A sertés parvovírus SAT– mutáns törzsének és víziszárnyas vírusok fertőzőképességének vizsgálata real-time PCR és immunfluoreszcens módszerekkel

Abstract

A short, alternative open reading frame (ORF) is located in the genome of the porcine parvovirus (PPV) overlapping the amino terminal region of the viral protein 2 (VP2) gene and encode the small alternatively translated protein (SAT). The SAT of the attenuated NADL-2 strain accumulates in the ER and its deletion induces “slow spreading” phenotype. We studied the effect of the deletion of SAT in the pathogen Kresse strain and analysed the signalling pathways involved in its function, using realtime PCR and immunofluorescence (IF) methods. The in vitro comparison of the wild type Kresse strain and its SAT– knockout mutant revealed that “slow spreading” is a general feature of the SAT– PPV viruses, and the prolonged cell integrity and late viral release are responsible for this feature. PPV infection induces ER stress in the infected cells regardless of SATp presence. It is demonstrated by the morphological changes of the ER, the expression of the unfolded protein response related proteins, the homeostasis restorer X-box binding protein 1 (XBP1) and the pro-apoptotic C/EBP homologous protein (CHOP). However, the presence of SAT protein accelerates the cell death and enhances the ER stress during infection as shown by the higher expression rate and the alteration of the localization of CHOP. The involvement of irreversible ER stress induction in the viral spreading was confirmed by treatment of infected cells by ER stress inducing chemicals (MG132, DTT and thapsigargin) that accelerated the egress both the wild type and the SAT– viruses. The short-term ultra violet irradiation had no beneficial effect on the copy number and the titer of the viruses underscoring the specificity of ER stress pathways in the acceleration process. Transfection experiments revealed that the expression of SAT alone cannot activate Xbp1 or CHOP expression, which raises the possibility that SAT exerts its effect in synchrony with other viral proteins. Furthermore, induction of the CHOP expression and the nuclear translocation of CHOP alone cannot be responsible for the biological effect of SAT protein, since nuclear CHOP could not complement the lack of SAT in a co-expression experiment. Most probably SAT interacts with the effect of other viral proteins, it causes severe ER stress, which results in the activation of more UPR pathways and/or other ER stress response routes in the infected cells. The influence of these pathways/proteins complementing the effect of the CHOP induced genes lead to earlier cell death and accelerated viral spread. Economically speaking, duck circovirus (DuCV), duck hepatitis A virus 1 (DHAV-1), goose haemorrhagic polyomavirus (GHPV), and goose parvovirus (GPV) are harmful pathogens of waterfowl, and do not or poorly replicate in immortalized cell lines. The AGE1.CR, AGE1.CR.pIX and AGE1.CS muscovy duck (Cairina moschata) originated cell lines have been developed previously for the production of human viral vaccines. These cell lines were studied to explore their suitability for propagating the aforementioned avian viruses. Our investigations verified that the AGE1 cell lines are permissive for the DHAV-1, GHPV and GPV and semi-permissive for the DuCV. In case of low multiplicity DAHV-1 and DuCV infection, the obtained growth curves were very similar in different cell lines, while at the GHPV and GPV infection, the shape of the curves depended on the type of the cell line substantially. The CRpIX proved to be the most sensitive for the DHAV-1 and GPV infection, while the CR was the most suitable for the GHPV replication. Despite the high sensitivity of the cell lines for viruses, their viral productivity remained relatively low for the investigated field isolates. However, optimization of virus infection and/or the adaptation of the viruses to the cells can increase viral productivity and make these cell lines suitable for vaccine development and production.