pathogenesis and transmission of the novel swine origin influenza virus a(h1n1) after experimental...

7/28/2019 Pathogenesis and Transmission of the Novel Swine Origin Influenza Virus a(H1N1) After Experimental Infection of Pigs

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Pathogenesis and transmission of the novel swine origin influenza virus A/H1N1 after1

experimental infection of pigs23

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5

Elke Lange1, Donata Kalthoff

2, Ulrike Blohm

1, Jens P. Teifke

1, Angele Breithaupt

1, Christina6

Maresch1, Elke Starick

2, Sasan Fereidouni

2, Bernd Hoffmann

2, Thomas C. Mettenleiter

3,7

Martin Beer2, and Thomas W. Vahlenkamp

1,*8

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1Institute of Infectology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems;11

2Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems;12

3Institute of Molecular Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany13

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Runnung title: Novel influenza A/H1N1 in pigs17

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JGV Papers in Press. Published July 10, 2009 as doi:10.1099/vir.0.014480-0


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Influenza virus A/H1N1 currently causing a pandemic contains gene segments with30

ancestors in North American and Eurasian swine lineages. To get insights into virus31replication dynamics, clinical symptoms and virus transmission in pigs we infected32

animals intranasally with influenza virus A/Regensburg/D6/09/H1N1. The inoculated33

pigs started virus excretion in nasal swabs at 1 day p.i. onwards and developed generally34

mild symptoms including fever, sneezing, nasal discharge, and diarrhea. Contact pigs35

became infected, shed virus and developed clinical symptoms similar to the inoculated36

animals. Plasma samples of all animals remained negative for viral RNA. NP- and H1-37

specific antibodies could be detected by ELISA 7 days p.i. CD4+

T-cells became activated38

immediately after infection and both CD4+

and CD8+

T-cell populations expanded 3 to 739

days p.i. coinciding with clinical signs. Contact chicken remained uninfected as judged40

by the absence of virus excretion, clinical signs and seroconversion.41

42


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Influenza A/H1N1 viruses were first isolated from swine in 1930 (Shope, 1931). From 193043

to the late 1990s these classical swine influenza viruses (SIV) circulated in pigs in the US and44remained relatively stable. This relative antigenic stasis of classical influenza A/H1N1 viruses45

in swine until 1998, during the time when significant antigenic drift of influenza H1 viruses in46

humans was observed, created a substantial antigenic gap between classical swine and human47

seasonal H1 viruses. Thus, swine became a reservoir of influenza H1 viruses with the48

potential to cause significant respiratory disease or even pandemics in humans (Garten et al.49

2009).50

Sporadic cross-species transfer of swine and avian influenza viruses to humans has been51

documented repeatedly during the last decades. Despite the development of severe clinical52

signs and fatal pneumonia in some of the patients the infections lacked the critical capacity to53

efficiently spread from human to human in order to pose a threat for medical care (van Reeth,54

2007; Irvine and Brown, 2009). Since its identification in April 2009 a novel swine origin55

influenza virus A/H1N1 containing a unique combination of gene segments from both North56

American and Eurasian swine lineages has continued to circulate in humans (Cohen, 2009;57

Garten et al., 2009). Homology analyses between the novel influenza virus A/H1N1 and its58

nearest relatives indicated that it may have been circulating undetected for an extended period59

of time (Smith et al., 2009). As of July 3rd

2009, there have been 89921 laboratory confirmed60

i 100 i l i i h 380 d h A k d i f h61


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submitted to GenBank (Accession numbers: FN401574, FN401575, FN401576, FN401577,77

FN401578, FN401579, FN401580, FN401581). From day 1 post infection (p.i.) 3 nave pigs78and 5 nave chicken were housed together with the infected animals in direct contact without79

any cages in the same room. From all pigs oropharyngeal swabs were taken daily and EDTA-80

blood samples were obtained on days 1, 2, 3, 5, 7, 10, 14 and 21 p.i. From the chicken cloacal81

and oropharyngeal swabs were sampled daily and all animals were assessed for disease signs82

following a clinical score which included nasal discharge, sneezing, salivation, diarrhea,83

fever, emaciation, lid edema and/or compromised general condition.84

Real time RT-PCR analysis of the swab samples using primers (http://offlu.net) designed to85

specifically amplify the HA gene of the novel influenza virus A/H1N1 could detect virus86

excretion by day 1 p.i. in 2 of the 5 inoculated pigs (Table 1). Possibly these two pigs were87

infected experimentally and subsequently passed the virus to the other pigs. Within 4 days p.i.88

the swab samples of all infected and contact pigs were positive. Positive RT-PCR results were89

detected until day 11 p.i. with intermittent days without detectable virus excretion in90

individual animals (Table 1). Virus could be reisolated from the swabs on MDCK cells with91

titers 101,6

TCID50 from day 3 p.i. onwards from the infected animals and from day 592

onwards from the contact pigs. The last positive virus isolations were obtained on day 11 p.i.93

All plasma samples remained negative for viral RNA. The experimentally infected pigs94

d l d li i l f d 3 i N f h i l di l d h 4 f95


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anti-H1 immune response was investigated using the HerdChek Swine Influenza Virus111

(H1N1) Antibody Test Kit (IDEXX, Switzerland). One contact animal reacted positive at day112

7 of the experiment. By day 14, 5 out of 8 animals had become positive in the H1 ELISA113

compared to 7 out of 8 animals in the NP ELISA (Table 1). Obviously the H1 ELISA detected114

antibodies against the novel influenza virus A/H1N1 but the sensitivity was lower compared115

to the NP ELISA.116

Immunological analysis revealed a transient increase of CD4+

and CD8+

T-cells with peak117

levels between days 5 and 6 p.i. (Fig. 1A) which coincided with the major clinical symptoms118

in the animals. A similar increase was also observed for B-cells in the peripheral blood (data119

not shown). Further characterisation of the CD4+

T-cells revealed an early activation of these120

cells within 24 to 48 h p.i. (Fig. 1B) characterized by a significant increase in the expression121

of CD25 in all infected animals. The three contact animals also showed CD4+

T-cell122

activation with peak levels 7 to 8 days p.i.123

In a second set of experiments pigs were infected similarly by the intranasal route with 106124

TCID50 of the same virus for pathomorphological examinations on day 2, 4, and 6 p.i. On day125

2 p.i. animals did not show any lung lesions, except a very slight hyperemia of the nasal126

turbinates. However, gross lesions in the lungs were observed at 6 day p.i. They were rubbery127

in texture and characterized by a diffusely noncollapsed parenchyma. Within the cranial lobes128

h l if l f b hi i l d h h h l b129


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avian influenza virus-like porcine influenza A/H1N1 viruses currently circulating in the145

European pig population. In addition, we observed diarrhea associated with the infection most146

probably due to the compromised general condition during the acute phase of the infection.147

Despite the fact that diarrhea was also seen in 30 % of the human patients infected in the148

recent years with triple-reassortant swine influenza A (H1) viruses in the US (Shinde et al.,149

2009), no diarrhea was observed in another study of experimental influenza virus A/H1N1150

infection of pigs (Brookes et al., 2009). It remains to be determined, whether diarrhea is more151

commonly associated with the novel influenza virus A/H1N1 infection in pigs. In the present152

study, virus transmission to contact pigs occurred rapidly. Already 3 days after contact all153

nave contact pigs started to shed virus. It can be concluded that pigs are susceptible to the154

novel influenza virus A/H1N1 and it has to be assumed that this virus will spread fast and155

efficiently if introduced into swine farms possibly establishing endemic infections. The case156

reports from Canada157

(http//:www.oie.int/wahis/public.php?page=weekly_report_index&admin=0; Vol. 22, no. 19,158

7 May, 2009) and Argentina159

(http://www.oie.int/wahis/public.php?page=single_report&pop=1&reportid=8238) about160

putative human-to-pig transmissions and also experimental studies of sequential passages of161

the virus in pigs (Brookes et al., 2009) support this observation. So far, pigs or other animals162

h b d d b i l d i h id i l d f h l163


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Acknowledgements179

We are especially thankful to Stephan Becker, Jennifer Uhlendorf, Mikhail Matrosovich and180

Markus Eickmann from the Institute of Virology, Philipps Universitt, Marburg (Germany)181

for their efforts in the initial isolation and providing the new influenza virus. We also like to182

thank all technical assistants and animal caretakers at the FLI for their excellent support and183

diligent care of the animals. We thank our colleague Timm C. Harder for critical reading of184

the manuscript.185

The experiments were performed with the support of the European network of excellence186

EPIZONE.187


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References188

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Brookes, S.M., Irvine, R.M., Nunez, A., Clifford, D., Essen, S., Brown, I.H., Van Reeth,190

K., Kuntz-Simon, G., Loeffen, W. et al. (2009). Influenza A (H1N1) infection in pigs. Vet191

Rec164(24):760761 (letter).192

193

Cohen, J. (2009). Swine Flu Outbreak: New Details on Virus's Promiscuous Past. Science194

324, 1127.195

196

Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A., Sessions, W.197

M., Xu, X., Skepner, E. et al. (2009). Antigenic and Genetic Characteristics of Swine-Origin198

2009 A(H1N1) Influenza Viruses Circulating in Humans. Science DOI:199

10.1126/science.1176225 [Epub ahead of print]200

201

Irvine, R.M & Brown, I.H. (2009). Novel H1N1 influenza in people - worldwide spread202

from an animal source? Vet Rec164, 577578.203

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Shope, R.E. (1931). Swine influenza: filtration experiments and etiology. J Exp Med 54,205

373 385206


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Zell, R., Motzke, S., Krumbholz, A., Wutzler, P., Herwig, V., Drrwald, R. (2008) . Novel222

reassortant of swine influenza H1N2 virus in Germany.J Gen Virol89, 271-276.223

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Figure legends226

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Fig. 1. Analysis of the CD4+

and CD8+

T-cells in the course of novel influenza virus A/H1N1228

infection. Shown are the percentages of CD4+

and CD8+

T-cells as percent of total peripheral229

blood leukocytes (A) and the expression of CD25 (alpha-chain of the IL-2 receptor) on CD4+230

T-cells in the infected and contact pigs (B).231

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Fig. 2. Pig lung at day 6 p.i. Within the left middle lobe there are multifocal areas of dark red233

discoloration and consolidation. The diffusely emphysematous and rubbery lung parenchyma234

did not collapse (A). Pig nasal cavity at day 6 p.i. The mucosa of the nasal turbinates and the235

nasopharynx are diffusely hyperaemic and show increased mucous secretion (B).236

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Table 1. Virus excretion in pigs after experimental infection with influenza virus A/Regensburg/D6/09/H1N1

Pig Days post infection

no. Type 0 ELISA 1 2 3 4 5 6 7 ELISA 8 9 10

1 infected - - (-) + +++ ++ +++ +++ ++ - + (-) + + -

2 infected - - (-) - - - + ++ - - - (-) - + -

3 infected - - (-) - - + ++ +++ + - ++(-) - - -

4 infected - - (-) + +++ +++ +++ +++ ++ ++ + (-) - + -

6 infected - - (-) - - - + ++ - - - (-) - ++ -

7 contact - (-) - - - + ++ - - - (-) - ++ -

8 contact - (-) - - - + ++ + - - (+) + ++ +

9 contact - (-) - - - + ++ - + - (-) +++ +++ +++

Real time RT-PCR results are given as: +++ (ct-values


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Figure 1

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Figure 2

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