Prevalence, genetics, and transmissibility in ferrets ofEurasian avian-like H1N1 swine influenza virusesHuanliang Yanga,1, Yan Chena,1, Chuanling Qiaoa,1, Xijun Hea, Hong Zhoub, Yu Sunb, Hang Yina, Shasha Menga,Liping Liua, Qianyi Zhanga, Huihui Konga, Chunyang Gua, Chengjun Lia, Zhigao Bua, Yoshihiro Kawaokac,2,and Hualan Chena,2

aState Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, People’sRepublic of China; bThe First Hospital of Harbin Medical University, Harbin 150070, China; and cDivision of Virology, Department of Microbiology andImmunology, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo108-8639, Japan

Contributed by Yoshihiro Kawaoka, November 19, 2015 (sent for review September 2, 2015; reviewed by Ruben Donis and Hans-Dieter Klenk)

Pigs are important intermediate hosts for generating novel in-fluenza viruses. The Eurasian avian-like H1N1 (EAH1N1) swineinfluenza viruses (SIVs) have circulated in pigs since 1979, andhuman cases associated with EAH1N1 SIVs have been reported inseveral countries. However, the biologic properties of EAH1N1SIVs are largely unknown. Here, we performed extensive in-fluenza surveillance in pigs in China and isolated 228 influenzaviruses from 36,417 pigs. We found that 139 of the 228 strainsfrom pigs in 10 provinces in China belong to the EAH1N1 lineage.These viruses formed five genotypes, with two distinct antigenicgroups, represented by A/swine/Guangxi/18/2011 and A/swine/Guangdong/104/2013, both of which are antigenically and genet-ically distinct from the current human H1N1 viruses. Importantly,the EAH1N1 SIVs preferentially bound to human-type receptors,and 9 of the 10 tested viruses transmitted in ferrets by respiratorydroplet. We found that 3.6% of children (≤10 y old), 0% of adults,and 13.4% of elderly adults (≥60 y old) had neutralization anti-bodies (titers ≥40 in children and ≥80 in adults) against theEAH1N1 A/swine/Guangxi/18/2011 virus, but none of them hadsuch neutralization antibodies against the EAH1N1 A/swine/Guangdong/104/2013 virus. Our study shows the potential ofEAH1N1 SIVs to transmit efficiently in humans and suggests thatimmediate action is needed to prevent the efficient transmissionof EAH1N1 SIVs to humans.

swine influenza | H1N1 | Eurasian lineage | evolution | pandemic potential

Pigs play a pivotal role in the ecology of influenza A viruses,being regarded as intermediate hosts for the generation of

novel and potentially dangerous influenza viruses for humans.Cellular receptors containing α-2,3–linked sialic acids (Sias)(avian-like receptors) and α-2,6–linked Sias (human-like recep-tors) in the pig trachea favor the productive replication of virusesfrom both the avian and mammalian lineages (1). Influenzaviruses of the subtypes H1N1, H1N2, and H3N2 are circulatingin pigs globally (2). Two lineages of H1N1 swine influenzaviruses (SIVs), classical H1N1 SIVs and Eurasian avian-likeH1N1 (EAH1N1) SIVs, have been circulating in pigs since 1918and 1979, respectively (3, 4). The classical H1N1 SIVs emergedin humans as a reassortant (2009/H1N1) and caused the 2009H1N1 influenza pandemic (5). The EAH1N1 SIVs have beendetected in pigs in many Eurasian countries (6) and havecaused several human infections in European countries andalso in China (7–11), where a fatal case was reported (11).EAH1N1 SIVs are reported to be most prevalent in pigs thathave been brought into Hong Kong since 2005 (12). However,the evolution and biologic properties of the EAH1N1 SIVs arelargely unknown.China is the largest pork-producing country in the world. Pigs

in China are not vaccinated against influenza, and therefore,influenza viruses can spread freely once they are introduced intopig herds. In this study, we performed active surveillance in pigs

and found that the EAH1N1 SIVs are predominant in the pigpopulation in China; we further found that the EAH1N1 SIVspose an imminent threat with regard to their ability to cause ahuman influenza pandemic.

ResultsSurveillance of SIVs in China. Nasal swabs were collected from36,417 pigs in slaughterhouses and on farms in 24 provinces inChina from August 2010 to March 2013 (SI Appendix, Table S1);228 SIVs were isolated from these samples, including 180 H1N1,44 H1N2, 2 H3N2, and 2 H9N2 SIVs. Partial sequence analysisof the hemagglutinin (HA) and neuraminidase (NA) genes of the180 H1N1 SIVs further classified them as 139 EAH1N1, 39 2009/H1N1-like, and two classical H1N1 SIVs (SI Appendix, TableS1). EAH1N1 SIVs was detected in ten provinces (SI Appendix,Table S1 and Fig. S1). The 2009/H1N1 and H1N2 SIVs weredetected in four provinces, respectively, and the classical H1N1,H3N2, and H9N2 SIVs were each detected in only one province(SI Appendix, Table S1). These results indicate that the EAH1N1is the predominant SIV circulating in pigs in China.

Significance

Animal influenza viruses continue to challenge human publichealth; surveillance of these viruses and evaluation of theirpandemic potential are important for future influenza pan-demic preparedness. Pigs are intermediate hosts for novel in-fluenza generation and transfer to humans. Two lineages ofH1N1 swine influenza viruses (SIVs), classical H1N1 SIVs andEurasian avian-like H1N1 (EAH1N1) SIVs, have been circulatingin pigs since 1918 and 1979, respectively. The classical H1N1SIVs emerged in humans as a reassortant and caused the 2009H1N1 influenza pandemic. Here, we found that, after long-term evolution in pigs, the EAH1N1 SIVs have obtained thetraits to cause a human influenza pandemic.

Author contributions: H. Yang, Y.C., C.Q., Z.B., and H.C. designed research; H. Yang, Y.C.,C.Q., X.H., H.Z., Y.S., H. Yin, S.M., L.L., Q.Z., H.K., and C.G. performed research; H. Yang,Y.C., C.Q., C.L., Z.B., Y.K., and H.C. analyzed data; and H. Yang, Z.B., Y.K., and H.C. wrotethe paper.

Reviewers: R.D., Center for Disease Control; and H.-D.K., Philipps-Universitat Marburg.

Conflict of interest statement: Y.K. has received speaker’s honoraria from Toyama Chem-ical and Astellas Inc.; has received grant support from Chugai Pharmaceuticals, DaiichiSankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., and Otsuka Pharma-ceutical Co., Ltd; and is a founder of FluGen.

Data deposition: Virus sequence data from this study were deposited in the Global Ini-tiative on Sharing Avian Influenza Data database http://platform.gisaid.org/ (accessionnos. EPI554268–EPI554539).1H. Yang, Y.C., and C.Q. contributed equally to this work.2To whom correspondence may be addressed. Email: kawaokay@svm.vetmed.wisc.edu orchenhualan@caas.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1522643113/-/DCSupplemental.

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Genetics of EAH1N1 SIVs. We fully sequenced the genome of 40representative EAH1N1 SIVs that came from different farms.The HA gene of the 40 SIVs shared 92.7–100% identity at thenucleotide level and formed two phylogenetic groups (Fig. 1A).The NA gene of these SIVs shared 91.1–100% identity at thenucleotide level and formed three phylogenetic groups (SI Ap-pendix, Fig. S2A).The six internal genes of the EAH1N1 SIVs showed distinct

diversity, with the basic polymerase 2 (PB2), PB1, acidic poly-merase (PA), nucleoprotein (NP), matrix (M), and nonstructuralprotein (NS) genes of the 40 viruses sharing 82.9–100%, 84.1–100%, 83.7–100%, 82.4–100%, 93.6–100%, and 80.1–100%identity, respectively, at the nucleotide level. The PB2 and NSgenes each formed three groups (two of which originated fromEAH1N1 and the third from 2009/H1N1) in the phylogenetictrees (Fig. 1B and SI Appendix, Fig. S2 B and G), and the PB1,PA, NP, and M genes each formed two groups (one of whichoriginated from EAH1N1 and the other from 2009/H1N1) intheir phylogenetic trees (Fig. 1B and SI Appendix, Fig. S2 C–F).On the basis of this genomic diversity, we divided these

EAH1N1 SIVs into five genotypes (Fig. 1B). The SIVs in gen-otypes 2 and 5 bear six and five internal genes, respectively, from

2009/H1N1 viruses (Fig. 1B). The two SIVs in genotypes 4 and 5have NS genes derived from the North American H1N2 triplereassortant SIVs rather than from the EAH1N1 SIVs (SI Ap-pendix, Fig. S2G). The genotype 1 SIVs were detected in 10provinces, whereas the viruses in genotypes 2, 3, 4, and 5 wereonly detected in one or two provinces (Fig. 1B).To understand the biologic properties of these EAH1N1 SIVs,

we selected 10 strains (red text in Fig. 1B), including six SIVs ofgenotype 1 and one SIV from each of genotypes 2, 3, 4, and 5,and tested their antigenic properties, receptor-binding prefer-ence, virulence, and transmissibility in mammals.

Antigenic Analysis. The antigenic match between a vaccine andthe circulating viruses is one of the most important factors indetermining the efficacy of the vaccine. When the prevalenthuman influenza viruses show a greater than fourfold differencein hemagglutination inhibition (HI) assay titer from the vaccinestrain, the immunity induced by the vaccine does not prevent thecirculation of such viruses in humans (13). We therefore selectedferret antisera against 2009/H1N1 virus A/Sichuan/1/2009 (SC/1/09), which is from the current H1N1 human influenza vaccinelineage, and against A/swine/Guangxi/18/2011 (SW/GX/18/11)

Fig. 1. Genetic relationships among the HA genes and genotype evolution of EAH1N1 SIVs. (A) Phylogenetic tree of HA. The unrooted tree was based onnucleotides 33–1733. Sequences of viruses with names in black were downloaded from available databases; viruses with names in colors were sequencedin this study. Scale bar indicates the number of nucleotide substitutions per site. (B) Genotypes of the EAH1N1 SIVs. The eight gene segments are indicatedat the top of each bar. The colors of the bars represent the groups in the trees of Fig. 1A and SI Appendix, Fig. S2. Viruses shown in red were selected forall other tests.

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and A/swine/Guangdong/104/2013 (SW/GD/104/13), which con-tain the HA of group 1 and 2, respectively, for antigenic analysis.The antiserum against SC/1/09 cross-reacted with the

EAH1N1 SIVs bearing the HA of group 1 and 2 with titers four-to eightfold and 64–128-fold, respectively, lower than the ho-mologous titer. The antiserum against SW/GX/18/11 reactedwell with the EAH1N1 SIVs bearing the HA of group 1, but itscross-reactive titers with 2009/H1N1 and the EAH1N1 SIVsbearing the HA of group 2 were fourfold and 32–128-fold, re-spectively, lower than the homologous titer. The antiserumagainst SW/GD/104/13 reacted well with the EAH1N1 SIVsbearing the HA of group 2, but its cross-reactive titers with theEAH1N1 SIVs bearing the HA of group 1 and with 2009/H1N1were 32–64-fold and 128-fold, respectively, lower than the ho-mologous titer (Table 1). We found 23 amino acid differences inthe HA antigenic sites among these viruses (SI Appendix, TableS2). Which amino acid contributes to these observed antigenicdifferences has yet to be determined. Our results indicate thatthe EAH1N1 SIVs formed two distinct antigenic groups andboth groups show a striking antigenic difference from the 2009/H1N1 virus.

Receptor-Binding Preference. We tested the receptor-bindingspecificity of the EAH1N1 SIVs by using a solid-phase bindingassay with four different glycans as described previously (14, 15).The SC/1/09, and an H5N1 virus, A/duck/Anhui/1/2006, wereincluded for comparison. The SC/1/09 and all 10 EAH1N1 SIVsbound to α-2,6–linked Sias with high affinity and to α-2,3–linkedSias with very low affinity (SI Appendix, Fig. S3 A–J and L),whereas the H5N1 virus bound to α-2,3–linked Sias with highaffinity and to α-2,6–linked Sias with very low affinity (SI Ap-pendix, Fig. S3K). These results indicate that the EAH1N1 SIVspreferentially bind to the human-type receptor.

Replication and Virulence in Mice. We investigated the virulence inmice of the EAH1N1 SIVs as described previously (16). The 10EAH1N1 SIVs replicated in the lungs of mice, nine SIVs weredetected in the nasal turbinate of mice, and two were also de-tected in the kidneys (SI Appendix, Fig. S4). No virus was de-tected in the spleen or brain of any mice. Mice infected withthese SIVs lost 7–29.1% of their body weight, and two SIVs,A/swine/Guangdong/30/2013 (SW/GD/30/13) and A/swine/Guang-dong/306/2013 (SW/GD/306/13), killed 40% and 60% of themice, respectively (SI Appendix, Fig. S4).

Replication and Transmission of EAH1N1 SIVs in Ferrets. Two ferretswere inoculated intranasally (i.n.) with 106.0 50% egg infectiousdose (EID50) of each virus, and organs from each ferret were col-lected on day 4 postinoculation (p.i.) for virus titration in eggs. All10 of the viruses replicated in the nasal turbinate, tonsils, and tra-chea of the ferrets (Fig. 2). Virus was detected in all lung lobes ofthe ferrets inoculated with six of the viruses (Fig. 2 A, B, D, F, H,and J) but was not detectable in some of the lung lobes of ferretsinoculated with the other four viruses (Fig. 2 C, E, G, and I). Viruswas detected in the kidneys of one ferret inoculated with A/swine/Hunan/26/2010 (SW/HuN/26), A/swine/Jiangsu/49/2012 (SW/JS/49/12), and SW/GD/30/13 (Fig. 2 A, E, andH). Virus was also detectedin the brain of one ferret in each of the groups that were in-oculated with SW/JS/49/12 and A/swine/Tianjin/47/2011 (SW/TJ/47/11) (Fig. 2 E andG) and in two ferrets in each of the groups thatwere inoculated with A/swine/Henan/232/2011 (SW/HeN/232/11)and A/swine/Heilongjiang/27/2012 (SW/HLJ/27/12) (Fig. 2 Cand D).Macroscopic lesions were observed in the lungs of ferrets in-

oculated with all of the SIVs, except for those inoculated with SW/GD/104/13 (Fig. 3 A and B). Pathological studies indicated thatmost of the lungs showed mild damage after infection with SW/GD/104/13 (Fig. 3C), and viral antigen was not detected (Fig. 3D). Bycontrast, the lungs of the other nine virus-infected ferrets showedsevere bronchopneumonia (Fig. 3 E–M), and viral antigen wasdetected in the epithelial cells of the bronchus and alveoli (Fig. 3N).

Table 1. Antigenic analysis of the EAH1N1 SIVs

Virus HA group

Ferret antisera

SC/1/09 SW/GX/18/11 SW/GD/104/13

SC/1/09 2009/H1N1 1280 320 10SW/GX/18/11 1 320 1280 20SW/GD/104/13 2 10 40 1280Test virusesSW/HuN/26/10 1 160 640 40SW/TJ/47/11 1 320 320 20SW/HeN/232/11 1 320 640 40SW/HLJ/27/12 1 320 1280 40SW/JS/49/12 1 160 640 40SW/HuN/30/13 1 160 1280 20SW/GD/30/13 1 320 640 20SW/GD/306/13 2 20 10 640

Ferrets were inoculated intranasally with 106 EID50 of the indicated virus,and sera were collected 3 wk postinoculation. Homologous titers are under-lined and bolded.

Fig. 2. Replication of EAH1N1 SIVs in ferrets. Ferrets (n = 2 per group) wereinoculated i.n. with 106.0 EID50 of each virus, and the indicated organs fromeach ferret were collected on day 4 p.i. for virus titration in eggs. Each colorbar represents the virus titer from an individual animal. The dashed bluelines indicate the lower limit of detection. (A) SW/HuN/26/10, (B) SW/GX/18/11, (C) SW/HeN/232/11, (D) SW/HLJ/27/12, (E) SW/JS/49/12, (F) SW/HuN/30/13,(G) SW/TJ/47/11, (H) SW/GD/30/13, (I) SW/GD/104/13, and (J) SW/GD/306/13.

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Respiratory droplet transmission was assessed in ferrets asdescribed previously (15, 16). The highly transmissible SC/1/09was included as a control (14, 17). Virus was detected in all ofthe directly infected animals (Fig. 4 A–K). In the exposed groups,virus was not detected in any of the animals exposed to the SW/GD/306/13-inoculated ferrets (Fig. 4J), but virus was detected inone ferret exposed to the ferrets that had been inoculated withSW/HuN/26/10, SW/HLJ/27/12, SW/GD/104/13, and SW/GD/30/13(Fig. 4 A, D, H, and I) and in two ferrets exposed to the ferretsthat had been inoculated with SW/JS/49/12 (Fig. 4E). Impor-tantly, like the 2009/H1N1 SC/1/09 virus (Fig. 4K), four EAH1N1SIVs—SW/GX/18/11, SW/HeN/232/11, A/swine/Hunan/30/2013(SW/HuN/30/13), and SW/TJ/47/11—transmitted to all three ex-posed ferrets (Fig. 4 B, C, F, and G). The ferrets that were in-oculated with these EAH1N1 SIVs experienced a 0.2–18.6% bodyweight loss, and the exposed ferrets lost up to 21% of their bodyweight (Table 2 and SI Appendix, Table S4). Seroconversion oc-curred in all of the virus-inoculated animals and in all exposedanimals that were virus-positive (Table 2).

Human Immunity to the EAH1N1 Viruses. Serum HI antibody titersof 40 (18, 19), or microneutralization (MNT) antibody titers of40 in children or at least 80 in adults, are associated with no lessthan a 50% reduction in risk of infection or disease with in-fluenza viruses in human populations (20). To investigatewhether humans have any existing immune protection againstthe EAH1N1 SIVs, we tested sera collected from children (<10 yold, n = 55), adults (25–53 y old, n = 52), and elderly adults (≥60 yold, n = 52), against three viruses (SC/1/09, SW/GX/18/11, andSW/GD/104/13) for both HI and MNT antibodies as describedpreviously (20, 21). We found that only 9.1%, 5.5%, and 0% ofthe children; 7.7%, 11.5%, and 3.8% of the adults; and 1.9%,3.8%, and 0% of the elderly adults had HI antibody titers ≥40to SC/1/09, SW/GX/18/11, and SW/GD/104/13, respectively (SIAppendix, Table S5). In addition, 12.7%, 3.6%, and 0% of thechildren had MNT antibody titers ≥40, and 1.9%, 0%, and 0% ofthe adults and 0%, 13.4%, and 0% of the elderly adults had MNTantibody titers ≥80 to SC/1/09, SW/GX/18/11, and SW/GD/104/13,respectively (SI Appendix, Table S5). These results indicate thatthe preexisting immunity in the human population may not be ableto prevent the spread of the EAH1N1 SIVs.

DiscussionHere, we detected different subtypes of SIVs in pigs in Chinafrom 2010 to 2013 and found that the predominant strains arethe EAH1N1 SIVs, which have evolved into five different geno-types through accumulated mutations or the acquisition of dif-ferent internal gene segments from viruses in other lineages. Wefurther determined that the EAH1N1 SIVs have formed twodistinct antigenic groups and exhibit markedly different antige-nicity from that of the 2009/H1N1 pandemic virus. Most im-portantly, we found that these SIVs preferentially or exclusivelybind human-like receptors and that some of them can transmitefficiently between ferrets by respiratory droplet.Several amino acid changes in PB2 are important for the

replication or virulence of influenza viruses in mammals (22–30).

Fig. 3. Lung lesions caused by EAH1N1 SIVs in ferrets. Macroscopic lesions inthe lungs and focal consolidations in dark red were observed in the lobes offerrets inoculated with all of the EAH1N1 viruses (A; the image is from anSW/HuN/30/13-inoculated ferret), except for SW/GD/104/13, which did notcause obvious macroscopic lesions in the lungs of ferrets (B). Only mild his-topathological changes were observed in the lungs of SW/GD/104/13-inoculatedferrets (C) (H&E staining), and viral antigen was not detected (D) (immu-nohistochemical staining), whereas the lungs of ferrets inoculated with SW/HuN/26/10, SW/GX/18/11, SW/HeN/232/11, SW/HLJ/27/12, SW/JS/49/12, SW/HuN/30/13, SW/TJ/47/11, SW/GD/30/13, and SW/GD/306/13 viruses showedsevere pathological lesions (E–M) (H&E staining). Viral antigen was de-tected in the epithelial cells of the bronchus and alveoli by means of im-munohistochemical staining (N, from the lung sample of a ferret inoculatedwith SW/HuN/30/13 virus). Images C and E–M were taken at 100× magni-fication; images D and N were taken at 400× magnification.

Fig. 4. Respiratory droplet transmission of EAH1N1 SIVs in ferrets. Each linerepresents the virus titer from an individual animal. The dashed lines in-dicate the values from the inoculated animals; the solid lines indicate thevalues from the exposed animals. The dashed black lines indicate the lowerlimit of detection. (A) SW/HuN/26/10, (B) SW/GX/18/11, (C) SW/HeN/232/11,(D) SW/HLJ/27/12, (E) SW/JS/49/12, (F) SW/HuN/30/13, (G) SW/TJ/47/11, (H) SW/GD/30/13, (I) SW/GD/104/13, (J) SW/GD/306/13, and (K) SC/1/09.

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A detailed comparison of the amino acid differences among theEAH1N1 SIVs indicated that all of these viruses have mamma-lian-adapting mutations in their PB2 genes (SI Appendix, TableS3). Therefore, although the EAH1N1 viruses do not cause se-vere disease in pigs and have different replication and virulencephenotypes in animal models, they may be able to replicate welland possibly cause appreciable disease in humans, as observedduring the 2009/H1N1 influenza pandemic (31).The human influenza vaccination coverage in China is less

than 2% (32). Although we cannot attribute the seropositivity ofhumans against H1N1 viruses to vaccination or natural infection,the overall seropositive rate to the EAH1N1 SIVs in humans islow. Two doses of the seasonal trivalent inactivated influenzavaccine could not substantially improve the levels of cross-reactive antibodies to the EAH1N1 virus in humans (10). Thus,preexisting immunity in humans may not be sufficient to over-come EAH1N1 SIV infection.Numerous studies have evaluated the pandemic potential of

animal influenza viruses (14–17, 22, 33–39). Although H5N1mutants and reassortants have been shown to be transmissible inferrets or guinea pigs (14, 33, 35, 36), none of the naturally oc-curring H5N1 viruses is transmissible via respiratory droplet inthese animal models. Certain H7N9 viruses are transmissible inferrets (15, 40), but their circulation so far is geographically re-stricted. The Centers for Disease Control and Prevention in theUnited States developed a risk assessment tool to evaluate the

pandemic potential of different influenza strains (41). By usingthis tool, we found that the EAH1N1 SIVs got the highest riskscore among six different subtypes of viruses (SI Appendix, TableS6), suggesting that the EAH1N1 SIVs may pose the highestpandemic threat among the influenza viruses currently circulat-ing in animals.

MethodsThis study was carried out in accordance with the recommendations in theGuide for the Care and Use of Laboratory Animals of the Ministry of Scienceand Technology of China (42). The protocols were reviewed and approvedby the Committee on the Ethics of Animal Experiments of the Harbin Vet-erinary Research Institute of the Chinese Academy of Agricultural Sciences.All experiments with live H1N1 viruses were conducted within the enhancedanimal biosafety level 2-plus facilities in the Harbin Veterinary Research In-stitute of the Chinese Academy of Agricultural Sciences. The researchers whowork with mice and ferrets wear N95 masks and disposable overalls; theyshower on exiting the facility.

The detailed methods for this study are provided in SI Appendix.

ACKNOWLEDGMENTS. We thank Susan Watson for editing the manuscriptand the Consortium for Functional Glycomics (Scripps Research Institute)for providing the glycans. This work was supported by the NationalNatural Science Foundation of China (31302108 and 31521005), the Foodand Agriculture Organization of the United Nations (CHN/2013/001/LOA),and the Japan Initiative for Global Research Network on Infectious Diseasesfrom the Japan Agency for Medical Research and Development.

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Table 2. Replication, virulence, and transmission of EAH1N1 SIVs in ferrets

Virus (genotype)

Maximum bodytemperature increase, °C

Maximum body weightloss, %

Seroconversion (HI antibodytiter range)

Inoculated Exposed Inoculated Exposed Inoculated Exposed

SW/HuN/26/10 (1) 2.2 1.4 0.8 5.6 3/3 (640–1280) 1/3 (1280)SW/GX/18/11 (1) 1.7 0.9 17.9 21.0 3/3 (1280–2560) 3/3 (640-5120)SW/HeN/232/11 (1) 1.9 1.2 18.6 14.0 3/3 (1280–2560) 3/3 (1280–2560)SW/HLJ/27/12 (1) 1.0 0.6 15.2 11.7 3/3 (1280) 1/3 (2560)SW/JS/49/12 (1) 2.1 1.2 5.6 12.3 3/3 (1280–2560) 2/3 (1280)SW/HuN/30/13 (1) 1.5 1.2 3.4 9.4 3/3 (2560–5120) 3/3 (1280–5120)SW/TJ/47/11 (2) 1.4 1.0 12.9 7.1 3/3 (1280) 3/3 (640–1280)SW/GD/30/13 (3) 1.0 0.5 3.2 4.6 3/3 (1280) 1/3 (640)SW/GD/104/13 (4) 1.0 0.6 1.8 1.0 3/3 (1280) 1/3 (1280)SW/GD/306/13 (5) 1.6 0.6 10.3 0.0 3/3 (320–1280) 0/3SC/1/09 (2009/H1N1) 2.2 2.3 10.1 21.2 3/3 (320–1280) 3/3 (640–1280)

Data shown are from the animal in that group with the maximum body temperature increase or maximum body weight loss. Thedata for all of the animals in each group are reported in SI Appendix, Table S4. Seroconversion was confirmed from ferret serum thatwas collected on day 21 postinoculation.

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