Paper-based archiving of biological samples

from fish for detecting betanodavirus

A.Navaneeth Krishnan,
T.Bhuvaneswari, P.Ezhil Praveena &
K.P.Jithendran

Archives of Virology
Official Journal of the Virology
Division of the International Union of
Microbiological Societies

ISSN 0304-8608
Volume 161
Number 7

Arch Virol (2016) 161:2019-2024

DOI 10.1007/s00705-016-2875-y

1 23
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1 23
 Author's personal copy
Arch Virol (2016) 161:20192024
DOI 10.1007/s00705-016-2875-y
BRIEF REPORT
Paper-based archiving of biological samples from fish
for detecting betanodavirus
A. Navaneeth Krishnan1 T. Bhuvaneswari1 P. Ezhil Praveena1
K. P. Jithendran1
Received: 11 December 2015 / Accepted: 22 April 2016 / Published online: 4 May 2016
Springer-Verlag Wien 2016
Abstract This study was carried out to evaluate the effi-
ciency of the Flinders Technology Associates (FTA) card
(Whatman) as a sampling device and storage platform for
RNA from betanodavirus-infected biological samples (viz.,
larvae, broodstock, cell culture supernatants and rearing
seawater spiked with infected materials). The study showed
that FTA cards can be used to detect betanodaviruses by
reverse transcription-polymerase chain reaction (RT-PCR).
The diagnostic efficiency of RT-PCR from all sample types
on FTA cards decreased after 21 days of storage at 4 C,
although the virus could be detected up to 28 days by
nested RT-PCR. The FTA card protocol thus provides a
supplementary method for quick and easy collection of
samples, preservation of RNA on a dry storage basis, and
detection of betanodavirus-infected fish.
Viral encephalopathy and retinopathy (VER), otherwise
known as viral nervous necrosis (VNN), is an infectious
neuropathological disease affecting more than 50 species
of marine and freshwater fish worldwide [12, 19]. Recur-
rent outbreaks of the disease, resulting in nearly 100 %
mortality within 2-3 days of onset of clinical signs during
production cycle of marine fish, are a global concern,
including in India [9]. The etiological agents of VER are
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-016-2875-y) contains supplementary
material, which is available to authorized users.
& K. P. Jithendran
kpjithendran@yahoo.com
1 In the present study, we report the use of the Flinders
Aquatic Animal Health and Environment Division, Central
Institute of Brackishwater Aquaculture, 75, Santhome High
Road, R.A Puram, Chennai 600 028, Tamil Nadu, India
members of the genus Betanodavirus of the family No-
daviridae. Based on phylogenetic analysis of the variable
region of the coat protein (CP) gene, striped jack nervous
necrosis virus (SJNNV), tiger puffer nervous necrosis virus
(TPNNV), barfin flounder nervous necrosis virus
(BFNNV), red spotted grouper nervous necrosis virus
(RGNNV), and a turbot betanodavirus strain (TNV) have
been proposed to represent separate species [24].
Persistent or carrier infection may develop to an acute
phase with biological and environmental stress factors and
facilitate vertical or horizontal transmission [1, 3, 8, 16] of
the virus, as biopsy samples of gonads of broodstock (ova
and milt) have been found positive for virus [17]. Screen-
ing of fish stock for VNN is vital for good management of
any fish hatchery. Non-lethal sampling of suitable tissues
from fish and maintaining the integrity of viral pathogens
within a minimum quantity of sample is a challenging task
for betanodavirus detection.
A paper-based technique has been in use for archiving
nucleic acids for molecular research, with DNA retaining
its quality longer than RNA. The Flinders Technology
Associates (FTA) card (Whatman) maintains the integ-
rity of nucleic acids for a long time and allows extraction
without any preservatives, thereby making it advantageous
for molecular epidemiological studies and characterization
of pathogens. This technology has been used by various
researchers for recovering DNA as well as RNA of
pathogens from humans, animals and plants, and it has
been a reliable tool in forensic sciences [57, 10, 11, 14,
15, 21, 23]. FTA cards have also been used in molecular
epidemiological studies of white spot syndrome DNA in
shrimp [22].
Technology Associates card-paper-based system designed
to fix and store nucleic acids directly from fresh tissues/
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 Author's personal copy
2020
medium to evaluate the stability and retrieval of RNA
from betanodavirus-infected samples (tissue homogenate,
cell culture supernatant, gonadal fluid-milt and seawater-
spiked with betanodavirus) originating from Asian sea-
bass (Lates calcarifer) and its subsequent use for
molecular screening by reverse transcription nested
polymerase chain reaction (RT-nPCR). Application of
this technology has potential for use as an alternate
storage and transport strategy for long-term surveillance
programmes, and also for developing a screening proto-
col for larvae and contaminated rearing water and non-
lethal sampling from broodstock to enhance biosecurity
and successful production of larvae in a marine fish-
breeding programme.
Four different types of samples were used as the source
of betanodavirus in the present study: larvae, broodstock,
VNN-infected cell culture supernatant and seawater (filter-
sterilized seawater spiked with larval homogenate). These
samples included i) a homogenate of seabass larval sam-
ples in PBS (1: 10 w/v) harvested from an outbreak of viral
nervous necrosis in seabass, ii) a pooled sample of milt
collected from a betanodavirus-positive seabass broodstock
fish, iii) a cell culture supernatant (in PBS) inoculated with
an isolate of betanodavirus (designated as BARL-LC01) in
the SISS cell line (courtesy of Dr. Sahul Hameed, C Abdul
Hakeem College, Melvisharam), at 96 h post-inoculation,
and iv) filtered seawater (salinity, 28 %) spiked with larval
homogenate (1:10000 w/v). Additionally, two control
samples were included: v) RNase-free water as a negative
and reagent control and vi) a homogenate of VNN-free
seabass larvae (negative control). All samples were stored
as 200-lL aliquots at -80 C until used for inoculation onto
FTA cards, while another set of aliquots was saved and
stored at -80 C for direct testing as fresh samples on the
day of RT-nPCR analysis.
The fresh samples (50 lL) were spotted onto FTA
Classic Cards according to the manufacturers protocol,
labeled and air-dried for 2 h at room temperature. These
cards were stored at 4 C in a refrigerator in multi-barrier
pouches until used for weekly tests on days 0, 7, 14, 21, 28
and 60 to compare the stability and yield of RNA after
long-term storage. Using a 3-mm Harris micro-punch
(Whatman), two discs were cut out from the centre of the
sampling area of each sample FTA card, and one disc was
placed directly into a PCR amplification tube, and another
one into a 1.5-mL tube for extraction of RNA as recom-
mended by the manufacturer. Caution was used to disinfect
the Harris punch using ethanol solution, and it was dried
using disposable wipes and used to punch two blank cards
before punching each sample card.
Two different protocols were used for the preparation of
cDNA template. In the first protocol, RNA extraction was
done using TRIzol Reagent (Invitrogen) as per the
123
A. Navaneeth Krishnan et al.
manufacturers protocol. The extracted RNA was quanti-
fied, and its purity was checked using a NanoDrop spec-
trophotometer (Implen, Germany). In the second protocol,
a single disc placed in an individual PCR tube for each of
the samples was used for disc-in-PCR after treating with
FTA purification reagent (Whatman) as per the manufac-
turers protocol prior to reverse transcription polymerase
reaction (RT-PCR). TE buffer was removed, and the disc
was used as RNA template for RT-PCR (cDNA
synthesis).
All samples were tested by RT-nPCR for the presence of
betanodavirus in fresh samples on day 0. For samples
applied to FTA cards, both types of RNA preparation
(extracted RNA and disc-in PCR) were used separately
for each sample at weekly intervals for one month (0, 7, 14,
21, 28 days) followed by another test 60 days after appli-
cation of samples to the FTA Classic Cards.
First-strand synthesis was done using a ProtoScript
First Strand cDNA Synthesis Kit (New England Biolabs).
The sequences of newly designed primers sets for first-step
and nested RT-PCR were as follows: BARL-F1 (5-
GTACGCAAAGGTGAGAAGAAA-3) and BARL-R1
(5-GTCCCAGATGCCCCA-3) for first-step PCR and
BARL-F2 (5-AACTGACAACGACCACACCTT-3) and
BARL-R2 (5-TGTGGAAAGGGAATCGTTG-3) for the
nested reaction. For one-step PCR, 1 lL of the cDNA
product of the step reverse transcription was used as the
template, whereas in the nested PCR, 1 lL of the primary
PCR product (1:5 dilution) was used. PCR was carried out
in 25-lL reaction mixtures containing 12.5 lL of ready-to-
use PCR master mix (Ampliqon Taq 2x Master Mix RED),
9.5 lL of nuclease-free water, and 10 pmol of forward and
reverse primers (1 lL each). The optimized thermal
cycling conditions for the first step were initial denatura-
tion at 95 C for 5 min followed by 39 cycles of denatu-
ration for 1 min at 95 C, annealing for 1 min at 60 C and
extension for 1 min at 72 C, and final extension at 72 C
for 5 min. For nested PCR, the conditions were initial
denaturation at 95 C for 5 min followed by 39 cycles of
denaturation at 95 C for 30 s, annealing at 60 C for 30 s
and extension at 72 C for 30 s, and final extension at
72 C for 5 min. The PCR products were visualized in a
1.2 % Tris-acetate-EDTA (TAE) agarose gel stained with
0.5 lg of ethidium bromide per mL using a Gel Doc XR?
Imaging system (Bio-Rad, USA).
RT-PCR targeting the betanodavirus capsid protein
sequences is now accepted as the gold standard for con-
firmatory diagnosis [18, 19]. In the present study, we
evaluated the FTA Classic Card for sampling and retrieval
of betanodavirus nucleic acid for diagnostic applications.
Samples applied on FTA cards include four different types
of samples known to be positive for betanodavirus and two
control samples known to be negative. Asian seabass
 Author's personal copy
Use of FTA cards for betanodavirus detection 2021

Table 1 Quality and quantity of RNA extracted from 3-mm discs from an FTA Classic Card
Sample Sample description A260/A280 Concentration (mean SD) of RNA per 3-mm disc (ng/lL) after application of sample,
no. (?, betanodavirus (on day 0; n=6
positive; -, n = 6)
betanodavirus (Mean SD) Duration (days)
negative) 0 7 14 21 28 60

1 Larval homogenate 1.850 0.03 1852.2 23.18 1657.5 6.28 1436.0 17.51 946.4 15.91 361.1 16.33 00
(?)
2 Cell culture, SISS (?) 1.745 0.02 1750.0 17.40 1499.0 4.90 1205.0 42.22 871.5 31.12 266.5 8.36 00
3 Milt (?) 1.848 0.02 1638.3 5.39 1372.5 2.95 1076.6 60.11 805.5 7.29 395.0 4.94 00
4 Seawater, spiked (?) 1.720 0.01 1758.2 4.02 1430.3 6.59 1150.3 34.49 716.8 4.22 271.7 6.65 00
5 RNase-free water (-) 00 00 00 00 00 00 00
6 Larval homogenate 1.853 0.02 1650.0 4.94 1341.0 14.77 1049.0 28.29 844.7 13.47 348.3 6.153 00
(-)
00, too low for detection

Fig. 1 Concentration (mean) of Concentraon (mean) of RNA (ng/l) in dierent samples

RNA in different samples (extracted from one FTA card disc of 3 mm diameter)
2000
1800
1600
1400 larval extract
RNA con. (ng/l)

1200 cell culture extract

1000
milt extraxt
800
600 seawater

400 RNA free water

200
healthy larval extract
0
0 7 14 21 28 60
No. of days

larvae, cell culture (SISS), milt and seawater, all containing
viruses and two sets of samples, seabass larvae (virus-free)
and RNase-free water served as controls. The concentration
(mean SD) of RNA (ng/lL) per disc isolated by the
TRIzol method using different samples on days 0, 7, 14,
21, 28 and 60 is shown in Table 1. The recovery of RNA
from a 3-mm disc punched out of an FTA card was rela-
tively satisfactory in terms of quality during the weekly
analysis, but the stability and concentration of total RNA
on the FTA disc was found to decrease significantly over a
period of three weeks, irrespective of the type of samples
applied on the card (Fig. 1). Of the two RNA preparation
protocols, both protocols employed in this study gave
consistent amplification of PCR products of predicted sizes
(902 bp and 313 bp in the first step and in nested PCR,
respectively) in all test sample discs. Both of these proto-
cols amplified the desired products by first-step PCR up to
21 days of storage, while virus detection by nested PCR
was possible up to 28 days of sample storage (Table 2,
Fig. 2). However, nested RT-PCR detection of betano-
davirus was not possible after 60 days of storage on an
FTA Classic Card at 4 C for any of the types of samples
tested in this study. No amplification was observed from
the respective negative control samples on each test point.
A laboratory isolate of betanodavirus (108 TCID50 mL-1
at 7 dpi) used as positive control (originally isolated from a
disease outbreak of VNN in seabass larvae) also gave the
desired product in both the first- and second-step PCR,
indicating the stability of viral RNA even after 60 days of
storage at -80 C (Fig. 2).
One of the inherent limitations of the disc-in-PCR
method used here was the inability to assess the quality and
quantity of RNA per disc and adjust the RNA concentra-
tion in the reaction mixture for PCR. Hence, carryover
PCR products from the first step reaction due to excess
template concentration and some nonspecific products

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2022 A. Navaneeth Krishnan et al.

Table 2 Results of RT-PCR using different biological samples applied on FTA Classic Cards at different time points (using 3-mm discs)
Sample no. Sample PCR type Duration (days)
(?, betanodavirus positive; -,
betanodavirus negative) 0 7 14 21 28 60

1 Larval homogenate (?) 1st step ? ? ? ? - -

Nested ? ? ? ? ? -
2 Cell culture, SISS (?) 1st step ? ? ? ? - -
Nested ? ? ? ? ? -
3 Milt (?) 1st step ? ? ? ? ? -
Nested ? ? ? ? ? -
4 Seawater, spiked (?) 1st step ? ? ? ? - -
Nested ? ? ? ? ? -
5 RNase free water (-) 1st step - - - - - -
Nested - - - - - -
6 Larval homogenate (-) 1st step - - - - - -
Nested - - - - - -
These PCR results apply to both protocols of RNA template preparation, i.e., RNA extraction from a single disc of 3-mm diameter and disc-in-
PCR without RNA extraction
? indicates doubtful diagnosis

were also noted in nested PCR reactions during first two
weekly tests on days 7 and 14. This was resolved by
dilution (1:10) of the template (first-step PCR product) in
subsequent nested reactions.
In recent years, there has been an increasing emphasis
on the use of non-lethal sampling methods that would
allow screening for betanodaviruses in fish stock, particu-
larly in breeders, without sacrificing the fish. Different
procedures used to analyze broodstock for nodavirus
include detection of serum antibodies in seabass [2],
detection of the virus by RT-PCR in gonads and eggs [13],
and detection by both techniques in barfin flounder [24]
and seabass [3] with contradictory results. It has been
suggested that the viruses may not always reside in the
reproductive organs of fish [17]; thus, PCR procedures may
not detect infection in brood fish if the gonad has not been
invaded. Some studies have concluded that blood samples
could be appropriate for selecting nodavirus-free spawners
of seabass and seabream only when RT-nPCR is used [4].
In these circumstances, the development of RT-nPCR
detection of betanodavirus in blood samples may represent
a significant improvement in speed and sensitivity, using
minimum amounts of sample. Studies using ante-mortem
sampling (blood) and post-mortem sampling (brain) of
Senegalese sole (Solea senegalensis) revealed that the
sensitivity and the negative predictive value of RT-nPCR
were slightly lower for blood samples than for brain sam-
ples and suggested a relative advantage of being able to
perform diagnosis on live fish albeit with lower sensitivity
for the selection of nodavirus-free broodstock [20].
In the present study, we demonstrated that viral RNA in
biological samples applied to an FTA card and stored at
4 C was successfully detectable up to four weeks by RT-
nPCR. However, the stability and concentration of total
RNA on the FTA disc was found to decrease significantly
over a period of three weeks, irrespective of the type of
sample applied. Similar observations have been reported
for other RNA viruses of humans, plants and poultry [14,
15]. Deterioration of RNA quality and yield has been an
important issue at ambient temperature, and it was reported
earlier to be a consequence of RNA denaturation due to
formation of nicks on RNA strands [15].
While using FTA card technology, a certain degree of
recovery loss of RNA can be expected, but the method was
sufficient to preserve and subsequently extract viral RNA in a
detectable quantity for almost a month. The stability of RNA
depends on the storage temperature and duration and the type
of biological specimen to a limited extent (unpublished
data). Hence, storage of FTA impregnated samples at lower
temperature and the use of a larger number of punched discs
per reaction might allow detection of the pathogen beyond
this period. In this study, a limited number of confirmed
samples that were positive for betanodavirus infection was
used for standardization, which may have caused some
degree of bias and overestimation of sensitivity. Although
less sensitive than traditional methods, the specificity of RT-
PCR using FTA cards was found to be the same as that
obtained in case of traditional methods using normal frozen
samples. FTA cards for non-lethal sampling of reproductive
fluids containing ova/milt or blood from broodstock can be
used to detect the carrier status of betanodavirus infection.
This has the clear advantage of making it possible to perform
diagnosis on live fish, albeit with lower sensitivity, for the
selection of nodavirus-free broodstock. However, further

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Use of FTA cards for betanodavirus detection 2023

M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 7 8 9 10 11 12

902 bp
1000 bp
313 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 7 8 9 10 11 12
1000 bp

902 bp
313 bp
0 day 07 day

M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 7 8 9 10 11 12

1000 bp 902 bp
313 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 7 8 9 10 11 12

1000 bp 902 bp
313 bp

14 day 21 day

M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 P 7 8 9 10 11 12 P

902 bp
1000 bp
313 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 P 7 8 9 10 11 12 P
902 bp
1000 bp 313 bp

28 day 60 day

Fig. 2 RT-PCR diagnosis of betanodavirus (by first-step and nested
PCR) using different samples applied onto an FTA Classic Card
(Whatman) after 0, 7, 14, 21, 28 and 60 days of application. The
upper part of the gel shows RT-PCR products obtained using RNA
extracted from a single disc of 3-mm diameter from an FTA card for
each type of sample. The lower part of each gel shows the products
obtained using the washed disc directly in an RT-PCR reaction
mixture (disc-in-PCR) without RNA extraction and subsequent use of
cDNA in PCR reactions. Lane M, 100-bp marker; lanes 1-6, first-step
PCR; 1, larval homogenate; 2, cell culture extract; 3, milt extract; 4,
seawater spiked with larval homogenate; 5, RNA-free water (control);
6, healthy larval homogenate (control). Lanes 7-12, nested PCR: 7,
larval homogenate; 8, cell culture extract; 9, milt extract; 10, seawater
spiked with larval homogenate; 11, RNA free water (control); 12,
healthy larval homogenate (control); P, positive control (included
60 days after sample application) as samples 1-12 were all PCR
negative

field studies are needed to understand its potential applica-
tion for quantifying the viral load and for batch screening of
larvae or broodstock of unknown disease status or subclinical
carriers in marine fish hatcheries to generate data on the
sensitivity and specificity of betanodavirus detection using
an FTA Classic Card.
Acknowledgments The authors are grateful to the Director, Central
Institute of Brackishwater Aquaculture (Chennai), for providing funds
and necessary facilities. The first author is grateful to National
Fisheries Development Board (Hyderabad) for a Senior Research
Fellowship under the National Surveillance Programme on Aquatic
Animal Diseases coordinated by the National Bureau of Fish Genetic
Resources (NBFGR), Lucknow (India).

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2024 A. Navaneeth Krishnan et al.

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