ZBiotech’s Lectin Array helps researchers generate useful insights into how viruses reshape the glycans on infected endothelial cells.

In a recent study, researchers investigated the effects of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) on porcine pulmonary microvascular endothelial cells (PPMVECs). PPMVECs are cells that line the microvascular system in pig lungs, playing crucial roles in maintaining the integrity and function of blood vessels. HP-PRRSV, specifically its strains HN and JXA1, is a highly contagious virus causing severe respiratory and reproductive issues in pigs, significantly impacting the swine industry.

Researchers isolated and cultured PPMVECs in vitro, infecting them with HP-PRRSV strains HN and JXA1 to study the virus’s impact on the cells’ surface carbohydrate chains. Scanning electron microscopy revealed that at 48 hours post-infection, the PPMVECs exhibited damaged cell membranes and a notable reduction or disappearance of the surface glycocalyx, a carbohydrate-rich layer critical for cellular protection and signaling.

Using lectin array to generate insights into how viral infection alters the glycan structures on cell surfaces

To further analyze glycocalyx changes observed through electron microscopy, the study employed a lectin microarray containing 26 lectins, each capable of binding specific carbohydrate residues. This high-throughput technique allowed for detailed glycosylation profiling of the infected cells. Researchers labeled the cells with Cy3 fluorescent dye, incubated them on the lectin microarray, and scanned the arrays using a LuxScan 10K-A microarray chip scanner. The fluorescence intensities were analyzed to identify variations in glycan motifs.

 

 

Summary of the glycan-binding specificities of 26 lectins used in the study. This collection of lectins can be used to profile most motifs found on mammalian glycans. The images are reproduced from Song, X., Wu, Y., Wu, X., Hu, G. & Zhang, T. Effects of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Infection on the Surface Glycoprofiling of Porcine Pulmonary Microvascular Endothelial Cells. Viruses 14, (2022).

The results indicated significant alterations in glycosylation post-HP-PRRSV infection. Specifically, bindings of eight lectins were significantly changed by the HP-PRRSV HN strain, while seven were affected by the JXA1 strain, with six common lectins between the two. Notably, lectins RCA-I, LEL, and STL were up-regulated, and lectins LCA, DSA, and PHA-E were down-regulated, findings were further confirmed by lectin fluorescence staining and flow cytometry.

 

 

Left: Scans of the lectin microarray. Each lectin was spotted in triplicate per block. Yellow frames and white frames marked indicated the lectin sites significantly increased and decreased compared to the normal control group, respectively. The negative controls showed no positive signal. NC: negative control.

Right: Fluorescence intensity analysis of the lectin microarray. Heat map and hierarchical clustering of the positive lectins in PPMVECs. Samples are listed in columns and lectins in rows.

The images are reproduced from Song, X., Wu, Y., Wu, X., Hu, G. & Zhang, T. Effects of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Infection on the Surface Glycoprofiling of Porcine Pulmonary Microvascular Endothelial Cells. Viruses 14, (2022).

These glycan changes suggest that HP-PRRSV infection disrupts the endothelial glycocalyx, leading to modifications in glycan composition that likely impact various cellular functions and contribute to viral pathogenesis. The up-regulated carbohydrate chains were mainly poly-N-acetyllactosamine, galactose, sialic acid, and fucose residues, while the down-regulated chains were primarily complex N-glycans.

In conclusion, the lectin microarray was instrumental in providing a detailed glycan profile of PPMVECs post-HP-PRRSV infection. This technology allowed researchers to identify specific changes in glycosylation, enhancing our understanding of the virus’s impact on endothelial cells. The study underscores the importance of glycan analysis in viral pathogenesis and highlights the potential of lectin microarrays as a valuable tool in virology research, offering insights that can aid in improving our understanding of host-pathogen interactions.