Antibody-induced endocytosis of viral glycoproteins, expressed on pseudorabies virus-infected monocytes protects these cells from complement-mediated lysis

Pseudorabies virus (PrV) can cause abortion in sows with an immune system activated by vaccination. Virus-carrying blood monocytes are essential for the spread of the virus from the respiratory tract to the pregnant uterus. Two major adaptive immune effector mechanisms should normally be capable of eliminating PrV-infected monocytes. First, newly synthesised viral proteins may be processed and coupled to the major histocompatibility complex class I (MHC I) which then is transported to the plasma membrane. This MHC I-antigen-complex can be recognised by cytotoxic T-lymphocytes (CTLs). Second, specific antibodies are capable of binding to newly synthesised viral envelope glycoproteins, which become expressed in the plasma membrane of the infected cell. Antibodies in association with complement or phagocytes may then result in the lysis of the infected cell. Addition of virus-specific antibodies to PrV-infected swine kidney cells in vitro is known to induce a redistribution of the plasma membrane-anchored viral glycoproteins. This redistribution finally leads to the release of the viral glycoproteins into the surrounding medium, leaving viable cells without visually detectable levels of viral glycoproteins on their plasma membrane. In the present study it was examined whether a similar phenomenon occurs in the natural carrier of the virus, the blood monocyte, in order to evaluate if this process may be significant to the immune evasion of the virus. Blood was collected from the vena jugularis from PrV-negative pigs and blood mononuclear cells were separated on Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). Blood monocytes were purified by plastic adhesion, and were cultivated for 24 h. Afterwards, the cells were inoculated with PrV strain 89V87 or Kaplan and incubated at 37\,^\circC with 5% CO$_2$ for 13 h. After washing of the cells, FITC-labelled virus-specific antibodies were added (0.1 mg IgG/ml), and the cells were incubated at 37\,^\circC for different time periods (0, 5, 10, 30 and 60 min) before fixation with 0.4% formaldehyde and analysis by fluorescence microscopy and/or confocal laser scanning microscopy. Shortly after the addition of antibodies, viral plasma membrane glycoproteins become aggregated (patches). These patches are then internalised by the cell, leaving an infected cell with no visually detectable levels of viral glycoproteins on its plasma membrane. Antibody-induced endocytosis is a fast and efficient process. Endocytosis started at 10 min post-antibody addition, and was completed in 65% of the infected cells at 1 h post-antibody addition. Furthermore, only very few quantities of viral glycoproteins on the plasma membrane (reached after 7 h PI) and very low concentrations of antibodies (0.04 mg IgG/mL) were needed to induce endocytosis. Genistein, a specific inhibitor of tyrosine kinase activity, was found to be a very efficient inhibitor of viral glycoprotein internalisation (100% inhibition at 50 $\mu$g/mL). We also evaluated the effect of viral glycoprotein internalisation on complement-mediated lysis of the infected monocytes. Monocytes were infected for 10 h, and incubated with virus-specific antibodies for 2 h ($\pm 100\%$ of the infected cells displayed internalised viral glycoproteins at this time point). The control cells were incubated with antibodies in the presence of $50\,\mu$g/mL genistein, or were incubated without antibodies. Afterwards, the cells were washed and incubated with different concentrations of guinea pig complement (0-10 IU) for 1 h. Afterwards, 20 $\mu$g/mL of the DNA-staining fluorochrome, propidium iodide, was added for 5 min. Propidium iodide specifically stains dead cells which allows to determine the percentage of dead cells by flow cytometry. Compared relatively to the viability of the cells incubated without either antibodies or the complement, viability of the cells, incubated with the complement for 1 h decreased slightly to 79% $\pm$ 12% for cells incubated without antibodies (no activation of the complement), and to 84% $\pm$ 4% for cells incubated with antibodies (internalised viral glycoproteins and antibodies). The viability dropped to 24% $\pm$ 11% for cells incubated with antibodies and genistein (there was no internalisation of viral glycoproteins and antibodies), which was not caused by toxic effects of genistein. We can therefore state that antibody-induced endocytosis of viral glycoproteins protects PrV-infected cells from complement-mediated lysis. When performing double labelling experiments, we observed that the MHC I co-aggregates and undergoes co-endocytosis with the viral glycoproteins. This may indicate that the addition of virus-specific antibodies to PrV-infected monocytes can hide these cells from both humoral and cellular immune responses. To investigate this hypothesis, we are currently constructing an in vitro assay to evaluate the effect of MHC I co-endocytosis on the capacity of cytotoxic T-lymphocytes to eliminate PrV infected monocytes. Furthermore, we are examining whether the observed processes also occur in vivo. Preliminary experiments, consisting of the injection of colostrum-free piglets with biotinylated PrV-specific antibodies, followed by PrV-inoculation, already showed that endocytosis of antibodies occurs in vivo in infected cells, e.g. in alveolar macrophages

Bitter-sweet symphony: glycan-lectin interactions in virus biology

Glycans are carbohydrate modifications typically found on proteins or lipids, and can act as ligands for glycan-binding proteins called lectins. Glycans and lectins play crucial roles in the function of cells and organs, and in the immune system of animals and humans. Viral pathogens use glycans and lectins that are encoded by their own or the host genome for their replication and spread. Recent advances in glycobiological research indicate that glycans and lectins mediate key interactions at the virus-host interface, controlling viral spread and/or activation of the immune system. This review reflects on glycan–lectin interactions in the context of viral infection and antiviral immunity. A short introduction illustrates the nature of glycans and lectins, and conveys the basic principles of their interactions. Subsequently, examples are discussed highlighting specific glycan–lectin interactions and how they affect the progress of viral infections, either benefiting the host or the virus. Moreover, glycan and lectin variability and their potential biological consequences are discussed. Finally, the review outlines how recent advances in the glycan–lectin field might be transformed into promising new approaches to antiviral therapy

Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains&nbsp;scarce. METHODS: Nine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 10 TCID of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS. RESULTS: Besides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome&nbsp;observed. CONCLUSIONS: These findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV&nbsp;infections.</p