Infective respiratory syncytial virus is present in human cord blood samples and most prevalent during winter months

Abstract

Background: Human respiratory syncytial virus (RSV) remains the most common cause of severe lower respiratory tract disease amongst infants, and continues to cause annual epidemics of respiratory disease every winter worldwide. Demonstrating placental transmission of viable RSV in human samples is a major paradigm shift in respiratory routes considered likely for RSV transmission.

Methods: Droplet digital PCR (ddPCR) was used to identify RSV present in cord blood mononucleocytes (CBM). CBMs testing positive for RSV were treated with phytohemagglutinin (PHA), PHA and nitric oxide (NO) or PHA, NO and palivizumab, and co-cultured with HeLa cell monolayers. Subsequent immuno-staining for RSV was used to visualize infective viral plaques.

Results: RSV was detected in 26 of 45 samples (57.7%) by ddPCR. CBM's collected in winter were more likely to test positive for RSV (17/21 samples, risk = 80%, OR = 7.08; 95% CI 1.80-27.80; p = 0.005) compared to non-winter months (9/24 samples, 37.5%). RSV plaques were observed in non-treated and treated co-cultured HeLa monolayers.

Conclusions: Demonstrating active RSV in CBMs suggests in utero transmission of infective virus to the fetus without causing overt disease. This is likely to have an important impact on immune development as well as future virus-host interactions, thereby warranting further investigation.

Fig 1. RSV N gene expression in cord blood samples measured by droplet digital real time PCR (ddPCR) using fluorescent probe detection.

Cord blood samples were assessed for RSV N gene expression using a commercially available RSV fluorescent probe real-time PCR detection kit, with droplets prepared using BioRad ddPCR fluorescent probe immersion oil and droplet generator. RNA purified from a pooled preparation of nasopharyngeal aspirates collected from patients diagnosed with RSV bronchiolitis were used as positive controls for RSVA and RSVB detection. Non-template control and cDNA prepared from non-infected HeLa cells were used as negative controls to set the appropriate background expression threshold (black line). All samples were run in duplicate with events above the threshold line considered positive (26/45 samples, 57.7%).

Fig 2. Birth season distribution for cord blood samples tested for RSV by ddPCR.

Of the 45 samples tested for RSV N gene expression by ddPCR, the largest proportion of positive samples was observed in those collected during winter months (17/26).

Fig 3

Relative risk (A) and expression (B) of RSV N gene detected in cord blood samples collected in winter and non-winter months. RSV N gene expression was more likely to be detected in samples collected during winter birth months (17/21, 81%; 95% CI 64.2–97.7) compared to cord blood samples from non-winter birth months (9/24, 37.5%; 95% CI 18.1–56.9, Fishers exact analysis). Of those samples where RSV was detected, no significant difference in RSV N gene expression was observed between winter and non-winter birth months (Mann-Whitney U analysis, p = 0.567).

Fig 4. RSV plaques detected in HeLa cultures co-cultured with cord blood mononucleocytes (CBM) matured with PHA and treated with soluble nitic oxide (NO) and palivizumab (PZB).

Cryopreserved cord blood samples were treated with PHA, ‘PHA with NO’ or ‘PHA with NO and PZB’ for 24hrs, then added to HeLa cells grown to 70% confluence. Significantly more RSV was detected in all HeLa monolayers co-cultured with CBMs regardless of treatment compared to HeLa monolayers alone (p<0.01). Non-treated CBMs co-cultured with HeLa cells had less RSV plaques then similar cultures treated with PHA and PHA with NO. While RSV was detected in co-cultures treated with ‘PHA, NO and PZB’ this was not significantly different to non-treated CBM co-cultures (Mann-Whitney U analysis, p = 0.193; n = 11 samples for all treatments).