Viral prevalence by gestational age and season in a large neonatal cord blood cohort

Abstract

Objective:

To investigate viral prevalence in a large neonatal cohort and determine the impact on pregnancy and birth outcomes.

Study design:

We prospectively collected 1044 neonatal samples from remnant neonatal cord blood RPR samples. We performed qRT-PCR/qPCR reactions for: adenovirus, anellovirus (alphatorquevirus and betatorquevirus), cytomegalovirus (CMV), Epstein-Barr virus (EBV), enterovirus, human herpesvirus 6 (HHV6), parechovirus, and parvovirus B19.

Result:

Overall viral prevalence was 5.6% with 58 positive samples. Alphatorquevirus (2%) and HHV6 (1.2%) were the two most prevalent viruses detected. Viral detection was most common in samples collected in the fall (September-November) and least common in those collected in winter (December–February). There was no statistical difference detected in viral prevalence or viral load by gestational age, preterm delivery, pre-eclampsia or chorioamnionitis.

Conclusion:

While there is seasonal variation in viral prevalence in neonatal cord blood samples, individual virus presence does not seem to effect pregnancy or birth outcomes.

Introduction

Preterm birth, parturition before 37 weeks gestation, is a major cause of neonatal morbidity and mortality. In 2018, 10.02% of all births in the United States were preterm [1]. Preterm birth, can occur secondary to intrauterine infection and the secondary inflammatory response to these microorganisms [2]. There is increasing recognition that the entire microbial community, rather than one discrete organism, is responsible for this inflammatory cascade [3]. While the contribution of the bacterial community to preterm labor is emerging [3,4], the contribution of viruses to preterm labor and term birth outcomes remains an area of continued investigation. Viruses can enter trophoblast cells, causing apoptosis, inflammation, and preterm delivery [5]. Additionally, there is now appreciation of transkingdom interactions between viruses and bacteria [6]. Specifically, experimental virus infection during pregnancy makes animals more susceptible to bacterial infection and preterm birth [7,8].

Data regarding viral prevalence in neonates at term and preterm gestation is limited. While several studies have detected viral nucleic acid in amniotic fluid obtained via amniocentesis for advanced maternal age, abnormal serum screening results, or suspected fetal anomalies [9–11], there have been few studies of viral nucleic acid detection in neonatal cord blood. A small cohort of matched maternal-preterm infant samples found low prevalence of common viruses in neonatal cord blood samples [12]. While most viruses are not well studied in neonates, cytomegalovirus (CMV) prevalence has been well studied in large cohorts of both term and preterm neonates. At the time of birth, CMV was detected in 0.7% of term neonates [13] and in 3% of preterm neonates [14]. These data suggest that viral prevalence may vary based on gestational age. We hypothesized that variation in viral prevalence contributes to preterm birth or adverse pregnancy outcomes.

To test our hypothesis that viral prevalence or viral load contributes to preterm birth or adverse pregnancy outcomes, we prospectively collected a cohort of neonatal cord blood samples over an 11 month period. We investigated viral prevalence and quantity of nine viruses: adenovirus, anellovirus (alphatorquevirus and betatorquevirus), CMV, Epstein-Barr virus (EBV), enterovirus, human herpesvirus 6 (HHV6),parechovirus, and parvovirus B19. Anelloviruses are a diverse and widely prevalent group of small circular single-stranded DNA viruses and are frequently detected in samples of maternal blood (prevalence 77–84%) and infant cord blood (0–48%) [12,15,16]. Anelloviruses may be markers for immune function as their viral loads correlate with CD4 counts in HIV patients and outcomes among transplant recipients [17–20]. The seven other viral targets were chosen for their role in early-onset sepsis, congenital infections or intrauterine infections.

Materials and methods

Subjects

We obtained neonatal cord blood plasma samples from the Barnes-Jewish Hospital (BJH) clinical laboratory, using 200 μL − 1050 μL of remnant samples from specimens collected for rapid plasma reagin (RPR) testing from September 2017 to July 2018, and frozen at −80 °C until used. Clinical metadata were abstracted from the medical record. This study was approved by the Washington University Human Research Protection Office.

Nucleic acid extraction

Samples were thawed and brought up to a total volume of 1050 μL with Dulbecco’s Phosphate Buffered Saline and then extracted on the Roche COBAS® Ampliprep (Roche Diagnostics, Indianapolis, IN) using the Roche total nucleic acid isolation kit. Extracted nucleic acid was stored at −80 °C prior to analysis.

qRT-PCR/qPCR reactions

We performed qRT-PCR/PCR assays to detect and quantify viral targets for each of the nine selected viruses: adenovirus, anellovirus (alphatorquevirus, and betatorquevirus), CMV, enterovirus, EBV, HHV6, parechovirus, and parvovirus B19 [12]. For each assay, the copy number sensitivity varied from 5 to 500 copies/reaction [12].

We generated a plasmid containing the region of interest for each assay. We performed serial dilutions of 5 × 10⁶ to 5 copies/reaction for each plasmid and ran samples in triplicate to generate a standard curve and to determine the sensitivity of each assay [12]. For each qRT-PCR assay, we generated in vitro transcribed RNA from the plasmid containing the region of interest using MEGAscript® T7 transcription kit (Thermo Fischer Scientific, Waltham, MA) per the manufacturer’s instructions. We performed serial dilutions (5 × 10⁶ to 5 copies/reaction) of the in vitro transcribed RNA to generate a standard curve and to determine the sensitivity of the assays [12].

We used a total reaction volume of 20 μL with a 5 μL sample input for each assay and utilized primer and probe concentrations of 900 nM and 250 nM, respectively. For the qPCR assays, we used Taqman® Fast Advanced Master Mix (Thermo Fischer Scientific, Waltham, MA), and we used Taqman® Fast Virus 1-Step Mix (Thermo Fischer Scientific, Waltham, MA) for the qRT-PCR assays.

All qPCR reactions included a 2 min polymerase activation stage at 95 °C and 40 cycles of 1 s at 95 °C for denaturation and 20 s at 60 °C for annealing/extension. The qRT-PCR reactions consisted of a reverse transcription stage of 5 min at 50 °C, RT inactivation stage for 20 s at 95 °C, and 40 cycles of 3 s at 95 °C for denaturation and 30 s at 60 °C for annealing/extension.

Samples were run in a 96-well plate format, with 8 water negative controls and 1 positive control per plate. A cycle threshold less than 40 signified a positive sample.

Statistical analyses

Significance was tested using Fisher’s exact test for categorical (e.g. presence or absence) data or MannWhitney U test or Kruskal-Wallis test for continuous data as the data were not normally distributed. Statistical analyses were performed using Graphpad Prism (v9.0.1). p-Values < .05 were considered statistically significant. All p-values were two-tailed.

Results

We analyzed 1044 total samples with gestational ages ranging from 26 to 42 weeks. Placental pathology was available for 456 neonates. We detected virus in 58 (5.6%) of 1044 cord blood plasma samples. One sample was positive for two viruses (alphatorquevirus and betatorquevirus) and one sample was positive for three viruses (alphatorquevirus, betatorquevirus, and HHV6). Individual viral prevalence ranged from 0.1% to 2% (Figure 1). The most common viruses detected were alphatorquevirus and HHV6. The average maternal age, gestational age of the neonate, and birth measurements were not different between infants with and without virus detected (Table 1). There were no differences in adverse pregnancy outcomes including pre-eclampsia, histologic chorioamnionitis, gestational diabetes, or intra-uterine growth restriction (IUGR) between samples positve for virus (Table 1) or by individual virus (Table 2).

Figure 1.

Viral prevalence in neonatal cord blood samples for nine viruses. Prevalence is shown in the total cohort, as well as by term and preterm birth status. There were no differences in overall viral prevalence or individual virus prevalence between term or preterm infants.

Table 1.

Demographic data comparing neonates with and without virus detected. Interquartile range or percent in parentheses.

	Positive samples n = 58 Median (IQR) or no. (%)	Negative samples n = 986 Median (IQR) or no. (%)	p-Value
Maternal Age (years)	27 (23–32)	28 (23–32)	.24
Gestational Age (weeks)	39 (37–39)	39 (37–39)	.67
Birthweight (grams)	3010 (2598–3488)	3140 (2730–3480)	.26
Length (cm)	50 (47.4–51)	50 (48–52)	.29
OFC (cm)	33.5 (31.7–35)	33.5 (32.5–34.5)	.40
Female	33 (56.9)	483 (48.9)	.4
5-minute APGAR	9 (9–9)	9 (9–9)	.40
C-section	19 (32.8)	315 (31.9)	.6
Preterm Labor	7 (12.1)	94 (9.5)	.49
AROM	32 (55.2)	608 (61.7)	.62
GBS Unknown	7 (12.1)	129 (13.1)	1
GBS Positive	16 (27.6)	240 (24.6)	.57
IUGR	6 (10.3)	87 (8.8)	.83
Pre-Eclampsia	8 (13.8)	149 (15.1)	.61
Gestational Diabetes	4 (6.9)	62 (6.5)	.33
Histologic Chorioamnionitis	5 (20.8)*	96 (20.4)*	.83
Lymphohistiocytic Villitis	1 (4.2)*	34 (7.2)*	.71
Decidual Vasculopathy	0 (0)*	30 (6.4)*	1

Fisher’s exact test and Mann-Whitney U-test was used to calculate p-values.

^*Placental pathology was available for 465 of infants.

Table 2.

Demographic and pregnancy co-morbidities for the seven most prevalent viruses.

‘Variable (Median (IQR) or n. (percent))	Alphatorque-virus (n = 21)	HHV6 (n = 13)	Parvovirus B19 (n = 7)	Enterovirus (n = 7)	CMV (n = 4)	Betatorque-virus (n = 4)	Parechovirus (n = 3)
Gestational Age	38 (37–39)	37 (34–40)	38 (38–39)	37 (36–39)	38 (38–38)	38 (38–40)	39 (39–39)
Birthweight	3.0 (2.6–3.4)	3.0 (2.2–3.5)	3.1 (2.8–3.3)	2.8 (2.6–3.3)	3.0 (2.6–3.2)	3.1 (2.7–3.7)	3.2 (3.1–3.4)
OFC	33.5 (32.5–35)	33.2 (32–35)	33.0 (31.6–34.5)	32.5 (30.8–34)	33.4 (32.9–34.2)	33.4 (32.4–35)	33 (32.2–34)
IUGR	2 (9.5)	0	0	1 (14.3)	1 (25)	2 (50)	1 (33)
Pre-Eclampsia	2 (9.5)	2 (15.3)	1 (14.3)	1 (14.3)	0	0	1 (33)
Gestational Diabetes	1 (4.8)	0	0	1 (14.3)	0	0	0
Preterm Labor	2 (9.5)	4 (30.7)	0	1 (14.3)	0	1 (25)	0

There are no statistically significant differences in outcomes by individual virus. .

Viruses were most frequently detected in cord blood from infants born at 32 weeks (14.3%) and 33 weeks (11.1%). Interestingly, no viruses were detected in cord bloods from children born less than 32 weeks (n = 11) or those born after 40 weeks (n = 31). There was no statistical difference in individual virus prevalence or viral loads for any agent by gestational age at birth.

Cord blood samples collected between the months of September and November most frequently were positive for virus (p = .027) (Figure 2). In contrast samples collected between December and February had the fewest number of positive samples. We next examined seasonal prevalence of each individual virus. There was an increase in HHV6 (p = .065) and enterovirus (p = .026) in the samples obtained during September to November (Figure 2). There was no statistically significant difference in viral load by season.

Figure 2.

Virus prevalence by season. The four most prevalent viruses are displayed, with the prevalence of all viruses tested (total) for each season.

Discussion

In this study we sought to identify prevalence of nine viruses in a large cohort of neonatal cord blood samples and to identify whether viral prevalence correlated with adverse pregnancy outcomes including prematurity, preterm labor, chorioamnionitis, or preeclampsia. We did not find differences in pregnancy outcomes for cord blood samples with or without viral nucleic acid detected nor for individual viruses. Our findings are consistent with those from a previous study reporting a smaller cohort of preterm infants and their mothers [12].

We detected an overall viral prevalence of 5.6%, with alphatorquevirus being the most prevalent at 2%. Alphatorquevirus has been demonstrated in cord blood between 0% and 48% [15,16], and our data are similar with those found in a preterm cohort (1.7%) [12]. While this virus is clearly present in a small number of neonates at birth, its impact on pregnancy or birth outcomes remains unknown [12,21]. HHV6 was the next most common virus with 1.2% of samples positive, and this is consistent with prior studies reporting HHV6 in 1–3.6% of cord blood samples [12,22]. Integration of HHV6 into the human genome is well described, so the positive samples we identified may reflect viral integration. This is less likely since these are plasma samples and we feel this more likely represents true viremia in the neonatal population [23,24].

Enterovirus and parvovirus B19 were the next most commonly detected viruses at 0.7% each. Enteroviral infections are often mild and self-limited in older children, but can present as a severe infection in neonates with shock and cardiovascular collapse [25]. We did not collect long term outcome measures on our cohort so we do not know if these infants developed severe disease, however, our findings add further evidence that enteroviral infections in neonates may be acquired in utero. Parvovirus B19 causes fetal infection by vertical transmission and 1.5% of pregnant women have seroconversion each year [26]. The risk to the fetus is most severe with infection in the first trimester and can result in non-immune hydrops secondary to severe anemia which may result in preterm delivery [26,27]. We demonstrate similar prevalence of parvovirus B19 among term and preterm gestations in our cohort, and our positive cases may represent asymptomatic neonates, as none were delivered for hydrops and all were well at birth. The prevalence of CMV was 0.4% and is consistent with prior reports, although all of our positives were term infants, and we did not find any positive results in preterm infants [13,14]

The overall prevalence of viral detection did not differ between term (5.3%) and preterm samples (6.6%) (p = .47). There were no positive samples from infants less than 32 weeks estimated gestation, although the number of infants born at less than 32 weeks was small (n = 11). Positive viral results were most frequently identified among infants born at 32 weeks (14.3%) and 33 weeks (11.1%), although these findings were not statistically significant. This finding suggests increasing viral prevalence at low gestational ages and warrants further investigation. Investigation in the extremely low birth weight population remains a challenge due to the difficulty obtaining sufficient cord blood sample volumes in these infants.

We found increased viral detection in infants born in the fall (September–November) as compared to other seasons (p = .026). Of note, we identified increased frequency of positive results in the summer and fall primarily due to enteroviruses reflecting their known seasonal pattern [25,28]. We found HHV6 prevalence was highest in the fall as well, although there is not a described season to HHV6 infections [22,29]. The enterovirus findings suggest that viral prevalence in neonates may follow the seasonal patterns seen in the general population. In contrast, parvovirus B19 is most prevalent in late winter and early spring which we did not identify in our cohort [26]. Further investigation into viral prevalence by season is warranted.

The strengths of this paper are the large cohort of term and preterm infants and the large number of pregnancy outcomes collected. To the best of our knowledge, this is the largest cohort to examine the prevalence of these nine viruses in term and preterm neonatal cord blood samples.

We acknowledge several limitations of this paper, such as absence of corresponding maternal samples to assess for viral presence in the mother-neonate dyad. In addition, as the samples were remnant samples from clinical specimens, there was variation in the time prior to sample freezing which may contribute to decay of nucleic acid (particularly RNA) and may decrease the sensitivity of our findings. We also had a limited number of samples from earlier gestational ages, which is likely secondary to the lack of sufficient remnant sample volume following clinical RPR analysis.

In conclusion, this work demonstrates that the nine viral targets we investigated occur in cord blood samples at low frequencies. We did not find an association with viral prevalence and adverse pregnancy outcomes including preterm birth, chorioamnionitis, preeclampsia, or birth measurements. We also did not find a difference in viral prevalence among term vs. preterm infants, although the increased prevalence of viral nucleic acid among samples from 32 and 33 week infants, while not statistically significant, was intriguing. We observed seasonal differences in viral prevalence in neonatal cord bloods. These data argue against the concept that a single virus drives pregnancy and neonatal outcomes. Future investigations of microbial drivers of adverse pregnancy outcomes should be focused on the complex interplay of viruses, specific bacteria, and bacterial communities.