Absence of clinical disease and contact transmission of HPAI H5NX clade 2.3.4.4 from North America in experimentally infected pigs

Background In the fall of 2014, highly pathogenic avian influenza (HPAI) subtype H5N8 clade 2.3.4.4 was introduced into North America by migrating waterfowl from Asia where, through reassortment, novel HPAI H5N2 and H5N1 viruses emerged. Objectives Assess the susceptibility of pigs to HPAI H5N1, H5N2, and H5N8 clade 2.3.3.3 from North America. Methods Pigs and trachea explants were inoculated with a representative panel of H5NX clade 2.3.4.4 HPAI viruses from North America. Nasal swabs, BALF, and sera were collected to assess replication and transmission in challenged and direct contact pigs by RRT‐PCR, virus isolation, hemagglutination inhibition, and ELISA. Results Limited virus replication was restricted to the lower respiratory tract of challenged pigs, though absent in the nasal passages and trachea cultures, as determined by RRT‐PCR in all samples. Seroconversion of inoculated pigs was detected by NP ELISA but was not reliably detected by antigen‐specific hemagglutination inhibition. Boost with adjuvanted virus was required for the production of neutralizing antibodies to assess cross‐reactivity between wild‐type avian strains. All RRT‐PCR and serology tests were negative for contact animals indicating a failure of transmission from primary inoculated pigs. Conclusions H5NX clade 2.3.4.4 strains can replicate in the lower respiratory tract of swine upon high titer inoculation, though appear to be incapable of replication in swine nasal epithelium in vivo or ex vivo in trachea explants in culture. Infected pigs did not produce high levels of serum antibodies following infection. Collectively, our data show HPAI H5NX clade 2.3.4.4 viruses to be poorly adapted for replication and transmission in swine.


| INTRODUCTION
In the fall of 2014, A/goose/Guangdong/1/1996 lineage clade 2.3.4.4 highly pathogenic avian influenza (HPAI) A viruses of the H5 subtype were introduced to the North American continent, presumably carried by infected migratory waterfowl, and subsequently isolated from wild birds in Western Canada and the Northwestern United States. [1][2][3][4] Following introduction to the continent, H5N8 clade 2.3.4.4 viruses reassorted with North American lineage avian influenza A viruses (IAV) producing reassortant H5N1 and H5N2 viruses. Genetically, the reassortant H5 viruses combined North American avian PB1, NP, and NA gene segments with PB2, PA, HA, MP, and NS from the Eurasian progenitor H5N8 virus. 1,5 Previous studies have shown that while North American H5NX viruses are low in virulence properties and non-transmissible in mice and ferrets, they remain highly pathogenic for many avian species. [6][7][8] As pigs have been proposed to be more permissive for replication of avian IAV compared to other mammalian species, 9,10 understanding whether H5NX clade 2.3.4.4 viruses can infect and transmit in swine is a primary concern in addressing the risk emerging IAV pose to agriculture and public health. H1N1, H1N2, and H3N2 IAV are enzootic respiratory pathogens of domestic swine with worldwide distribution, causing mild infection of the upper and lower respiratory tract. In North America, the circulating IAV subtypes H1N1, H1N2, and H3N2 are genetically diverse products of multiple reassortments, combining the viral gene segments of avian, human, and swine influenza lineage IAV. 11 The novel gene constellations resulting from reassortment in swine provide ample genetic diversity for the emergence of viruses capable not only of increased replication and pathogenesis in swine like the triple reassortant H3N2, 12,13 but the potential ability to infect other species, including humans, exemplified by the 2009 H1N1 pandemic. 14,15 Further, pH1N1 IAV are frequently detected as a reverse zoonotic agent, with repeated introductions into swine facilitating the replacement of classical swine lineage MP gene segment with one of pandemic lineage, in addition to vastly increasing the genomic complexity of IAV genotypes circulating in North American swine populations. [16][17][18] It remains to be seen if the expanded diversity of internal gene constellations of IAV now endemic in domestic swine herds will be detrimental to agricultural production and/or human health.
To assess the ability of H5NX clade 2.  were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs. The allantoic fluid of inoculated eggs was collected, and 50% egg infective dose (EID 50 ) titers were determined as previously described. 19

| Inoculation of swine
A total of 85 three-week-old weaned pigs were used in this study.

| Virus replication and shedding
Nasal swabs were collected at 1, 3, and 7 dpi from donor, contact, and control animals and stored in 2 mL of minimum essential medium (MEM). Whole lungs were taken from five necropsied pigs at 3 and 5 dpi and from three primary inoculated pigs and all five contact animals at 21 dpi. Lavages were performed on excised lungs using 50 mL of MEM. Broncho-alveolar lavage fluid (BALF) was collected as previously described. 21 Both nasal swab and BALF samples were used to assess the presence or absence of IAV via reverse transcriptase realtime polymerase chain reaction (RRT-PCR) as previously described. 22 Samples positive by RRT-PCR were subjected to virus isolation in embryonated chicken eggs.

| Serological analysis
Seroconversion of challenged and contact animals was assessed using a commercial enzyme-linked immunosorbent assay (ELISA) and the hemagglutination inhibition (HI) assay. Whole blood was collected, and the serum was separated by centrifugation at 290 g for 10 minutes. Swine serum was then diluted 1:4 with receptor destroying enzyme (RDE) (Denka-Seiken, Tokyo, Japan) and incubated for 18 hours at 37°C. Next, RDE was heat inactivated by incubation at 56°C for 30 minutes. HI assays were performed in U bottom 96-well plates (Corning, Tewksbury, MA) by diluting 50 μL RDE-treated sera 1:2 in PBS, incubating with 25 μL 4 hemagglutinating units (HAU) at room temperature for 1 hour, followed by a 30-min incubation with 0.5% turkey red blood cells at room temperature. ELISA for IAV nucleoprotein (NP) was performed using FlockChek AI MultiS-Screen Antibody Test kit following manufacturer's instructions (IDEXX Laboratories, Inc., Westbrook, ME, USA).

| Statistical analysis
GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis of RRT-PCR data. Two-way analysis of variance was used to compare groups. P ≤ .05 was considered to be statistically significant.

| Viral replication in upper and lower respiratory tract and in trachea explants
Nasal swabs were collected from all experimentally infected pigs on 1, 3, 5 dpi, and BALF was collected from 5 animals at 3 and 5 dpi ( Table 1) Virus isolation was attempted on BALF fluid samples, and virus was cultivated from at least one pig from all virus groups of primary inoculated pigs on 3 and 5 dpi (Table 1).
To further assess the ability of H5NX viruses to replicate in the re-

| Serology
Serum was collected on 21 dpi in primary pigs and 19 dpc in direct contact pigs and assayed for antibodies against IAV hemagglutinin (HA) and nucleoprotein (NP

| DISCUSSION
These studies were undertaken to characterize the replication and  Ex vivo inoculation of swine trachea explants was conducted to further assess the replication kinetics of HPAI H5NX in porcine respiratory tissue. Trachea explants maintain the normal in vivo tissue architecture and sialic acid receptor distribution, abundant alpha-2,6linked (human) and a complete absence of alpha-2,3-linked (avian) receptors. 29,30 Our findings are consistent with previous works where IAV isolated directly from avian species are incapable of replication