Susceptibility of primary chicken intestinal epithelial cells for low pathogenic avian influenza virus and velogenic viscerotropic Newcastle disease virus

Highlights

• Primary duodenal chicken IEC culture are a suitable model for host-pathogen interaction.

• Differences in IFN mRNA expression pattern between Newcastle disease virus and avian Influenza virus.

• Significant upregulation of IFN λ after LPAIV and NDV-infection highlights its possible role as an antiviral cytokine in the gut.

• Difference in mRNA expression of interferon stimulated genes IFIT5 and ISG12.

Abstract

Avian influenza virus (AIV) and Newcastle disease virus (NDV) share a high tropism for the avian respiratory epithelium and may cause severe clinical disease associated with high mortality. Both viruses have different pathotypes, which may lead to differences in the severity of the disease. Respiratory epithelial cells were shown to be the primary target cells for infection and replication. Nevertheless, intestinal epithelial cells (IECs) were also suggested as target cells for both viruses in avian species. Most studies on AIV and NDV focused on the respiratory tract, while information regarding the virus-host interaction at the intestinal epithelial cell interface is lacking. We established a primary chicken IEC culture model. Primary chicken embryo fibroblast cultures (CEFs) were used for comparison. IECs and CEFs were infected with a low infectious dose (LID; multiplicity of infection, MOI, of 0.01) or high infectious dose (HID, MOI of 1), of low pathogenic AIV (LPAIV) H9N2 or velogenic viscerotropic NDV (vvNDV) Herts 33/56. Virus replication, mRNA expression pattern of the type I and type III interferon (IFN) and related genes IFIT5 (interferon-induced protein with tetratricopeptide repeats 5) and ISG12 (interferon stimulated gene 12) were investigated at four, 16, and 24 h post infection (hpi). The results suggest high susceptibility of primary chicken IECs for these AIV and NDV strains. Replication rates and expression pattern of IFNs as well as related genes differed between the infecting viruses as well as cell culture systems. Both viruses induced an IFN λ-increase of more than 30-fold in IECs, while IFN-α and IFN-β mRNA expression was either downregulated or only slightly increased with up to 10fold changes for the latter at 24 h post LPAIV-infection. These results suggest a possible role of IFN λ in the control of viruses at the gut epithelial surface. LPAIV induced upregulation of IFIT5 as well as ISG12 expression in a dose and time dependent manner, while vvNDV infection only led to slight upregulation of IFIT5 and downregulation of ISG12, indicating differences in the down-stream regulation of the antiviral immune response between investigated viruses. Overall, our data demonstrate that IECs are a suitable model to investigate selected parameters of virus-host interaction for AIV and NDV and may be used to study other strains as well as other host species.

Introduction

Avian influenza viruses (AIV) and Newcastle disease viruses (NDV) may induce high morbidity and mortality rates in susceptible poultry flocks and therefore lead to high economic losses (Alexander et al., 2012, Capua and Alexander, 2010). AIV and NDV are frequently isolated from the gastrointestinal tract (GIT) of various wild waterfowl, which are considered as the main reservoir for both viruses, often without causing severe or any clinical signs (Curran et al., 2013, Gilchrist, 2005, Kim et al., 2007, Kuiken et al., 1998).

AIV belongs to the family Orthomyxoviridae. Based on the virus surface proteins hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are classified into 18 HA and 11 NA subtypes (Tong et al., 2012, Tong et al., 2013). Subtypes H1 to H16 have been detected in birds, whereas up to date subtypes H17N10 and H18N11 have only been identified in bats (Wu et al., 2014). Based on their pathogenicity, AIV can be classified into highly pathogenic (HPAIV) and low pathogenic avian influenza virus strains (LPAIV) (Webster et al., 1992). HPAIV cause systemic infections in poultry (Swayne and Pantin-Jackwood, 2006), whereas LPAIV induce local infections in the gastrointestinal and respiratory tract (Post et al., 2013).

NDV belongs to the family Paramyxoviridae, genus Avulaviridae, serotype Paramyxovirus 1. Based on the intracerebral pathogenicity index (ICPI), ND viruses are classified into four pathotypes: 1) apathogenic NDV, causing no clinical signs (e.g. vaccine strain VG/GA isolated from the intestine of healthy turkeys), 2) lentogenic NDV (ICPI ≤ 0.7), causing mild local infections of the respiratory tract, 3) mesogenic NDV (ICPI 0.7–1.5), which causes respiratory or neurological signs in young chicks and mild signs in adult birds and 4) velogenic NDV (ICPI ≥ 1.5), which may induce high morbidity and mortality in all age groups (Cattoli et al., 2011, Meulemans et al., 2015). Both mesogenic and velogenic NDV strains lead to systemic infection.

Attachment and entry of both viruses requires the presence of sialic acids (SA) on target cells (Connaris et al., 2002, Gambaryan et al., 2002). AIV and NDV preferentially bind to SAα(2,3)-Gal receptors. This is the predominating sialic acid found in epithelial cells of the small intestine of chickens, whereas SAα(2,6)-Gal receptors predominate in the chicken trachea (Pillai and Lee, 2010, Yu et al., 2011). Binding of either virus to an epithelial cell leads to immediate activation of innate immune responses in order to control and inhibit virus spread and growth. This involves the recognition of pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs). In chicken, the PRR melanoma differentiation-associated gene-5 (MDA-5) plays an important role in this process (Karpala et al., 2011). Subsequently, intracellular signaling cascades are induced, thereby leading to the release of soluble factors such as interferons (IFNs) and cytokines, an upregulation of IFN-stimulated genes (ISG) and the establishment of an antiviral state (Munir et al., 2005). ISGs IFIT5 (interferon-induced protein with tetratricopeptide repeats 5) and ISG12 (IFN stimulated gene 12) belong to the most common ISGs involved in the mediation of antiviral effects by inhibiting cell proliferation, migration and translation initiation (Cheriyath et al., 2011).

To date, the involvement of chicken ISGs in virus infections is still unclear (Goossens et al., 2013). IFIT5 is the only member of the IFIT family described in avian species and little is known about the exact molecular pathways during viral infections in the chicken gut (Ranaware et al., 2016, Vanderven et al., 2012, Zhou et al., 2013). Vanderven et al. (2012) detected high levels of IFIT5 and ISG12 in relative transcripts in the intestine of ducks following in vivo infection with Influenza virus (Vanderven et al., 2012). Wang et al. (2015) demonstrated high levels of IFIT5 in stomach, small and large intestine of healthy ducks (Wang et al., 2015). ISG12 is a nuclear export protein and also may play a role during viral infection, which has not fully been characterized so far. It may be involved in the induction of apoptosis because of its destabilizing properties of the mitochondrial membrane (Cheriyath et al., 2011)

Cornelissen et al. (2012) demonstrated that both IFN α and IFN β mRNA expression is upregulated in the chicken gut immediately after in vivo infection with LPAIV (Cornelissen et al., 2012). Post et al. (2012) compared gene expression patterns after LP- and HPAIV infection in the ileum. Similar pathways were stimulated by both low and highly pathogenic AIV in all cells of this gut section (Post et al., 2012).

In vitro and in vivo infection with virulent NDV led to a rapid and strong induction of IFN α and other cytokines, such as IL-6 and IL-1β (Rue et al., 2011, Sick et al., 2000, Sick et al., 1998). Measurements of gene expression patterns upon NDV infection revealed upregulation of numerous ISGs and signal transducer and activator of transcription-1 (STAT-1α/β) in chicken embryo fibroblasts (CEFs) (Liu et al., 2014, Munir et al., 2005, Röll, 2013) while no information is available for epithelial cells of the gut.

AIV and NDV studies have mainly been conducted in vivo. Mechanisms of virus-host interaction at the epithelial cell interface are difficult to elucidate in an animal experiment under in vivo conditions. Tracheal organ cultures (TOCs) are an alternative and have been successfully used as an in vitro model to understand virus-host interaction (Sid et al., 2016, Petersen et al., 2012). Yet, little is known about the virus-host interaction in avian intestinal epithelial cells (IECs). In vitro studies involving intestinal epithelial cell culture models originating from the chicken gut are limited in number or were used to investigate only bacteria-IEC interaction, which is why the interaction of AIV and NDV with the avian intestinal tract is still poorly understood (Alitheen et al., 2012, Punyadarsaniya et al., 2015, Van Deun et al., 2008).

The highly organized gastrointestinal system and sensitivity of IECs towards any kind of manipulation are the main limitations researchers are facing when establishing a primary IEC culture (Bermudez-Brito et al., 2013).

The aim of this study was to investigate the virus-host interaction in primary chicken intestinal epithelial cells and to compare possible differences between AIV and NDV. Based on previous investigations and preliminary results comparing lento-, meso- and velogenic NDV strains (Kaiser and Rautenschlein, unpublished data), we selected to compare a velogenic NDV and LPAIV H9 (Kaiser et al., unpublished data; Petersen et al., 2012). The selected NDV strain is known to cause severe gut lesions with necrotic boutons in vivo, while H9N2 does not induce macroscopic lesions in the gastrointestinal tract of infected birds (Brown et al., 1999, Post et al., 2013).

Although these viruses differ in their in vivo pathogenicity, both were shown to be strong IFN inducers in TOCs (Reemers et al., 2009). For this purpose, we established a primary chicken IEC culture model and infected IECs with a LPAIV H9N2 or velogenic viscerotropic NDV (vvNDV) strain. We compared virus replication rates, virus titers at certain time points, the induction of type I and type III IFN mRNA expression and ISGs IFIT5 and ISG12 as parameters of innate antiviral immunity (Kapczynski et al., 2013, Zhang et al., 2013). Chicken embryo fibroblasts (CEFs) were also infected with AIV and NDV as a reference for virus replication as well as for induction of selected innate immune parameters.