Specific and quantitative detection of Human polyomaviruses BKPyV and JCPyV in the healthy Pakistani population

Abstract

Background: The BK Polyomavirus (BKPyV) and JC polyomavirus (JCPyV) infections are widespread in human population and have been associated with severe kidney and brain disorders, respectively. The viruses remain latent primarily in reno-urinary tract, reactivating only in case of a compromised immune system. The seroepidemiology and molecular prevalence of BKPyV and JCPyV have been widely studied both in healthy and immunocompromised patients worldwide. However, data regarding the prevalence of these viruses in the immunocompetent or apparently healthy Pakistani population is lacking. Herein, we present the first ever report on quantitative prevalence of BKPyV and JCPyV in the peripheral blood of a randomly selected cohort of healthy Pakistani population.

Methods: A total of 266 whole blood samples were examined. The subjects were divided into three age groups: ≤ 25 years (young), 26-50 years (middle) and ≥ 51 years (elder). Absolute real time PCR assay was designed to quantify the BKPyV and JCPyV viral copy numbers in the range of 10⁶ to 10⁰ copies/mL.

Results: Overall, BKPyV was detected in 27.1% (72/266) individuals while JCPyV in 11.6% (31/266) indicating significant difference (p < 0.005) in the distribution of these two viruses. The prevalence of BKPyV significantly decreased from 51% (49/96) in young age group to 8.2% (7/85) in eldest age group. Whereas, JCPyV positivity rate slightly increased from 8.3% (8/96) in young age group to 11.8% (10/85) in elder age group. The median viral load was calculated as 6.2 log and 3.38 log copies/mL of blood for BKPyV and JCPyV, respectively. Notably, no significant difference in viral load of either of the subtypes was found between different age groups.

Conclusion: The current study provides an important baseline data on the prevalence and viral load of circulating BKPyV and JCPyV in Pakistani population. The prevalence and viral load of BKPyV was comparatively higher than JCPyV. The prevalence of BKPyV significantly decreased with increase in age while JCPyV positivity rate slightly increased with increasing age. Viral load of both BKPyV and JCPyV was not correlated with the individual ages.

Keywords: BKPyV; Healthy blood; JCPyV; Pakistan; qPCR.

Fig. 1

Amplification and standard curves for BKPyV dilutions. A standard curve was generated using duplicate ten-fold serial dilutions of purified LT-BKPyV-pcDNA3 template. The linear range was set to lie between 10⁶ to 10⁰ copies/reaction. The assay efficiency was checked by (a) amplification plots of BKPyV standards dilution (b) Melt curves analysis for BKPyV target gene with a peak at 74 °C (c) standard curve analysis for BKPyV dilutions. The assay efficiency was calculated 110% with R2 = 0.98. d The amplified product of each dilution was analyzed on agarose gel. Lane 1: molecular mass marker; Lane 2–8: dilution (copy number 10⁶ to 10⁰); Lane 9: negative control

Fig. 2

Amplification and standard curves for JCPyV dilutions. A standard curve was generated using duplicate ten-fold serial dilutions of purified LT-JCPyV-pcDNA3 template. The linear range was set to lie between 10⁶ to 10⁰ copies/reaction. The assay efficiency was checked by (a) amplification plots of JCPyV standards dilution (b) Melt curves analysis for JCPyV target gene with a peak at 77 °C (c) standard curve analysis for JCPyV dilutions. The assay efficiency was calculated 109% with R2 = 0.99. d The amplified product of each dilution was analyzed on agarose gel. Lane 1: molecular mass marker; Lane 2–8: dilution (copy number 10⁶ to 10⁰); Lane 9: negative control

Fig. 3

Real time assay for the quantification of BKPyV in healthy blood samples. The qPCR was performed by using 30 ng DNA of each sample and virus copy number was determined by comparing with standard curve of known BKPyV dilutions. a Amplification plots of some representative positive samples for BKPyV. b The amplified product of these representative positive samples was analyzed by gel electrophoresis. The amplified product showed band of 151 bps corresponding to BKPyV LT. Lane 1: molecular mass marker; Lane 2–13: representative positive samples for BKPyV; Lane 14: negative control

Fig. 4

Real time assay for the quantification of JCPyV in healthy blood samples. The qPCR was performed by using 30 ng DNA of each sample and virus copy number was determined by comparing with standard curve of known JCPyV dilutions. a Amplification plots of some representative positive samples for JCPyV. b The amplified product of these representative positive samples was analyzed by gel electrophoresis. The amplified product showed band of 177 bps corresponding to JCV LT. Lane 1: molecular mass marker; Lane 2–14: representative positive samples for JCPyV; Lane 15: negative control

Fig. 5

The prevalence of BKPyV and JCPyV in the blood of healthy Pakistani population. Overall 266 blood samples from healthy individuals of Pakistan were analyzed for the presence of BKPyV and JCPyV. a The bar graph shows the overall and age-wise prevalence of BKPyV in the studied population. b The bar graph shows the overall and age wise prevalence of JCPyV in the studied population

Fig. 6

The age specific prevalence and copy number of BKPyV and JCPyV in the blood of healthy Pakistani population. Overall 266 blood samples from healthy individuals of Pakistan were analyzed for the presence of BKPyV and JCPyV. The virus copy numbers were also calculated for positive samples. The virus load in the positive samples was expressed as log copy number/mL. a Box-whisker plots show the BKPyV positive samples and range of virus copy number in the positive samples of overall and three age groups (≤25 years, 26–50 years and ≥ 51 years). b Box-whisker plots show the JCPyV positive samples and range of virus copy number in the positive samples of overall and three age groups (≤25 years, 26–50 years and ≥ 51 years)

Fig. 7

A comparison of the prevalence as well as viral between BKPyV and JCPyV in the blood of healthy Pakistani population. Overall 266 blood samples from healthy individuals of Pakistan were analyzed for the presence of BKPyV and JCPyV. a The bar graph shows the overall difference among the prevalence of BKPyV and JCPyV in the studied population. b Box-whisker plots compares the BKPyV and JCPyV range of virus copy number in the positive samples of overall population