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  Table of Contents  
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 464-468

A real-time polymerase chain reaction-based approach for qualitative estimation of viral RNA in organ tissues of coxsackievirus A-16-infected neonatal mice

1 Enteric Viruses Group, ICMR-National Institute of Virology, Pune, Maharashtra, India
2 ICMR-National Institute of Virology, Mumbai Unit, Mumbai, Maharashtra, India

Date of Submission21-Mar-2020
Date of Decision14-Jul-2020
Date of Acceptance26-Aug-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Varanasi Gopalkrishna
ICMR-National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune - 411 001, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_20_117

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 ~ Abstract 

Hand, foot and mouth disease (HFMD) is a paediatric disease associated with enteroviruses (EVs). Among EVs, coxsackievirus A-16 (CVA-16) strain is currently in circulation and causing outbreaks in India. Neonatal mice (Institute of Cancer Research) strains were infected with CVA-16 strain isolated from HFMD patients to conduct pathological and molecular studies. Infected organs were harvested as per time points. A real-time polymerase chain reaction was used for qualitative estimation of viral RNA in organ tissues of infected mice. Skeletal muscle, brain tissue and cardiac tissues were the major target sites of CVA-16 tropism. The first-ever study was conducted on CVA-16 strains using the current approach in India.

Keywords: Institute of Cancer Research ( ICR), neonatal mice, organ tissues, real-time polymerase chain reaction, viral RNA

How to cite this article:
Tikute SS, Pawar SD, Kawale SS, Sharma DK, Gopalkrishna V. A real-time polymerase chain reaction-based approach for qualitative estimation of viral RNA in organ tissues of coxsackievirus A-16-infected neonatal mice. Indian J Med Microbiol 2020;38:464-8

How to cite this URL:
Tikute SS, Pawar SD, Kawale SS, Sharma DK, Gopalkrishna V. A real-time polymerase chain reaction-based approach for qualitative estimation of viral RNA in organ tissues of coxsackievirus A-16-infected neonatal mice. Indian J Med Microbiol [serial online] 2020 [cited 2021 Jan 17];38:464-8. Available from:

 ~ Introduction Top

Hand, foot and mouth disease (HFMD) is a childhood viral disease mostly observed in children aged below five years.[1] In general, the disease is self-limiting but may develop to cause serious complications such as brainstem encephalitis, aseptic meningitis, acute flaccid paralysis and pericardial disease.[2] The symptoms initiate with mild-to-moderate grade fever, mouth ulcers, pharyngitis and rashes on hand and vesicle formation on hand, elbow, knee, sole and buttocks. The disease is caused due to enteroviruses (EVs) of the family Picornaviridae. Among Enterovirus (EV) genus, enterovirus A-71 (EV-A71) and coxsackievirus A-16 (CVA-16) are the major etiological agents associated with the disease. Along with EV-A71 and CVA-16, other EV types such as CVA-10, CVA-6 and E-9 have also been found associated in sporadic cases.[3]

In India, cases of HFMD were diagnosed earlier based on clinical features of diseased patients.[4],[5] Subsequently, studies conducted from different geographical regions in India revealed CVA-16 as the major viral etiological agent in circulation as identified by molecular approach, i.e., reverse transcription–polymerase chain reaction (RT-PCR). However, no studies have so far been conducted to understand the pathogenicity of CVA-16 strains using neonatal mice and also qualitative estimation of viral RNA in different organ tissues of CVA-16-infected mice in order to understand the viral tropism. Keeping in view of the lacunae and importance, a qualitative real-time PCR-based approach was developed for the estimation of CVA-16 viral RNA in organ tissues of infected neonatal mice strains (ICR- Institute of Cancer Research) infected neonatal mice. The study is helpful to assess the viral tropism towards vital organ tissues of neonatal ICR mice infected with CVA-16 strain isolated from HFMD patient's samples. A real-time PCR-based approach for qualitative estimation of viral RNA in vital organ tissues of CVA-16-infected from clinical samples of HFMD patient. neonatal mice was conducted for the first time in India.

 ~ Materials and Methods Top

Clinical specimens

In India, HFMD cases were reported from different geographical regions. Clinical specimens (n = 68) such as throat swabs, vesicular swabs, rectal swabs and stool samples were collected from HFMD patients reported from Kerala (southern India), Bhubaneshwar and Kolkata (eastern India). The age group of the patients ranged from 10 months to 3.5 years. The project was approved by both the Institutional Biosafety and Animal Ethical Committees. Prior informed consent from the children's parents/guardian was obtained before collecting specimens. Clinical specimens (n = 68) were subjected to EV-RNA detection by RT-PCR of primers targeting 5'NCR region. Molecular typing of the EV-positive samples was carried out by VP1 gene amplification followed by cycle sequencing.

Virus isolation and propagation

EV-positive samples as detected by RT-PCR and typed by VP1 gene amplification were subjected to virus isolation. Isolation of the virus was carried out in susceptible RD cell line in 24 well culture plates (Nunc, Roskilde, Denmark). Clinical specimens were taken as undiluted and 1:10 dilution and inoculated onto RD cell lines with appropriate cell controls. RD cell line was grown in minimum essential medium (MEM, HiMedia, India) along with 10% foetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin. The infected cultures were incubated at 37°C and supplemented with 5% CO2. The cultures were observed daily for any morphological changes and harvested accordingly. The cultures showing cytopathic effect (CPE) were passaged out up to the 4th passage level (P-4). Confirmation of the isolate was carried out by testing the presence of EV-RNA and further typed by VP1 gene amplification. The isolated virus strain was confirmed as CVA-16 based on cycle sequencing of the amplimers. The CVA-16 strain was isolated from the clinical specimen (vesicular swab) collected from a 3½-year-old HFMD male patient belonged to Kolkata (West Bengal), eastern region of India. The patient was presented with rashes on hands, feet, buttocks and face with mouth ulcers. The virus isolate obtained was further propagated by scaling up in RD cell line using T-25, T-75 and T-225 cm2 tissue culture flasks (Corning, USA). The viral lysate obtained at P-4 level was harvested and culture flasks were freeze-thawed thrice, and lysate was centrifuged at 5000 rpm at 4°C for 20 min to remove the cell debris. The virus titre was estimated in RD cell cultures using a 96-well plate (Nunc) by performing 10-fold dilutions from 101 to 108/ml in MEM. Subsequently, 100 μl of each dilution was inoculated onto cell cultures in a 96-well culture plate along with cell controls fed with plain MEM. The culture plates were kept for virus adsorption for 1 h at 37°C, and to this, 900 μl of MEM was added to each well. Culture plate was observed for 4–5 days for any development of CPE.

Mouse experiments

The neonatal mouse strain (ICR-strain) was selected to conduct experiments and to understand the virus tropism towards different infected mouse organs such as brain, heart, lungs, liver, spleen, kidney, intestine and skeletal muscle.[6],[7]. The study was approved by the Institutional Animal Ethical Committee and the Institutional Biosafety Committee. The CVA-16 isolate confirmed with 2 logs of 105 TCID50/ml in 80–100 μl was administered via intraperitoneal route in neonatal ICR mice.[6],[8],[9] Control group mice were administered with normal tissue culture fluid. Four groups (one group contained eight neonates and mother) of neonatal ICR mice were included for the experiment along with the control group. Experimental and control group mice were observed twice a day for any clinical signs and symptoms of the disease.[10] The mice from both the groups were harvested on day 1, day 3, day 5, day 7 and day 9 of post-inoculation day (PID).[11] Organs such as brain, heart, lung, liver, spleen, intestine, kidney and skeletal muscle were collected aseptically and stored in RNA later at appropriate temperature.

Detection of enterovirus RNA in organ tissues of infected neonatal mice

Eighty to hundred milligrams of organ tissues from both the experimental and control groups was used for preparation of organ tissue suspensions. MEM was used as a diluent for preparing these suspensions. Organs recovered from the control group mice were initially processed, followed with experimental mouse organs for suspensions to avoid cross-contamination. RNA extraction was carried out from these homogenized suspensions as per time points using QIAamp Viral RNA Kit (QIAGEN, Germany). Detection was carried out by RT-PCR.[12]

Molecular typing of enteroviruses

The EV-positive samples were further typed by full-length VP1 gene amplification using SuperScript II One-step RT-PCR Platinum Taq HiFi Kit (Invitrogen, USA).[13],[14],[15] The primer sequences used in the study are mentioned in [Table 1].
Table 1: Primer sequences used for VP1 gene typing

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Estimation of enterovirus RNA in experimental neonatal mouse organs using real-time polymerase chain reaction

Estimation of EV-RNA in organ tissues of CVA-16-infected experimental mice was carried out by real-time PCR. Organ tissues obtained from the brain, heart, lung, liver, spleen, intestine, kidney and skeletal muscle at different time points, i.e., day 1, day 3, day 5, day 7 and day 9 PID of infected mice, were subjected for EV-RNA estimation. Organ suspensions were prepared from the tissues using MEM as a diluent, as mentioned earlier. RNA extraction was carried out from the homogenised suspensions, as described earlier. Extracted viral RNA was further subjected to qualitative real-time PCR using ITD kit (supplied by the World Health Organization, Geneva). The procedure was carried out in 7500 Real-Time PCR System (Applied Biosystems, USA) by RT inactivation at 95°C for 1 min, denaturation at 95°C for 15 s and annealing at 50°C for 45 s, and a total of 40 cycles were carried out. standardization of real-time PCR assay was performed using viral RNA extracted from CVA-16 isolate which was used initially for mouse experiments. The cycle threshold (CT) values obtained by the assay of different organ tissues at different time points were compared with the standard CT values for interpretation.

 ~ Results Top

Detection and molecular typing of enteroviruses

Twenty-eight of the 68 clinical specimens (41.2%) tested were detected positive for EV-RNA by RT-PCR of primers targeted 5'NCR. Of these, 16 of 28 (57.2%) EV positives were further typed and confirmed as CVA-16 based on VP1 gene amplification and sequence confirmation.

Virus isolation

All the sixteen CVA-16-type confirmed positive samples were successfully isolated in RD cell cultures. The isolates were further passaged in RD cell line up to passage level 4 (P-4) and propagated in T25, T75 and T225 cm2 tissue culture flasks. Electron microscopic observation revealed the presence of picornavirus-like particles. Serial tenfold dilution of the virus titration was conducted using 96-well plates, which showed 105/ml of virus titre.

Mouse inoculation experiments

Neonatal ICR mouse strains were inoculated with 2 logs of TCID50/ml (80–100 μl) of the CVA-16 isolate by IP route, and infected mice were observed twice in a day for development of any clinical signs or symptoms. The mice started showing signs and symptoms of the disease and succumbed to the infection on day 3 PID. Further, weight loss, hunched posture, difficulty in movement and limb paralysis were observed. Infected mice were harvested as per time points, as mentioned earlier along with control mice.

Molecular studies

In order to determine the distribution and spread of the CVA-16 in different organs of mice, tissue suspensions were made from the infected mouse organs and were subjected for EV-RNA detection by RT-PCR as per different set time points. The study revealed that the presence of EV was observed in organ tissues of the brain and heart on day 1 of PID, while in other organ tissues such as liver, kidney, intestine and skeletal muscle, the presence of virus was observed on day 3 onwards. [16,17]

Qualitative estimation of viral RNA in organ tissues of neonatal mice by real-time polymerase chain reaction

Real-time PCR for estimation of EV-RNA in different organ tissues of ICR neonatal mice is as follows.

Viral RNA estimated in skeletal muscle tissues showed the CT values of 13.054 and 12.88 respectively, on day 3 and 5 PIDs. However, 21.24 and 21.72 CT values were observed on day 7 and 9 PIDs. An higher amount of viral RNA was detected on day 5 of PID. Viral RNA detected in brain tissues showed, CT values of 26.58 and 27.0 on day 3 and 5 PIDs respectively. The positive control used in the ITD kit indicated 24.74 CT value [Figure 1].
Figure 1: Qualitative estimation of enterovirus RNA in skeletal muscle and brain tissues of infected mice using real-time polymerase chain reaction

Click here to view

The CT value obtained in real-time PCR in skeletal muscle tissue RNA on day 5 PID indicates an elevated copy number of EV-RNA. The cardiac muscle and brain tissue RNA, respectively, showed a comparatively low copy number of viral RNA by indicating 25 and 26.58 CT values and also suggested a higher copy number of EV-RNA detected on day 5 of PID.

The qualitative EV-RNA estimated by real-time PCR indicated that experimental neonatal mice inoculated with CVA-16 isolate showed active replication of EV in the vital organs from day 3 PID onwards. The mouse organs such as brain, heart and skeletal muscle are the major targeted sites of viral tropism. However, other infected organ tissues showed lower EV-RNA levels, as indicated by their CT values in organ tissues of infected neonatal [Table 2] and [Figure 2] after mouse strain.
Table 2: Real-time polymerase chain reaction estimation of enterovirus RNA in different organ tissues of Neonatal mice at different time points

Click here to view
Figure 2: Overview of qualitative enterovirus RNA estimated in different organ tissues of Infected Neonatal mice at various time points by real-time polymerase chain reaction

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 ~ Discussion Top

A real-time PCR-based approach for qualitative estimation of viral RNA in organ tissues of CVA-16-infected neonatal mouse strain was developed in order to understand the tropism of CVA-16 towards organs of infected mice showing pathogenicity in causing disease. In this study, virus-infected mouse organs such as brain, heart and skeletal muscle are the major target organs that showed pathological changes and tend to be more affected; these were followed by intestine and kidney. Qualitative estimation of EV-RNA by real-time PCR in various organ tissues indicated that skeletal muscle tissue on day 3 PID reveals higher EV-RNA followed by brain and cardiac tissues on day 5 and day 7 PID, respectively. These findings further suggest the viral tropism in different target organs of CVA-16-infected neonatal mice. Studies have been conducted earlier on CVA-16 for vaccine evaluation in a neonatal mouse model and CVA-16 infection for antiviral evaluation using a murine model.[10.11]

Although studies are available from other parts of the world, such studies are very much lacking from the Indian context where CVA-16 is the major EV strain predominantly in circulation and causing HFMD outbreaks. In the present study, a real-time PCR which is a very sensitive and useful method over conventional PCR was applied for qualitative estimation of CVA-16 viral RNA in organ tissues of infected neonatal mice. The study helped in identifying the major target organs towards CVA-16 viral tropism in neonatal mice. Such type of studies was conducted for the first time in the Indian context.

The outcome of the current study and leads obtained will be further useful in development and evaluation of different antivirals against CVA-16 strains circulating in India using neonatal mice models and also for vaccine evaluation experiments. Over and above, systemic hospital-based surveillance studies on HFMD and identifying the predominant circulating EV strains across the country and understanding pathogenicity of the strains using the current approach will be very much useful in development of intervention strategies to control HFMD, a childhood viral disease of global public health concern.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 ~ References Top

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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