Virology. Ji-Young Min 1, Grace L Chen 1, Celia Santos, Elaine W Lamirande, Yumiko Matsuoka, Kanta Subbarao. Contents lists available at ScienceDirect

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1 Virology 408 (2010) Contents lists available at ScienceDirect Virology journal homepage: Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets Ji-Young Min 1, Grace L Chen 1, Celia Santos, Elaine W Lamirande, Yumiko Matsuoka, Kanta Subbarao Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, USA article info abstract Article history: Received 9 July 2010 Returned to author for revision 23 August 2010 Accepted 10 September 2010 Keywords: 2009 pandemic influenza Classical swine H1N1 influenza Cross-protection The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/ia/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/ia/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus. Published by Elsevier Inc. Introduction In April 2009, the Centers for Disease Control and Prevention (CDC) reported the first two cases of human infection with a novel influenza H1N1 virus in the United States (2009a,c) and over the course of the next several weeks, the virus spread around the world (WHO, prompting the World Health Organization (WHO) to declare a global influenza pandemic on June 11, 2009 (WHO, swineflu/en). Antigenic and genetic characterization of the novel influenza H1N1 virus indicate that the virus contains a unique combination of gene segments from viruses that had been circulating in pigs in North American and Europe; 6 of the 8 gene segments (3 polymerase genes, HA, NP and NS) were derived from a triple reassortant North American H1N2 swine influenza virus and the NA and M gene segments were derived from a Eurasian lineage swine H1N1 virus (Garten et al., 2009). The 2009 pandemic H1N1 viruses isolated from human cases are antigenically equivalent to one another and are similar to North American swine H1N1 viruses, but distinct from seasonal human influenza H1N1 viruses (Garten et al., 2009). The 2009 pandemic H1N1 virus is not the first swine-origin H1N1 virus to infect humans. In the past 35 years, more than 50 sporadic Corresponding author. LID, NIAID, NIH 33 North Drive, MSC 3203, Room 3E.13C.1 Bethesda, MD 20892, USA. Fax: address: ksubbarao@niaid.nih.gov (K. Subbarao). 1 J.-Y. Min and G. Chen contributed equally to this work. cases of swine influenza virus infection have occurred in humans, mostly caused by classical swine influenza viruses (Hay et al., 2001; Kendal et al., 1977; Myers et al., 2007; Shinde et al., 2009; Thompson et al., 1976; Webster et al., 1992). Signs and symptoms of infection with classic swine influenza virus in humans are often indistinguishable from those of infection with human influenza viruses (Myers et al., 2007). The most notable instance occurred in 1976, after the isolation of a swine H1N1 influenza virus (A/New Jersey/1976) from a military recruit at Fort Dix, NJ. There were 12 additional cases identified by virus isolation or serologic methods and one military recruit died of the disease (Gaydos et al., 1977; Hodder et al., 1977). Concerns of the pandemic potential of the A/New Jersey/1976 H1N1 influenza virus led to the implementation of a mass vaccination program, which resulted in the administration of ~45 million doses of inactivated vaccine in the U.S (Dowdle, 1997). The vaccine campaign was halted because the pandemic did not materialize and adverse events were reported in association with the vaccine. Since then, sporadic cases of swine influenza infection associated with clinical disease have occurred in people exposed to sick pigs (O'Brien et al., 1977) and were occasionally fatal (Rota et al., 1989); only limited, non-sustained human-to-human transmission of swine influenza viruses had been reported before April 2009 (Robinson et al., 2007; Top and Russell, 1977; Wells et al., 1991). Because the hemagglutinin of the 2009 pandemic H1N1 virus is a derivative of and is antigenically related to classical swine influenza H1N1 viruses, we investigated the ability of swine influenza viruses to protect ferrets and mice from challenge with the 2009 pandemic H1N1 virus. We selected A/California/7/2009 (H1N1) and A/ /$ see front matter. Published by Elsevier Inc. doi: /j.virol

2 J.-Y. Min et al. / Virology 408 (2010) California/4/2009 (H1N1) viruses that were isolated from two unrelated cases of febrile respiratory illness in children who resided in adjacent counties in southern California and were the first swineorigin human 2009 pandemic H1N1 viruses identified in the United States (2009a,c). Hemagglutination inhibition (HAI) assays reveal that the 2009 pandemic H1N1 viruses (A/California/7/2009 and A/ California/4/2009 viruses) are antigenically similar and distinct from currently circulating seasonal H1N1 viruses (Garten et al., 2009). The two classical swine influenza viruses we included in the study were A/swine/Iowa/1931 (H1N1), a reference classical swine H1N1 virus closely related to the first isolate A/swine/Iowa/15/1930 and A/New Jersey/8/1976 (H1N1), an isolate from the limited outbreak in 1976 in Fort Dix, NJ. This comparison was made to gain insight into the degree of protective immunity that may be conferred by prior infection with influenza viruses that were derived from pigs or were antigenically related to swine influenza viruses. Results We first established the kinetics of replication and pathogenesis of the 2009 swine-origin pandemic H1N1 influenza viruses (CA/7/09 and CA/4/09) and two classical swine influenza viruses (sw/iw/31 and NJ/76) in mice and ferrets and then used these models to assess the extent of homologous and heterologous protection provided by these H1N1 viruses. Level of viral replication in mice The kinetics of replication of the H1N1 influenza viruses are displayed in Fig. 1. With the exception of the NJ/76 virus, all of the H1N1 viruses replicated to high titer in the upper respiratory tract with the sw/ia/31 being the highest (Fig. 1A). In the upper respiratory tract, mean peak titers on day 3 p.i. of 10 5,10 4.9, and TCID 50 /g were observed in mice inoculated with the CA/7/ 09, CA/4/09, NJ/76 and sw/ia/31 viruses, respectively. All of the H1N1 viruses replicated to high titer in the lower respiratory tract of mice (Fig. 1B); peak titers achieved following inoculation of the viruses were as follows: TCID 50 /g on day 2 p.i. in mice inoculated with CA/7/09; TCID 50 /g on day 4 p.i. following inoculation of CA/4/09; TCID 50 /g on day 2 p.i. following inoculation of NJ/76; and TCID 50 /g on day 2 p.i. in mice inoculated with sw/ia/ 31virus. Virus was detected in the brain of mice infected with CA/7/ 09 and CA/4/09 on day 2 p.i. at mean titers of and TCID 50 / g, respectively (Fig. 1C). We cannot conclude definitively that the 2009 swine-origin pandemic H1N1 influenza viruses (CA/7/09 and CA/4/09) spread to the brain of mice because olfactory bulbs were harvested along with the brain and it is possible that the detection of virus in these samples may represent direct infection from the nasal cavity rather than disseminated or systemic spread. Virus was not detected in the brain of mice that received the NJ/76 virus. Although virus was not detected in the brain of mice that were infected with the low dose (10 TCID 50 ) of sw/ia/31 (data not shown), virus was detected in the brain of mice that received a higher dose (10 5 TCID 50 ) of sw/ia/31, at mean titers of , and 10 2 TCID 50 /g on days 2, 3 and 4 p.i., respectively (Fig. 1C). The presence of virus at three time points following infection indicate that sw/ia/31 virus is capable of neuroinvasion and replication in the central nervous system. Clinical illness and level of viral replication in ferrets None of the viruses caused lethargy, sneezing, ruffled fur, decreased interest in food, or nasal discharge in ferrets and weight loss was not observed (Fig. 2A). A slight increase in body temperature was detected on day 3 especially with sw/ia/31 infection, however the temperatures remained within the normal range for ferrets (Fig. 2B). Both the 2009 H1N1 viruses (CA/7/09 and CA/4/09) and Fig. 1. Level of replication of H1N1 viruses in mice. Groups of 4 mice were inoculated i.n. with 10 5 TCID 50 /50μl of CA/7/09, CA/4/09, NJ/76, and sw/ia/31 viruses. Virus titers in the nasal turbinates (A), lungs (B), and brains (C) of 4 mice per group sacrificed on days 2 (black bars), 3 (open bars), 4 (grey bars), and 7 (hatch bars) p.i., respectively are expressed as mean±se log 10 TCID 50 /g of tissue. The dashed horizontal line indicates the lower limit of detection. the classical swine H1N1 viruses (sw/ia/31 and NJ/76) replicated to high titers in the upper respiratory tract of ferrets on 1, 3, and 5 days p. i. (Fig. 2C). With the exception of the NJ/76 virus, all of the H1N1 viruses replicated to high titers in the lower respiratory tract (Fig. 2D); peak replication of the CA/7/09 virus was detected on day 3 and CA/4/09 and sw/ia/31 viruses on day 5 p.i. The peak titer of the NJ/76 virus was statistically significantly lower than the other H1N1 viruses (pb0.05, Kruskal Wallis). Virus was not detected in the brain of ferrets inoculated with these viruses (data not shown). Effect of prior infection with H1N1 viruses on subsequent challenge with CA/7/09 virus in mice In order to assess the extent of homologous and heterologous protection provided by these antigenically related viruses, we evaluated the effect of prior infection with 2009 pandemic H1N1 viruses (CA/7/09 and CA/4/09) or classical swine influenza viruses (NJ/76 and sw/iw/31) on subsequent challenge with the CA/7/09 virus. Following primary infection with H1N1 viruses, serum neutralizing antibody titers against the CA/7/09 virus and classical

3 130 J.-Y. Min et al. / Virology 408 (2010) Fig. 2. Evaluation of clinical signs and viral replication following inoculation of H1N1 viruses in ferrets. Groups of 3 ferrets were inoculated i.n. with 10 6 TCID 50 /0.2ml of H1N1 CA/7/ 09 ( ), CA/4/09 (*), NJ/76 ( ), sw/ia/31( ) viruses. Mock-infected ferrets ( ) received L-15. Percentage change in body weight (A) and average body temperature (B) following i. n. inoculation of each indicated virus were monitored daily for 14 days in ferrets. Virus titers in the nasal turbinates (C) and lungs (D) of 3 ferrets per group sacrificed on days 1 (black bars), 3 (open bars), and 5 (grey bars) p.i., respectively are expressed as mean ±SE log 10 TCID 50 /g of tissue. The dashed horizontal line indicates the lower limit of detection. swine viruses (NJ/76 and sw/iw/31 viruses) were measured in sera collected on day 25 post-inoculation (Table 1) and the extent of replication of the CA/7/09 challenge virus is presented in Fig. 3. Six of the eight mice inoculated with the CA/7/09 virus as the primary infection were euthanized between days 7 and 10 post-inoculation due to severe weight loss and only two mice in the group survived long enough for challenge; subsequent studies (data not shown) indicate that these mice received a dose that was approximately the LD 50 of the CA/7/09 virus ( TCID 50 ). Serologic analysis was therefore limited to the sera from the two surviving mice. Because CA/ 7/09 and CA/4/09 viruses are antigenically similar, we used the CA/ 7/09 virus for serologic assays. Primary infection with each of the H1N1 viruses elicited a modest to robust homologous antibody response in 100% of the mice (Table 1, titers in bold). The NJ/76 virus induced a lower homologous neutralizing antibody response than the other H1N1 viruses, though the virus replicated to comparable titers in the lungs of mice. Primary infection with CA/7/09 and CA/4/09 viruses provided complete protection from subsequent homologous challenge in both the upper and lower respiratory tract of mice (Fig. 3A and B). Primary infection with NJ/76 and sw/ia/31 viruses elicited low titers of cross-neutralizing antibody against the CA/7/09 virus in 7 of 8 mice. Consistent with the low homologous neutralizing antibody response, the NJ/76 virus induced a lower titer of crossneutralizing antibody response than the sw/ia/31 virus. Although the level of cross-neutralizing antibody was low, primary infection with sw/ia/31 virus conferred significant protection from CA/7/09 virus Table 1 Serum neutralizing antibodies elicited in mice following intranasal infection with H1N1 viruses. Primary infection a Serum neutralizing antibody response against indicated virus CA/7/09 NJ/76 sw/ia/31 GMT (range) b # with detectable antibody/total c GMT (range) # with detectable antibody/total GMT (range) # with detectable antibody/total CA/7/ d 2/2 ND e ND e CA/4/ ( ) 8/8 ND e ND e NJ/76 34 (10 63) 7/8 58 (20 113) 8/8 16 (10 25) 3/8 sw/ia/31 41 (10 101) 7/8 29 (16 57) 8/8 281 ( ) 8/8 a Mice received 10 5 TCID 50 of indicated virus i.n. and serum was obtained on day 25 p.i. b Values represent geometric mean antibody titers from 2-8 mice per group. Antibodies were not detectable in pre-infection sera. Homologous antibody titers are in bold. An undetectable serum neutralizing antibody titer was assigned a value of 10. c Numbers of mice elicited neutralizing antibody response from each group are indicated. d Serum neutralizing antibody titer in two of the eight mice in the group that survived following primary infection with CA/7/09. e ND: not determined.

4 J.-Y. Min et al. / Virology 408 (2010) none induced cross-reactive antibodies against sw/ia/31. The sw/ia/ 31 virus elicited low titers of cross-neutralizing antibodies against the CA/7/09 pandemic H1N1 virus (Table 2) and a cross-hai antibody response was detected in 1 of 3 animals. These data indicated that sw/ IA/31 was antigenically distinct from the 2009 pandemic H1N1 virus. The significance of cross-hai and cross-neutralizing antibody titers that exceed homologous titers is unknown. Primary infection with each of the H1N1 viruses, including sw/ia/31 provided robust protection from replication of the CA/7/09 virus in the respiratory tract of ferrets; the titer of the challenge virus was below the limit of detection when assayed on day 5 post-challenge. The titer of the challenge virus was measured on day 5 post-challenge because virus titers in the lungs of ferrets on day 5 were similar to titers achieved on day 3 (Fig. 2D). The titer of the challenge virus in the lungs and nasal turbinates of mock-infected animals was TCID 50 /g and TCID 50 /g, respectively and the absence of detectable virus in the other groups was statistically significant (pb0.05, Mann Whitney U test). Discussion Fig. 3. Effect of prior infection with H1N1 viruses on subsequent challenge with CA/7/ 09 virus in mice. Groups of 4 mice were inoculated i.n. with 10 5 TCID 50 /50μl of CA/7/09 (horizontal hatch bars), CA/4/09 (grey bars), NJ/76 (diagonal hatch bars), and sw/ia/ 31 (open bars) viruses. Mock-infected mice (black bars) received L-15. On day 28 p.i., the mice were challenged with 10 5 TCID 50 /50μl of CA/7/09 virus. Virus titers in the nasal turbinates (A) and lungs (B) of 4 mice per group sacrificed on 2 and 4 days postchallenge are expressed as mean±se log 10 TCID 50 /g of tissue. The dashed horizontal line indicates the lower limit of detection. challenge in the upper respiratory tract of mice on 2 days postchallenge (Fig. 3A) (pb0.05 Kruskal Wallis, compared with mockinfected animals) and NJ/76 virus infection resulted in a reduction in virus titer compared with mock-infected mice, though the difference did not achieve statistical significance. In the lower respiratory tract, primary infection with sw/ia/31 and NJ/76 viruses conferred partial protection; the reduction in titer was statistically significant for sw/ IA/31 compared with mock-infected animals (pb0.05, Kruskal Wallis) (Fig. 3B). Effect of prior infection with H1N1 viruses on subsequent challenge with the CA/7/09 virus in ferrets The extent of homologous and heterologous protection provided by antigenically related H1N1 viruses was also evaluated in ferrets. Primary infection with the H1N1 viruses induced robust homologous HAI and neutralizing antibody responses (Table 2, titers in bold). The CA/7/09, CA/4/09 and NJ/76 viruses induced robust HAI and neutralizing antibodies against the CA/7/09 and NJ/76 viruses but In order to better understand the pathogenesis of the swine-origin pandemic 2009 H1N1 virus, several investigators have compared these viruses with seasonal influenza viruses. However, CA/7/09, the virus recommended by the WHO as the reference virus for vaccine development, has not been evaluated extensively in animal models. We evaluated the ability of this virus to cause disease in mice and ferrets and compared it with CA/4/09 (H1N1) as well as a progenitor classical swine H1N1 virus, sw/ia/31, and NJ/76, a swine influenza virus that caused a limited outbreak of human infections at a military camp in We used egg grown viruses, as have other investigators (Maines et al., 2009; Munster et al., 2009). Our goal was to establish the kinetics of replication in these models so that we could evaluate the extent of cross-protection conferred by classical swine H1N1 viruses against the 2009 pandemic H1N1 virus. Consistent with findings of other investigators (Itoh et al., 2009; Maines et al., 2009), we observed that the pandemic 2009 H1N1 influenza viruses (CA/7/09 and CA/4/09) replicated efficiently in the respiratory tract of mice without prior host adaptation. Statistically significant differences were not observed in the replication kinetics of the 2009 pandemic and classical swine H1N1 viruses in either the upper or lower respiratory tract of mice. The replication kinetics and pathogenicity of the swine-origin pandemic 2009 H1N1 viruses in ferrets in our study were also consistent with findings of others (Itoh et al., 2009; Maines et al., 2009; Munster et al., 2009). However, the replication of the pandemic H1N1 influenza viruses in ferrets was not associated with significant signs or symptoms of illness in our experiments. In our study, female and male ferrets aged 8 12 weeks were inoculated intranasally with 10 6 TCID 50 of virus in a volume of 0.2 ml while other investigators who infected older ferrets with a larger inoculum reported clinical illness (Maines et al., 2009). We used a smaller volume of inoculum because we have previously shown that intranasal administration of virus in larger volume Table 2 Serum neutralizing and HAI antibodies elicited in ferrets following intranasal infection with H1N1 viruses. Primary infection a Geometric mean (range) serum neutralizing antibody titers against indicated virus b Geometric mean (range) serum HAI antibody titers against indicated virus c CA/7/09 NJ/76 sw/ia/31 CA/7/09 NJ/76 sw/ia/31 CA/7/ ( ) 2560 ( ) (80 160) 5 CA/4/ ( ) 941 ( ) ( ) 80 (40 160) 5 NJ/ ( ) 2463 ( ) (40 160) 127 (80 160) 5 sw/ia/31 37 (32 40) 1881 ( ) 1280 ( ) 8 (5 20) 64 (40 160) 101 (40 320) a Ferrets received 10 6 TCID 50 of the virus i.n. and serum was obtained 4 weeks later. Values represent geometric mean antibody titers from three ferrets per group. Antibodies were not detectable in pre-infection sera. Homologous antibody titers are in bold. Ranges of antibody titers are in parentheses. b An undetectable serum neutralizing antibody titer was assigned a value of 10. c An undetectable HAI antibody titer was assigned a value of 5.

5 132 J.-Y. Min et al. / Virology 408 (2010) (0.5 ml) can result in a pulmonary inflammatory response in the absence of significant local replication of the inoculated virus (Jin et al., 2007). In both mice and ferrets, the pandemic 2009 H1N1 influenza viruses induced a robust homologous antibody response. Although NJ/76 and sw/ia/31 viruses are antigenically different from the CA/ 7/09 virus (Garten et al., 2009), primary infection with the classical swine H1N1 viruses provided partial protection from subsequent challenge with the CA/7/09 virus in mice. Despite the relatively low degree of amino acid sequence homology between the HAs of the pandemic 2009 H1N1 viruses and the classical swine viruses (CA/7/ 09 vs. NJ/76 and sw/ia/31 91% and 90%, respectively), primary infection with the classical swine H1N1 viruses elicited some crossreactive neutralizing activity and provided robust protection from subsequent challenge with the CA/7/09 virus in ferrets. Primary infection with influenza viruses stimulates mucosal and systemic antibodies as well as cellular immune responses and these responses contribute to protection. However, our analysis was limited to serum antibody responses, which are the most well established correlate of protection. The difference in the level of cross-protection conferred by the swine influenza viruses in mice and ferrets in our study are consistent with the level of cross-reactive antibodies induced in the two species; higher titers of cross reactive antibodies were induced in ferrets associated with robust protection from challenge while lower titers of cross reactive antibodies in mice were associated with partial protection. Our findings are consistent with the observations of Manicassamy et al., 2010 and Kash et al., 2010 who found that antibodies elicited against 1918-like or classical swine H1N1 vaccines fully protect mice from lethal challenge with the 2009 pandemic H1N1 virus. A proportion of people, especially older individuals, have antibodies that cross-react with the 2009 pandemic H1N1 virus (2009b; Greenberg et al., 2009; Hancock et al., 2009; Itoh et al., 2009; Kash et al., 2010; McCullers et al., 2010); these individuals were likely exposed to H1N1 viruses that were more closely related to classical swine H1N1 influenza viruses than recent seasonal H1N1 viruses are. The epidemiology of the 2009 H1N1 pandemic shows that the elderly are less affected by severe morbidity and mortality (Chowell et al., 2009), suggesting that they are protected from severe disease by cross-reactive immunity. Sera from a small sample of individuals who had received the swine flu vaccine in 1976 had cross-reactive antibodies to the 2009 H1N1 virus (Hancock et al., 2009). Our findings in animal models support these observations and suggest that the elderly and the ~45 million people who had prior infection with classical swine influenza virus and who received the swine flu vaccine in 1976 would be protected from severe disease caused by the pandemic 2009 H1N1 influenza viruses (CA/7/09). Materials and methods Viruses The swine-origin 2009 H1N1 viruses, A/California/7/2009 (CA/7/ 09) and A/California/4/2009 (CA/4/09) used in this study were kindly provided by Drs. Ruben Donis and Alexander Klimov from the Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA. The A/swine/Iowa/1931 (sw/ia/31) (H1N1) and A/ New Jersey/8/1976 (NJ/76) (H1N1) viruses were kindly provided by Drs. Jeffery Taubenberger and Brian Murphy, respectively, from the National Institutes of Health, Bethesda, MD. The CA/4/09 virus was amplified in Madin-Darby canine kidney (MDCK) cells and the other viruses were propagated in the allantoic cavity of 9 to 11-day old embryonated specific pathogen-free (SPF) hen's eggs (Charles River, Wilmington, MA), incubated at 37 C, harvested 48 h post-inoculation and tested for hemagglutinating activity. Infectious allantoic fluids were pooled, divided into aliquots and stored at 80 C until use. The fifty percent tissue culture infectious dose (TCID 50 ) for each virus was determined by serial titration of virus in MDCK cells and calculated by the method developed by Reed and Muench (1938). Animal experiments were approved by the National Institutes of Health Animal Care and Use Committee. Evaluation of viral replication in the respiratory tract of mice and antibody response to the H1N1 viruses in mice Level of replication. To evaluate the level of replication of the viruses in the respiratory tract of mice, we administered 50μl containing 10 5 TCID 50 of each of the H1N1 viruses or 50μl of Leibovitz-15 (L-15, Invitrogen-GIBCO) intranasally (i.n.) to 4- to 6-week-old female BALB/c mice (Taconic Farms, Inc., Germantown, NY) lightly anesthetized with isoflurane. Based on previous data (Memoli et al., 2009) establishing the lethality of sw/ia/31 for mice, one group of mice also received 50 μl of 10 TCID 50 of sw/ia/31. On days 2, 3, 4 and 7 following inoculation, lungs, nasal turbinates and brains (including olfactory bulbs) were harvested from 4 mice in each group. In the mice that received 10 5 TCID 50 of sw/ia/31, tissues were harvested on days 2, 3 and 4 following inoculation; in mice that received 10 TCID 50 of sw/ia/31, tissues were harvested on days 2, 4 and 7 following inoculation. Organs were weighed and homogenized in L-15 medium (Invitrogen-GIBCO) containing antibiotic antimycotic (penicillin, streptomycin, and amphotericin B) (Invitrogen-GIBCO) to make 5% (wt/vol) (nasal turbinates) or 10% (wt/vol) (lungs, brains) tissue homogenates. Tissue homogenates were clarified by centrifugation and titrated in 24- and 96-well tissue culture plates containing MDCK cell monolayers and TCID 50 was calculated by the method developed by Reed and Munch (Reed and Muench, 1938). Titers are expressed as log 10 TCID 50 /g of tissue. Evaluation of antibody response. Neutralizing antibody titers in pre and post-infection (p.i.) sera were determined in a microneutralization (MN) assay as described earlier (Joseph et al., 2008). Serial twofold dilutions of serum were prepared starting from a 1:10 or 1:20 dilution following receptor destroying enzyme (RDE) treatment and heat-inactivation. Equal volumes of serum and virus were mixed and incubated for 60 min at room temperature. The residual infectivity of the virus serum mixture was determined in MDCK cells in four replicates for each dilution. Neutralizing antibody titers were defined as the reciprocal of the highest dilution of serum that completely neutralized the infectivity of 100 TCID 50 of the virus as determined by the absence of CPE at day 4, as previously described (Joseph et al., 2008). The small volume of mouse sera precluded testing in a hemagglutination inhibition (HAI) assay. Evaluation of viral replication and antibody response to H1N1 viruses in ferrets Level of replication. The ability of H1N1 viruses to replicate in ferrets was compared in groups of 8- to 12-week-old ferrets (Triple F Farms, Sayre, PA) that were seronegative to seasonal H3N2, H1N1 and swineorigin 2009 H1N1 viruses. Each ferret was lightly anesthetized with isoflurane and inoculated i.n. with 10 6 TCID 50 of each of the H1N1 viruses in a volume of 0.2 ml (0.1 ml per nostril). On days 1, 2, and 3 post-inoculation, groups of three ferrets were euthanized and nasal turbinates, left lower lobe of the lung, and portions from the anterior, middle and posterior part of the brain were harvested and 10% w/v tissue homogenates were titrated on MDCK monolayers, as previously described (Joseph et al., 2008). Titers are expressed as log 10 TCID 50 /g of tissue.

6 J.-Y. Min et al. / Virology 408 (2010) Evaluation of clinical signs. In a separate study, groups of three ferrets were infected i.n. with each of the H1N1 viruses to evaluate symptoms/ clinical signs and antibody responses to the H1N1 viruses. The infected animals were monitored daily for 14 days for clinical signs of influenza infection, body temperature and weight. Ferrets were evaluated for nasal symptoms including nasal rattling, sneezing, nasal discharge, and mouth breathing and level of activity (Reuman et al., 1989). Evaluation of antibody response. Serum samples collected prior to dosing and on days 14 and 28 p.i. were tested for HAI antibodies by standard methods using 4 HA units of virus in V-bottom 96-well microtiter plates with 0.5% turkey erythrocytes. Neutralizing antibody titers were also evaluated in a microneutralization assay, as described above. Cross protection studies in mice Groups of four 6- to 8-week-old BALB/c mice (Taconic Farms, Inc., Germantown, NY) were infected with 50 μl containing 10 5 TCID 50 of H1N1 viruses or L-15 medium (mock-infected) and were challenged with 50 μl containing 10 5 TCID 50 of the CA/7/09 virus i.n. 27 days later. The nasal turbinates, lungs, and brain tissue were harvested from these mice at days 2 and 4 following challenge and were homogenized and virus titers were determined in MDCK cells. Cross protection studies in ferrets Groups of three 8- to 12-week-old ferrets infected with 10 6 TCID 50 of H1N1 viruses or L-15 medium (mock-infected) were challenged with 10 5 TCID 50 of the CA/7/09 virus i.n. 28 days later. The nasal turbinates, lungs, and brain tissue were harvested from these ferrets 5 days following challenge and were homogenized and virus titers were determined in MDCK cells. We selected this time point for harvest of tissues because the difference in titer on days 3 and 5 was not statistically significant and day 5 was a later time point. Acknowledgments We thank Jadon Jackson and the staff of the Comparative Medicine Branch, NIAID for technical support for animal studies performed at the NIH. We thank Dr. Catherine Luke for invaluable discussion throughout this work. This research was supported in part by the Intramural Research Program of the NIH, NIAID. References Chowell, G., Bertozzi, S.M., Colchero, M.A., Lopez-Gatell, H., Alpuche-Aranda, C., Hernandez, M., Miller, M.A., Severe respiratory disease concurrent with the circulation of H1N1 influenza. N Engl J. Med. 361 (7), Dowdle, W.R., Pandemic influenza: confronting a re-emergent threat. The 1976 experience. J. Infect. Dis. 176 (Suppl 1), S69 S72. Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A., Sessions, W.M., Xu, X., Skepner, E., Deyde, V., Okomo-Adhiambo, M., Gubareva, L., Barnes, J., Smith, C.B., Emery, S.L., Hillman, M.J., Rivailler, P., Smagala, J., de Graaf, M., Burke, D.F., Fouchier, R.A., Pappas, C., Alpuche-Aranda, C.M., Lopez-Gatell, H., Olivera, H., Lopez, I., Myers, C.A., Faix, D., Blair, P.J., Yu, C., Keene, K.M., Dotson Jr., P.D., Boxrud, D., Sambol, A.R., Abid, S.H., St George, K., Bannerman, T., Moore, A.L., Stringer, D.J., Blevins, P., Demmler-Harrison, G.J., Ginsberg, M., Kriner, P., Waterman, S., Smole, S., Guevara, H.F., Belongia, E.A., Clark, P.A., Beatrice, S.T., Donis, R., Katz, J., Finelli, L., Bridges, C.B., Shaw, M., Jernigan, D.B., Uyeki, T.M., Smith, D.J., Klimov, A.I., Cox, N.J., Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325 (5937), Gaydos, J.C., Hodder, R.A., Top Jr., F.H., Soden, V.J., Allen, R.G., Bartley, J.D., Zabkar, J.H., Nowosiwsky, T., Russell, P.K., Swine influenza A at Fort Dix, New Jersey (January February 1976). I. Case finding and clinical study of cases. J. Infect. Dis. 136 (Suppl), S356 S362. Greenberg, M.E., Lai, M.H., Hartel, G.F., Wichems, C.H., Gittleson, C., Bennet, J., Dawson, G., Hu, W., Leggio, C., Washington, D., Basser, R.L., Response after one dose of a monovalent influenza A (H1N1) 2009 Vaccine Preliminary Report. N Engl J. Med. 361 (25), Hancock, K., Veguilla, V., Lu, X., Zhong, W., Butler, E.N., Sun, H., Liu, F., Dong, L., Devos, J.R., Gargiullo, P.M., Brammer, T.L., Cox, N.J., Tumpey, T.M., Katz, J.M., Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus. N Engl J. Med. 361 (20), Hay, A.J., Gregory, V., Douglas, A.R., Lin, Y.P., The evolution of human influenza viruses. Philos. Trans. R. Soc. Lond. B Biol. Sci. 356 (1416), Hodder, R.A., Gaydos, J.C., Allen, R.G., Top Jr., F.H., Nowosiwsky, T., Russell, P.K., Swine influenza A at Fort Dix, New Jersey (January February 1976). III. Extent of spread and duration of the outbreak. J. Infect. Dis. 136 (Suppl), S369 S375. Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y., Tamura, D., Sakai-Tagawa, Y., Noda, T., Sakabe, S., Imai, M., Hatta, Y., Watanabe, S., Li, C., Yamada, S., Fujii,K.,Murakami,S.,Imai,H.,Kakugawa,S.,Ito,M.,Takano,R.,Iwatsuki-Horimoto,K., Shimojima, M., Horimoto, T., Goto, H., Takahashi, K., Makino, A., Ishigaki, H., Nakayama, M.,Okamatsu,M.,Warshauer,D.,Shult,P.A.,Saito,R.,Suzuki,H.,Furuta,Y.,Yamashita, M., Mitamura, K., Nakano, K., Nakamura, M., Brockman-Schneider, R., Mitamura, H., Yamazaki,M.,Sugaya,N.,Suresh,M.,Ozawa,M.,Neumann,G.,Gern,J.,Kida,H., Ogasawara, K., Kawaoka, Y., In vitro and in vivo characterization of new swineorigin H1N1 influenza viruses. Nature 460 (7258), Jin, H., Manetz, S., Leininger, J., Luke, C., Subbarao, K., Murphy, B., Kemble, G., Coelingh, K.L., Toxicological evaluation of live attenuated, cold-adapted H5N1 vaccines in ferrets. Vaccine 25 (52), Joseph, T., McAuliffe, J., Lu, B., Vogel, L., Swayne, D., Jin, H., Kemble, G., Subbarao, K., A live attenuated cold-adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets. Virology 378 (1), Kash, J.C., Qi, L., Dugan, V.G., Jagger, B.W., Hrabal, R.J., Memoli, M.J., Morens, D.M., Taubenberger, J.K., Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus. Influenza Other Resp. Viruses 4 (3), Kendal, A.P., Goldfield, M., Noble, G.R., Dowdle, W.R., Identification and preliminary antigenic analysis of swine influenza-like viruses isolated during an influenza outbreak at Fort Dix, New Jersey. J. Infect. Dis. 136 (Suppl), S381 S385. Maines,T.R.,Jayaraman,A.,Belser,J.A.,Wadford,D.A.,Pappas,C.,Zeng,H.,Gustin, K.M., Pearce, M.B., Viswanathan, K., Shriver, Z.H., Raman, R., Cox, N.J., Sasisekharan, R., Katz, J.M., Tumpey, T.M., Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science 325 (5939), Manicassamy, B., Medina, R.A., Hai, R., Tsibane, T., Stertz, S., Nistal-Villan, E., Palese, P., Basler, C.F., Garcia-Sastre, A., Protection of mice against lethal challenge with 2009 H1N1 influenza A virus by 1918-like and classical swine H1N1 based vaccines. PLoS Pathog. 6 (1), e McCullers, J.A., Van De Velde, L.A., Allison, K.J., Branum, K.C., Webby, R.J., Flynn, P.M., Recipients of vaccine against the 1976 swine flu have enhanced neutralization responses to the 2009 novel H1N1 influenza virus. Clin. Infect. Dis. 50 (11), Memoli, M.J., Tumpey, T.M., Jagger, B.W., Dugan, V.G., Sheng, Z.M., Qi, L., Kash, J.C., Taubenberger, J.K., An early 'classical' swine H1N1 influenza virus shows similar pathogenicity to the 1918 pandemic virus in ferrets and mice. Virology 393 (2), MMWR Morb Mortal Wkly Rep (2009a). Outbreak of swine-origin influenza A (H1N1) virus infection - Mexico, March-April (17), MMWR Morb Mortal Wkly Rep (2009b). Serum cross-reactive antibody response to a novel influenza A (H1N1) virus after vaccination with seasonal influenza vaccine. 58(19), MMWR Morb Mortal Wkly Rep (2009c). Swine influenza A (H1N1) infection in two children Southern California, March-April (15), Munster, V.J., de Wit, E., van den Brand, J.M., Herfst, S., Schrauwen, E.J., Bestebroer, T.M., van de Vijver, D., Boucher, C.A., Koopmans, M., Rimmelzwaan, G.F., Kuiken, T., Osterhaus, A.D., Fouchier, R.A., Pathogenesis and transmission of swineorigin 2009 A(H1N1) influenza virus in ferrets. Science 325 (5939), Myers, K.P., Olsen, C.W., Gray, G.C., Cases of swine influenza in humans: a review of the literature. Clin. Infect. Dis. 44 (8), O'Brien, R.J., Noble, G.R., Easterday, B.C., Kendal, A.P., Shasby, D.M., Nelson, D.B., Hattwick, M.A., Dowdle, W.R., Swine-like influenza virus infection in a Wisconsin farm family. J. Infect. Dis. 136 (Suppl), S390 S396. Reed, L.J., Muench, H., A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, Reuman, P.D., Keely, S., Schiff, G.M., Assessment of signs of influenza illness in the ferret model. J. Virol. Methods 24 (1 2), Robinson, J.L., Lee, B.E., Patel, J., Bastien, N., Grimsrud, K., Seal, R.F., King, R., Marshall, F., Li, Y., Swine influenza (H3N2) infection in a child and possible community transmission, Canada. Emerg. Infect. Dis. 13 (12), Rota, P.A., Rocha, E.P., Harmon, M.W., Hinshaw, V.S., Sheerar, M.G., Kawaoka, Y., Cox, N. J., Smith, T.F., Laboratory characterization of a swine influenza virus isolated from a fatal case of human influenza. J. Clin. Microbiol. 27 (6), Shinde, V., Bridges, C.B., Uyeki, T.M., Shu, B., Balish, A., Xu, X., Lindstrom, S., Gubareva, L. V., Deyde, V., Garten, R.J., Harris, M., Gerber, S., Vagasky, S., Smith, F., Pascoe, N., Martin, K., Dufficy, D., Ritger, K., Conover, C., Quinlisk, P., Klimov, A., Bresee, J.S., Finelli, L., Triple-reassortant swine influenza A (H1) in humans in the United States, N Engl J. Med. 360 (25), Thompson, R.L., Sande, M.A., Wenzel, R.P., Hoke Jr., C.H., Gwaltney Jr., J.M., Swineinfluenza infection in civilians. Report of two cases. N. Engl. J. Med. 295 (13), Top Jr., F.H., Russell, P.K., Swine influenza A at Fort Dix, New Jersey (January February 1976). IV. Summary and speculation. J. Infect. Dis. 136 (Suppl), S376 S380. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., Evolution and ecology of influenza A viruses. Microbiol. Rev. 56 (1), Wells, D.L., Hopfensperger, D.J., Arden, N.H., Harmon, M.W., Davis, J.P., Tipple, M.A., Schonberger, L.B., Swine influenza virus infections. Transmission from ill pigs to humans at a Wisconsin agricultural fair and subsequent probable person-toperson transmission. JAMA 265 (4),