Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses

Meng, Fanxiang; Punyadarsaniya, Darsaniya; Uhlenbruck, Sabine; Hennig-Pauka, Isabel; Schwegmann-Weßels, Christel; Ren, Xiaofeng; Dürrwald, Ralf; Herrler, Georg

doi:10.1186/1297-9716-44-110

Cited by 33 publications

(56 citation statements)

References 33 publications

(45 reference statements)

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“…To analyze the adaptation potential of H9N2 strains to growth in the target cells of a new host, we chose precision-cut lung slices (PCLS) as a culture system for differentiated airway epithelial cells. Replication of swine influenza viruses in PCLS was found to reflect the virulence properties of the respective virus strains (33). Following three passages in porcine PCLS, the P3 virus eventually differed from P0 to P2 viruses by a shorter growth cycle but not 50 .…”

Section: Discussionmentioning

confidence: 99%

“…Each bronchus in the microscopic field was virtually divided into 10 segments, each of which was monitored for the presence or absence of ciliary activity (32,33). Slices which showed 100% ciliary activity at the beginning were selected for the experiments.…”

Section: Methodsmentioning

confidence: 99%

“…In this respect, H9N2 viruses resembled low-virulence SIV. In contrast, infection of PCLS by virulent SIV results in complete ciliostasis (33). A difference was noted only for replication efficiency.…”

mentioning

confidence: 86%

“…PCLS were prepared from lungs of 3-month-old healthy crossbred pigs as described previously (32,33,63,64).…”

Section: Methodsmentioning

confidence: 99%

“…culture system for differentiated respiratory epithelial cells from the porcine lung that, upon infection by swine influenza viruses (SIV), reflects the viral virulence properties (32,33). To investigate the adaptation of H9N2 avian influenza viruses to growth in the respiratory epithelium of pigs, we passaged an avian influenza virus, A/chicken/Saudi Arabia/CP7/98 (H9N2), three times (P1, P2, and P3) in PLCS.…”

mentioning

confidence: 99%

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Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice

Yang

Punyadarsaniya

Lambertz

et al. 2017

J Virol

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The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precisioncut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized ␣2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with ␣2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190ϩHA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 86%

“…PCLS were prepared from lungs of 3-month-old healthy crossbred pigs as described previously (32,33,63,64).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice

Yang

Punyadarsaniya

Lambertz

et al. 2017

J Virol

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Precision-cut intestinal slices as a culture system to analyze the infection of differentiated intestinal epithelial cells by avian influenza viruses

Punyadarsaniya

Winter

Mork

et al. 2015

Journal of Virological Methods

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Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species.

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Comparison of mono- and co-infection by swine influenza A viruses and porcine respiratory coronavirus in porcine precision-cut lung slices

Krimmling

Schwegmann-Weßels

2017

Research in Veterinary Science

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Coronaviruses as well as influenza A viruses are widely spread in pig fattening and can cause high economical loss. Here we infected porcine precision-cut lung slices with porcine respiratory coronavirus and two Influenza A viruses to analyze if co-infection with these viruses may enhance disease outcome in swine. Ciliary activity of the epithelial cells in the bronchus of precision-cut lung slices was measured. Co-infection of PCLS reduced virulence of both virus species compared to mono-infection. Similar results were obtained by mono- and co-infection experiments on a porcine respiratory cell line. Again lower titers in co-infection groups indicated an interference of the two RNA viruses. This is in accordance with in vivo experiments, revealing cell innate immune answers to both PRCoV and SIV that are able to restrict the virulence and pathogenicity of the viruses.

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Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses

Cited by 33 publications

References 33 publications

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice

Precision-cut intestinal slices as a culture system to analyze the infection of differentiated intestinal epithelial cells by avian influenza viruses

Comparison of mono- and co-infection by swine influenza A viruses and porcine respiratory coronavirus in porcine precision-cut lung slices

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