2.4 Discussion
2.4.1 Infection of different chiropteran cells by TGEV, SIV, VSV, and IBV
Viral host range depends on the interaction of the viral receptor binding protein and the presence of the corresponding receptor on the surface of the host cell. In general coronaviruses have a narrow host range. Most of them infect one or just a few species (KUO et al. 2000). Porcine aminopeptidase N is the species specific receptor for TGEV. The TGEV spike protein is essential for pAPN binding (LAUDE et al. 1986;
GODET et al. 1994; KUBO et al. 1994) and membrane fusion (DE GROOT et al.
1989). ST cells endogenously express pAPN, the specific receptor for TGEV, on their cell surface. After TGEV infection the virus is able to enter the cells and to replicate within the cytoplasm. We demonstrated that infection of the chiropteran cell lines HypNi/1.1, MyDauDa/46, Tb1Lu, and PipNi/1 depends on the presence of pAPN.
After pAPN-expression the virus can enter these cells and is able to replicate.
Receptor-dependent entry by TGEV was also shown by Hoffmann et al. using HypNi/1.1 and Tb1Lu as well as a variety of other chiropteran cell lines (HOFFMANN
Fig. 2-16 Localization of TGEV envelope (E) protein 1 dpi. Chiropteran cells co-transfected with pAPN and compartment marker cDNA. Cells were treated with Triton-X-100. Compartment marker for ERGIC/Golgi: green; envelope protein: red; nucleus: blue. PFA fixation 24 hpi.
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et al. 2013). In Myotis daubentonii and Pipistrellus species viral RNA of the genus Alphacoronavirus has been detected (GLOZA-RAUSCH et al. 2008). Therefore, in our study we did not only analyze HypNi/1.1 and Tb1Lu cells – like Hoffmann et al. – but also the PipNi/1 and MyDauDa/46 cell lines. In addition to pAPN-dependent entry we demonstrated that viral replication in those cell lines was possible although to a different extent. By connecting the release of infectious TGEV with the measured expression level of pAPN for each cell line, no relation was observed (e.g. comparing HypNi/1.1 and PipNi/1 cells). This shows that the receptor expression level does not correlate with the outcome of infectious virus in every tested cell line. To find out, if the infection rate of the tested chiropteran cells is similar also when infected with other viruses (e. g. virus replication is always more successful in HypNi/1.1 cells compared to the other chiropteran cells), they were infected with SIV-H3N2, VSV, and IBV. The SIV-H3N2 is an influenza A virus which binds to alpha-2,6-linked sialic acids to enter the cells. All tested cell lines were susceptible to SIV-H3N2 which is consistent with the endogenous expression of alpha-2,6-linked sialic acids. PipNi/1 cells showed a higher receptor expression level as well as a higher amount of released virus particles compared to the other chiropteran cell lines. VSV belongs to the family Rhabdoviridae and interacts with LDL (Low-Density Lipoprotein) during virus entry (FINKELSHTEIN et al. 2013). The tested cell lines were all susceptible for VSV. Tb1Lu and PipNi/1 cells showed a similar density of LDL receptor on their cell surface although Tb1Lu cells had a higher release (>101 pfu/ml) of virus particles.
Regarding VSV the receptor expression level does not correlate with the infection rate. Concerning IBV, a Gammacoronavirus, all tested cell lines were susceptible.
Infected ST, HypNi/1.1, PipNi/1, and MyDauDa/46 cells were observed via immunofluorescence but no re-infection of Vero cells was visible when the supernatants were used for titration although the amount of expressed alpha-2,3-linked sialic acids in the bat cells was rather high. Reasons could be on the one hand that the virus was not able to replicate successfully in these cells or that the virus assembly, maturation or budding step was impaired. On the other hand the amount of released infectious virus particles may have been too low for a successful re-infection and/or the presence of a certain – so far unknown – specific protein receptor is necessary for efficient virus entry. Only in Tb1Lu, BHK-21, and Vero cells, a measurable amount of IBV was able to replicate and to exit the cells as infectious virus particles.
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The abundance of specific cellular receptors leads to virus species restriction (KUO et al. 2000). A specific cell line in which all tested virus species can replicate more successfully than in the other cells was not observed. HypNi/1.1 cells showed the highest virus titer after TGEV infection compared to the other tested chiropteran cell lines but not when infected by SIV-H3N2, VSV or IBV. This indicates that beside the presence of a specific receptor other cellular as well as viral factors may influence replication, maturation as well as the budding process.
2.4.2 Localization of TGEV proteins within chiropteran cell lines
To further analyze possible factors that influence the efficiency of the TGEV infection in chiropteran cells the localization of the viral structural proteins S, M, N, and E in the different cell lines were compared.
Within the tested chiropteran cells different patterns of the TGEV S and M expression could be observed. In HypNi/1.1 and in Tb1Lu cells, the S protein as well as the M protein were expressed in the whole cytoplasm whereas in PipNi/1 and MyDauDa/46 cells S and M were accumulated near the nucleus, ERGIC and Golgi compartment. A difference in distribution of the TGEV S protein was also observed by Hoffmann et al.
(HOFFMANN et al. 2013) after infection of HypNi/1.1 and EpoNi/22.1 cells. As in the present study, the TGEV S protein was distributed all over the cell in HypNi/1.1 cells but in EpoNi/22.1 it was restricted to dot-like structures (HOFFMANN et al. 2013).
The TGEV assembly process takes place in the cytoplasm at the ERGIC. After budding viral particles are transported to the plasma membrane via the secretory pathway. The restricted localization of the TGEV S and M proteins to the ERGIC 24 h after infection in PipNi/1 and MyDauDa/46 cells indicates impairment in virus assembly. Additionally, retention of S and M protein within PipNi/1 and MyDauDa/46 cells due to an interaction with host factors or due to the reduction in interaction with host factors could be reasons for the reduction in released infectious viral particles.
Within the coronaviral lipid bilayer the TGEV N is associated with the viral genome and forms a nucleocapsid (BOSCH et al. 2005). The TGEV E protein is involved in the coordination of virus assembly and release (MCBRIDE & MACHAMER 2010).
Both proteins, N and E, had similar expression patterns in the different chiropteran cell lines. In all tested cells, the N protein was expressed in the whole cytoplasm while the E, the TGEV structural protein with the lowest abundance in the virus
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particle, was accumulated near the nucleus. These findings suggest that the observed differences in the efficiency of TGEV replication in chiropteran cells are restricted to TGEV S and M protein expression as well as viral protein interaction with host factors.