note18"><p>NA, not applicable.</p></fn></table-wrap-foot></table-wrap></sec><sec id="ss16"><title>Chicken embryo lethality test</title><p>Highly pathogenic avian influenza viruses are typically lethal to chicken embryos and death can be observed as early as 24 hours post‐inoculation. To determine the lack of pathogenicity of Anhui/PR8 virus as compared to its wild type counterpart, 10‐day‐old chicken embryos were inoculated with reassortant or wild type viruses. High proportions of viable embryos were noted in groups at 24 hours after inoculation with relatively high doses of Anhui/PR8 virus and at 48 hours the CELD<sub>50</sub> for the reassortant virus was ≥10<sup>5·3</sup> PFU/0·1 ml (data not shown and <xref rid="t5" ref-type="table-wrap">Table 5</xref>). In contrast, wild type A/Anhui/01/2005 virus was highly lethal to embryos at very low viral doses of virus; the CELD<sub>50</sub> was ≤10<sup>−0·39</sup> PFU/0·1 ml at 48 hours after inoculation (<xref rid="t5" ref-type="table-wrap">Table 5</xref>), in agreement with previous reports.<xref rid="b15" ref-type="ref"><sup>15</sup></xref></p><table-wrap id="t5" xml:lang="en" orientation="portrait" position="float"><label>Table 5</label><caption><p> Virulence assessment in chicken embryos and chickens</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 align="left" valign="bottom" rowspan="1" colspan="1">Virus</th><th align="left" valign="bottom" rowspan="1" colspan="1"> CELD<sub>50</sub>*</th><th align="left" valign="bottom" rowspan="1" colspan="1">Route</th><th align="left" valign="bottom" rowspan="1" colspan="1"> Dose<sup>†</sup></th><th align="left" valign="bottom" rowspan="1" colspan="1">IVPI</th><th align="left" valign="bottom" rowspan="1" colspan="1">Morbidity</th><th align="left" valign="bottom" rowspan="1" colspan="1">Mortality (MDT)</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">Anhui/PR8</td><td align="left" valign="top" rowspan="1" colspan="1">≥10<sup>5·3</sup></td><td align="left" valign="top" rowspan="1" colspan="1">IV</td><td align="left" valign="top" rowspan="1" colspan="1">10<sup>8·8</sup></td><td align="left" valign="top" rowspan="1" colspan="1">0·0</td><td align="left" valign="top" rowspan="1" colspan="1">0/8</td><td align="left" valign="top" rowspan="1" colspan="1">0/8</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">A/Anhui/01/2005</td><td align="left" valign="top" rowspan="1" colspan="1">≤10<sup>−0·39</sup></td><td align="left" valign="top" rowspan="1" colspan="1">IV</td><td align="left" valign="top" rowspan="1" colspan="1"> 10<sup>8·8</sup></td><td align="left" valign="top" rowspan="1" colspan="1"> 3·0</td><td align="left" valign="top" rowspan="1" colspan="1"> 8/8</td><td align="left" valign="top" rowspan="1" colspan="1"> 8/8 (1 d)</td></tr></tbody></table><table-wrap-foot><fn id="t5_note21"><p>*CELD<sub>50</sub>: 50% chicken embryo lethal dose; PFU/ml.</p></fn><fn id="t5_note22"><p><sup>†</sup>Dose administered expressed in EID<sub>50</sub> (50% egg infectious dose) per 0·1 ml.</p></fn><fn id="t5_note23"><p>IV, intravenous; IVPI, intravenous pathogenic index; MDT, mean death time; d, days.</p></fn></table-wrap-foot></table-wrap></sec><sec id="ss17"><title>Pathogenicity testing in chickens</title><p>To determine the pathogenic potential of the reassortant virus in birds, groups of chickens were inoculated by either the i.v. or i.n. routes with 10<sup>8·8</sup> or 10<sup>6</sup> EID<sub>50</sub>, respectively, of Anhui/PR8 virus or its wild type parent. All chickens inoculated with the wild type parental virus, A/Anhui/01/2005, died with a mean death time of 1 day after inoculation by the i.v. route and 2·1 days by the i.n. route (<xref rid="t5" ref-type="table-wrap">Table 5</xref>). Mortality was 100% in the groups inoculated with wild type virus via either route. Postmortem examinations revealed lesions typical of HPAI infection, including petechial hemorrhages on viscera, hemorrhage of comb and wattle, and severe lung congestion and hemorrhage with interstitial pneumonia (data not shown) as reported previously.<xref rid="b28" ref-type="ref"><sup>28</sup></xref> In contrast, all chickens inoculated with Anhui/PR8 virus remained healthy throughout the 14‐day observation period. Virus was isolated from both oral and cloacal swabs from birds inoculated with the wild type virus, but not from animals inoculated with the reassortant (data not shown). A/Anhui/01/2005 virus was also isolated from brain, lung, heart and kidney tissues of the inoculated chickens. In contrast, no virus was detected in either oral or cloacal swabs or from any of the above tissues in chickens inoculated with Anhui/PR8 virus (data not shown). Our results demonstrate the lack of pathogenicity of the H5N1 reassortant pre‐pandemic candidate vaccine virus in chickens, indicating that this virus does not pose a major threat to domestic or wild birds.</p></sec><sec id="ss18"><title>Safety testing in ferrets</title><p>Ferrets infected with HPAI H5N1 viruses may display signs of lethargy, severe weight loss, and respiratory or neurological symptoms.<xref rid="b29" ref-type="ref"><sup>29</sup></xref>, <xref rid="b30" ref-type="ref"><sup>30</sup></xref> To determine the pathogenic potential of the reassortant virus, six 8‐month‐old ferrets were inoculated with Anhui/PR8 or PR8‐RG virus. None of the ferrets infected showed severe signs of disease over the 14 d.p.i. observation period, although they did show a 1–3% weight loss. A peak mean viral titer of 10<sup>3·5</sup> EID<sub>50</sub>/ml was detected in nasal washes collected from ferrets infected with Anhui/PR8 virus at 5 d.p.i. but virus was cleared from nasal washes by 7 d.p.i. in two of three ferrets. Viral titers ranged from 10<sup>2·3</sup> to 10<sup>5·5</sup> EID<sub>50</sub>/ml in nasal washes from PR8‐RG virus‐infected ferrets on 1, 3, and 5 d.p.i. and one ferret at 7 d.p.i. (data not shown). Virus isolation studies from inoculated animals are presented in <xref rid="t6" ref-type="table-wrap">Table 6</xref>. Anhui/PR8 virus was detected at day 3 p.i. in the nasal turbinates and in the olfactory bulb of one of three ferrets but virus was not detected in the lungs, spleen, brain (posterior to the olfactory bulb) or whole blood of any of the inoculated ferrets, demonstrating that the reassortant vaccine candidate virus is not pathogenic to ferrets, a critical safety parameter for H5N1 vaccines.</p><table-wrap id="t6" xml:lang="en" orientation="portrait" position="float"><label>Table 6</label><caption><p> Detection of infectious virus in ferret tissues at 3 days post‐inoculation</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><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1">Virus</th><th align="left" valign="bottom" rowspan="1" colspan="1">Nasal turbinates</th><th align="left" valign="bottom" rowspan="1" colspan="1">Lungs</th><th align="left" valign="bottom" rowspan="1" colspan="1">Spleen</th><th align="left" valign="bottom" rowspan="1" colspan="1">Brain</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">Anhui/PR8</td><td align="left" valign="top" rowspan="1" colspan="1">1/3*</td><td align="left" valign="top" rowspan="1" colspan="1">0/3</td><td align="left" valign="top" rowspan="1" colspan="1">0/3</td><td align="left" valign="top" rowspan="1" colspan="1">0/3</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">PR8‐RG</td><td align="left" valign="top" rowspan="1" colspan="1">3/3</td><td align="left" valign="top" rowspan="1" colspan="1">1/3</td><td align="left" valign="top" rowspan="1" colspan="1">0/3</td><td align="left" valign="top" rowspan="1" colspan="1">0/3</td></tr></tbody></table><table-wrap-foot><fn id="t6_note15"><p>*Number of animals with infectious virus/number tested.</p></fn></table-wrap-foot></table-wrap></sec><sec id="ss19"><title>Antigenic analysis of reassortant virus</title><p>To determine if the Anhui/PR8 reassortant virus had retained the antigenic characteristics of the A/Anhui/01/2005 donor virus we performed reciprocal HI assays using ferret antisera to each of these viruses (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). For both wild type and reassortant viruses, homologous sera resulted in the same relative titers of hemagglutination inhibition. But more importantly, the convalescent or post‐infection ferret serum to the A/Anhui/01/2005 virus inhibited hemagglutination by Anhui/PR8 virus to a similar titer as that against the immunizing wild type virus (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). Likewise, the antisera to Anhui/PR8 virus inhibited hemagglutination by A/Anhui/01/2005 virus to a similar extent as the immunizing virus. A significant difference in antigenicity is typically defined as HI titer difference of at least fourfold. Therefore, these results indicate that within the limits of sensitivity of the HI assay, Anhui/PR8 virus maintained the antigenic characteristics of the parental virus.</p></sec></sec><sec id="ss20"><title>Discussion</title><p>In contrast to the predominance of only a few major clades of each subtype of seasonal human influenza viruses,<xref rid="b31" ref-type="ref"><sup>31</sup></xref>, <xref rid="b32" ref-type="ref"><sup>32</sup></xref> multiple clades of H5N1 co‐circulate in birds in Asia, Africa, and Europe. Evolutionary studies of H5N1 virus suggest that although some lineages have become extinct, there are multiple lineages still co‐circulating that can be grouped in diverse clades.<xref rid="b9" ref-type="ref"><sup>9</sup></xref> Phylogenetic analysis is crucial to understand how H5N1 evolves and phylogenetic divergence often goes hand in hand with changes in antigenicity which determine vaccine strain selection. Inactivated influenza vaccines are thought to elicit strain‐specific protection.<xref rid="b33" ref-type="ref"><sup>33</sup></xref>, <xref rid="b34" ref-type="ref"><sup>34</sup></xref> Therefore, co‐circulation of multiple antigenically distinct H5N1 virus clades presents a formidable challenge to pandemic preparedness efforts involving vaccine stockpiles. Any of the viruses in the various H5N1 virus clades could potentially initiate a pandemic.<xref rid="b10" ref-type="ref"><sup>10</sup></xref> By matching the pre‐pandemic vaccine candidate viruses to major circulating H5N1 virus antigenic groups, an updated library of vaccine reference viruses is readily available for immediate use. Such a repository enables rapid development of seed viruses for manufacturing of pre‐pandemic vaccine for stockpiles or for expedited production of vaccine<xref rid="b10" ref-type="ref"><sup>10</sup></xref>, <xref rid="b14" ref-type="ref"><sup>14</sup></xref> and immunization in case of pandemic emergency.</p><p>H5 viruses of four antigenically distinct clades have been isolated from humans since 2004. Clades 1, 2.2, 2.1.3, and 2.3.4 have been isolated from clinical cases and public health efforts have concentrated in the derivation of reassortant viruses belonging to these clades.<xref rid="b10" ref-type="ref"><sup>10</sup></xref> The first candidate H5N1 reassortant viruses were derived from clades 1 and 2.1 viruses. Later, the isolation of viruses belonging to clades 2.2 and 2.3.4 in humans motivated the generation of additional reassortant viruses. The A/Anhui/01/2005 virus belonging to clade 2.3.4 and isolated from a pregnant woman in the Anhui province of china, was chosen as a representative reference strain to make the Anhui/PR8 reassortant virus.<xref rid="b17" ref-type="ref"><sup>17</sup></xref> Clade 2.3.4 viruses are predominant in China (and also Vietnam, Thailand, and Laos) and have resulted in the majority of human cases in the country.</p><p>The preparation of a pre‐pandemic vaccine candidate must be coupled with the attenuation of the pathogenic potential of its parental virus strain in ferrets, chickens, and chicken embryos. We set out to characterize whether, in fact, Anhui/PR8 virus could be used as a safe and effective pre‐pandemic candidate vaccine seed virus. The WHO has developed scientific guidance for the development of seed viruses.<xref rid="b35" ref-type="ref"><sup>35</sup></xref> The Anhui/PR8 reassortant virus demonstrated high‐yield characteristics when grown in eggs, which is an important feature needed for the successful manufacturing scale‐up process. This reassortant virus contains six internal genes from the egg adapted PR8 donor virus (PB2, PB1, PA, NP, M, and NS) which is responsible for the high‐yield characteristic. It also contains the HA and NA genes from A/Anhui/01/2005 virus and sequence analysis confirmed that the HA of Anhui/PR8 virus has a modified cleavage site, lacking a four basic amino acid sequence necessary for systemic virus replication. As a result, Anhui/PR8 virus resembles low pathogenic avian influenza viruses in its cleavage motif and displayed a strict dependence on trypsin supplementation for the formation of plaques on CEF cells. In agreement with these <italic>in vitro</italic> studies, the Anhui/PR8 virus was not lethal to chicken embryos unless it was inoculated at very high doses.</p><p>The survival of 100% of the chickens inoculated with Anhui/PR8 virus and the absence of clinical signs indicated that the reassortant virus is not pathogenic for chickens according to the classification of avian influenza established by the World Organization for Animal Health (OIE).<xref rid="b25" ref-type="ref"><sup>25</sup></xref>, <xref rid="b35" ref-type="ref"><sup>35</sup></xref> In addition, the reassortant virus safety test in ferrets showed that none of the infected animals developed substantial signs of disease over the 14 d.p.i. experimental period. All of the above data indicate that the Anhui/PR8 virus meets the criteria to be classified as a low pathogenic avian influenza virus that complies with national and international safety standards.</p><p>Importantly, the Anhui/PR8 virus elicited an antibody response in ferrets that could not distinguish the reassortant virus from wild type virus and <italic>vice versa</italic>, indicating that the antigenic properties of the parental A/Anhui/01/2005 virus were maintained in the reassortant virus. In addition, antisera against Anhui/PR8 virus also neutralized other clade 2.3.4 H5N1 viruses (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). All the manipulations required for the cloning of A/Anhui/01/2005 HA and NA genes, and its rescue and subsequent propagation in eggs were performed in dedicated facilities, using qualified Vero cells certified for vaccine use and reagents under a quality management system free of animal raw materials.<xref rid="b35" ref-type="ref"><sup>35</sup></xref> In addition, quality tests showed that the Anhui/PR8 virus was not pathogenic to chicken embryos (<xref rid="t5" ref-type="table-wrap">Table 5</xref>), did not display significant amounts of plasmid DNA or sterility issues (data not shown). These findings support the proposed use of this virus for development of seeds and production of inactivated pre‐pandemic vaccine for use in clinical trials.</p></sec><sec id="ss22"><title>Author disclosure statement</title><p>The authors state that there are no competing financial interests.</p></sec></body><back><ack id="ss21"><title>Acknowledgements</title><p>The authors would like to thank Aleksandr Lipatov and Li‐Mei Chen for critical reading of the manuscript and Xiu‐Feng Wan for suggestions. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.</p></ack><ref-list id="ss23"><title>References</title><ref id="b1"><label>1</label><mixed-citation publication-type="journal" id="cit2"><string-name><surname>Horimoto</surname><given-names>T</given-names></string-name>,
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