INTRODUCTION:

JC virus (JCV) is a member of the Polyomaviridae family, including naked DNA viruses with icosahedral capsids and small, circular, and double-stranded DNA genomes (1). Progressive multifocal leukoencephalopathy (PML) is a distinct disease entity, first described in 1958 based on its characteristic demyelinating patterns (2).

Progressive multifocal Leukoencephalopathy (PML) is a severe demyelinating disease of the central nervous system. It is caused by JC virus (JCV), a polyoma virus found worldwide. As seroprevalence is high, at up to 80%, latent persistent infection is assumed(3).

Diagnosis of PML reside on the demonstration of demyelinating lesions in brain biopsy in patients suffering from typical signs and symptoms supported by viral isolation in cerebrospinal fluid (CSF) and blood(4). However, it is not always possible to take brain biopsy either due to lack of patients will or the inavailability of well equipped centers to perform and to histologically read brain biopsy. One the other hand serological detection of viral antibodies lacks the desired sensitivity since many published articles gave different rate of serological detection of ant-JC viral antibody (5-7).

Molecular methods utilizing polymerase chain reaction (PCR) techniques have been suggested to aid in the isolation and characterization of JC virus in patients suspected with PML(8). Nevertheless, the sensitivity of this technique was far less than that described for serological tests in most published literatures. PCR methods have been applied to various samples including blood, urine and CSF; however, none of them considered a gold standard to replace the brain biopsy in establishing the diagnosis of PML caused by JC virus infection(9).

Several authors have tried to cultivate and isolate the JC virus using different cell lines but most literatures favored human kidney cell line(10-12) (Saribas. For this reason the current study was tailored to involve Real time PCR applied on blood samples and also human kidney cell line in order to cultivate the JC virus.

The rarity of local Iraqi literatures about PML and JC virus, in addition to the inconsistent sensitivity of serological method in establishing the diagnosis of PML, this study was designed.

The aim of the present study was to find out the validity of RT-PCR and human kidney cell line cultures as adjuvant to aid in the diagnosis of PML.

MATERIALS AND METHODS:

Patients and samples:

The present cross sectional study included 61 Iraqi patients, diagnosed as having multifocal leukoencephalopathy according to clinical and radiologic evidences. Their age range was from 16 to 65 years and they were 10 males and 51 females. Blood samples were obtained from all patients and subjected to PCR analysis for viral DNA detection and for serologic identification of anti-JC virus antibody. Real-Time PCR was performed for detection of JC polyomavirus by using the specific primers and probe for large T antigen (LTA) gene. The primers and probe were designed in this study by using the complete sequence of John Cunningham virus (JC virus)large T antigen (LTA) gene, complete cds (GenBank: AF015527.1) using NCBI-Genbank and Primer3 plus design, and these primers and probe were used in Real-Time PCR assay for rapid detection of JC virus in human blood. Also another PCR primer for VP1 gene was used in PCR amplification that used in DNA sequence assay and phylogenetic tree analysis. The primers were provided by (Bioneer. Company, Korea).

Human Kidney cell line (Vero) was supplied by Iraqi Biotechnology Company (iRAQBiotech) Baghdad, Iraq. cells were Cultured as an adherent monolayer in complete medium at 37°C in a humidified atmosphere. Cells become confluent in 2–5 days. They passaged at a ratio of 1_3once they become confluent. Cells were treated with 4 ml diluted JCV sample (enoughto just cover the surface of the dish). The procedure of adding JCV sample was by adding the virus at first for 2 hrs at 37C to allow virus attachment and penetration. The flasks were gently rocked every 15 min to ensure proper distribution of the inoculum. After that, serum free medium was added, after five days cells were examined under inverted microscope for characteristics featured of JCV cytopathic formation.

Statistical analysis:

Statistical analysis was carried out using SPSS version 23. Variables were expressed as number, percentage, mean, standard deviation (SD), range, median and inter-quartile range (IQR). The level of significance was considered at P<0.05.

RESULTS:

The current cross sectional study included 61 patients with clinical diagnosis of progressive multifocal leukoencephalopathy (PML) based on clinical, radiological (MRI) and cerebrospinal fluid (CSF) findings.

Demographic characteristics of the study group:

The study included patients with an age range of 16 to 65 years old and the mean age of all patients enrolled in the present study was 37.41 ± 9.76. Majority of cases (37 out of 61) were less than 40 years old which forms 60.7% of the entire sample of PML patients. The study sample included 10 male patients and 51 female patients and the male to female ratio was 1: 5.1. Patients who were with jobs and or students accounted for 37.7% of cases, whereas, patients without job (retired and or housewives) accounted for 62.3%, as shown in table 1.

Table 1:Demographic characteristics of the study sample

Characteristics	Mean ±SD	Range (minimum – maximum)	Number (%)
Age (years)	37.41 ± 9.76	49 (16 -65)
Male patients			10 (16.4)
Female patients			51 (85.6)
Patients having Job or student			23 (37.7)
Patients without job			38 (62.3)

Molecular study:

The RT-PCR results of amplification plots of large T antigen (LTA) gene in JC polyoma virus based on TaqMan hybridization probe were shown in figure (1), in which, the positive amplification samples showed crossing up the threshold cycle number. The Real-Time PCR endpoint analysis of large T antigen (LTA) gene in JC polyoma virus was shown in figure (2) that demonstrated some positive and negative amplification.Real-Time PCR threshold cycle number of large T antigen (LTA) gene in JC polyoma virus was shown in figure (3). The figure showed that the positive samples (14 out of 61) were apparent at 12.80-22.15 qPCRcycle whereas the negative showed as non-amplification (NA).

Figure 1:Real-Time PCR amplification plots of large T antigen (LTA) gene in JC polyoma virus based on TaqMan hybridization probe. The positive amplification samples crossed up the threshold cycle number.

RFU: Relative Fluorescence Units

Figure 2:Real-Time PCR endpoint analysis of large T antigen (LTA) gene in JC polyoma virus with some positive and negative amplification.

Figure 3:Real-Time PCR threshold cycle number of large T antigen (LTA) gene in JC polyoma virus. Where, the positive samples appeared 12.80-22.15 qPCRcycle and the negative showed noamplification (NA).

The results of DNA sequencing are shown in figure (4) and figure (5) were labeled in the gene bank as Homology sequence identity of JC polyomavirus VP1 gene Iraq isolate 1 to NCBI-Blast JC polyomavirus type 1 VP1 gene and Homology sequence identity of JC polyomavirus VP1 gene Iraq isolate 2to NCBI-Blast JC polyomavirus type 1 VP1 gene.

Figure (6) showed the Phylogenetic tree analysis based on envelope protein (VP1) gene partial sequence that used for JC polyomavirusGenotyping identification. The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version). The local JC polyomavirus iraq1 and iraq2 isolates showed close relation to NCBI-Blast JC polyomavirus type 1(VP1) geneisolate (AF368028.1) .Whereas, other NCBI-Blast JC polyomavirustypes were different and out of tree.

Table (2) shows the probability of substitution (r) from one base (row) to another base (column)[1]. For simplicity, the sum of r values is made equal to 100. Rates of different transitional substitutions are shown in bold and those of transversionsal substitutions are shown in italics. The nucleotide frequencies are 29.48% (A), 17.73% (T), 17.47% (C), and 35.32% (G). The analysis involved 9 nucleotide sequences. Codon positions included were 1st. All ambiguous positions were removed for each sequence pair. There were a total of 355 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Table 2:Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution

	A	T	C	G
A	-	4.29	4.23	10.8
T	7.14	-	15.77	8.55
C	7.14	16	-	8.55
G	9.02	4.29	4.23	-

Analysis of PCR Results and correlation with serum anti JC virus antibody results:

Real time PCR analysis of blood samples obtained from patients showed a positive detection rate of 14 out of 61 (23%).According to patients registered data, the two step enzyme linked immunosorbant assay (ELISA), already performed at hospital laboratory, for the serologic detection of antibodies against JCV virus in the sera of patients enrolled in the present study showed a positive rate of detection of 40 out of 61 patients (65.6%). This rate of detection is much higher than the detection rate of viral antigen in the blood using RT-PCR which was (23%). One proportion Z test showed significant difference between the rate of detection of JC virus in the blood of patients by PCR and rate of detection of ant-viral antibody in sera of patients (P<0.05). However, 10 patients who were sero-negative showed evidence of viremia by PCR blood analysis for JC viral DNA, suggesting that the sensitivity of serology is not optimum.

Results of cell line culture and viral inoculation:

The monolayred cell lines that were infected with blood samples from patients were followed up weekly and changes were studied and reported. Figure (7) represents photomicrographs of human kindeny cell line 2 weeks after viral inoculation. Cellular detachments and cellular debris are readily recognized (arrows a and b). These changes may be due to the cytopathic effects of the inoculated virus. Further follow up showed more pronounced cellular detachments and formation of cell aggregates, as shown in figure (8) which represents human kidney cell line, 6 weeks after viral inoculation.

In order to be sure that these cytopathic effects are due to viral and not other cause, samples were taken from the cultured cell lines 6 weeks after being inoculated with patients blood and then they were analyzed using RT-PCR technique. The results documented the presence of viral DNA and this supported the belief that the cytopathic effect was due to viral infection rather than other environmental or artifactual effects.

Figure (7): Human kidney cell line; 2 weeks after JCV inoculation. Note the ctyopathic effect in the form of cellular detachment (arrow a) and cellular debris (arrow b).

Figure (8):Human kidney cell line; 2 weeks after JCV inoculation. Note the ctyopathic effect in the form of cellular detachment (arrow a) and cellular debris (arrow b).

DISCUSSION:

There is enough evidence in the published literatures to question the sensitivity of serological methods adopted to diagnose JC virus infection(13). In one of the studies, Viscidiet al., in 2011 found that 29% of patients with JCV viremia were seronegative, supporting the notion that the assay was not very sensitive.Viscidiet alsupposed that immune suppression is the cause behind lack of suitable antibody response to be detected by methods that rely on serology (14). It is a matter of fact that patients with acquired immune deficiency syndrome frequently develop PML due to JC virus infection (15, 16). So the lack of sufficient sensitivity was a great motive toward seeking another reliable technique to diagnose PML due to JC virus with excellent sensitivity.The frequency of JCV exposure in the population determined by antibody studies has been reported between 33% and 91%. These differences have been largely ascribed to differences in assay methods, although other factors may be important(17).

The second important reason for conducting this study was the lack of local Iraqi studies that deal with PML and JC virus infection.

The development of quantitative polymerase chain reaction (qPCR) assays provides a specific, sensitive and quantitative method to measure JC viral load(18). Pal et al designed sets of primer/probes for BKV, JCV and SV40 specific for both the early and late coding regions of each virus. These were characterized and it was shown that they could detect between 1–10 copies of their cognate genomes while failing to react with as many as 10⁸ copies of DNA from the other viruses. Thus qPCR assays have the capacity to enumerate small numbers of viral genomes in the presence of large amounts of unrelated DNA (18).

In the present study two JC viral isolates were isolated from blood samples of Iraqi patients suffering from clinical manifestation of PML. These isolates were identified by DNA sequencing and registered by gene bank under the names JC polyomavirus iraq1 and JC polyomavirus iraq2.

The identification of two distinct DNA sequences may support the findings of several authors that mutations in JC virus capsid viral protein 1 (VP1) is the initial step in the pathogenesis of PML (3). JCV establishes a chronic asymptomatic infection in the urinary tract in approximately 50% of the population and is excreted in urine in approximately one-third of healthy subjects(19, 20). The mechanisms leading to PML are mostly unknown, yet involve JCV reactivation in the context of compromised immune control. The major capsid viral protein 1 (VP1) is likely a key player in pathogenesis, as it mediates immune responses, as well as cell attachment(21) and viral cell entry(22) through sialic acid cell receptors. The VP1 gene is highly polymorphic; 13 distinct geographically associated subtypes have been identified(23). However, there is no clear evidence of an association between any JCV subtype and PML.

The laboratory work of the current study was successful in identification of JC virus genome in blood samples of patients with PML; however the rate of detection depending on RT-PCR technique was significantly lower than the rate of detection of anti-viral antibody in the sera of the patients, 23 % versus 65.6% and this may be explained by low rate of viremia in those patients at time of blood sample withdrawal.

Berger et al., (2013a)(13) analysed blood samples of 67 patients with PML and found seropositivity, assessed by ELISA, rate of 59.7%. This rate of serologic detection is in accordance with the findings of the present study (65.6%). Viscidiet al., in 2011 carried out a study to compare the serological detection rate with blood viral DNA rate in 83 patients with PML and found that serology detected the anti-JC viral antibody in 58% whereas, RT-PCR detected viral DNA in blood samples of 17% of the 83 patients with PML (14). These findings again supports the findings of the present study in that serological rate of detection is higher than the rate of viral DNA isolation in blood samples; however, the finding of viremia in serologically negative patients, as it was shown by the present study and the study carried out by Burger et al. (13) suggest the combination of both techniques in order to get optimum results for diagnosing JC virus infection.

Qiaoet al., 2016 tested the presence of JC virus DNA in blood samples of JC310 renal transplant patients and found that viremia was detected in 33.5% of patients, while serological results were far more higher than this rate (24). Again, these findings supports the results of the present stud that serology is more sensitive than PCR technique in detecting lab evidence for JC virus in blood of patients suspected to have PML.

In the current study, inoculation of human kidney cell line monolayer by blood samples of patients with PML showed evidence of cytopathic effect that was proved by PCR analysis of samples taken from culture media 6 weeks following inoculation.

In infected individuals, JCV can persist and replicate asymptomatically in the urinary tract and possibly other organs. In immunocompetent individuals, the virus is rarely found outside of the urinary tract. However, under conditions of severe immunosuppression or treatment with specific immunomodulating drugs, the virus may establish a lytic infection in oligodendrocytes, leading to PML.(25, 26).

For that reason it was suggested to use human kidney cell line to study the ability to isolate JC virus form blood samples of patients suspected to have PML. In addition it is well known than nervous tissue cell lines are very difficult to grow and need long time in order to get a well established monolayer (27).

A lot of published literatures have documented the ability to cultivate JC virus in kidney cell lines (10-12). For that reason we chosed the human kidney cell line and succeded in cultivating the JC virus following iinoculation of these cell lines with blood samples from patients with PML. So we recommend this method to be adopted to support the diagnosis when serology and molecular methods fails to establish the diagnosis.

In conclusion: Despite being of low sensitivity molecular isolation of JC virus DNA applied on blood samples improves the sensitivity of routine serologic methods and that human kidney cell line may add to the diagnostic workup of JV induce PML.

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Received on 05.02.2018 Modified on 19.04.2018

Research J. Pharm. and Tech 2018; 11(8): 3392-3398.

DOI: 10.5958/0974-360X.2018.00625.X