INTRODUCTION:

Puppy newborn deaths are caused by canine parvo viral enteritis, a virus that is extremely
infectious and deadly. The causative agent is a virus called canine parvovirus 2 (CPV-2). A 5.2 kb
long, tiny, non-enveloped, negative-sense ss-DNA virus that encodes two structural (VP1 and
VP2) and two non-structural (NS1 and NS2) proteins. [1-2] The primary capsid protein, known
as VP2, is essential for controlling antigenicity, host range, and tissue tropism. This virus, which
first appeared in the 1970s and expanded around the world afterward, is currently thought to
be endemic in the dog population due to the prevalence of many antigenic variations, including
CPV-2a, CPV-2b, and CPV-2c as well as the novel CPV-2a/2b. [2] An early and rapid diagnosis is
necessary in order to implement biosecurity measures for preventing the spread of the illness
to the vulnerable population due to the acute and highly infectious nature of the disease, and
also to start receiving supportive care in order to lower morbidity and death. Therefore, a
straightforward, affordable, field-based test is crucial for the quick identification of canine
parvo viral enteritis.[3] Numerous diagnostic assays have been created for the detection of CPV,
but they each have advantages and disadvantages in terms of price, sensitivity, specificity, and
ease of use that may prevent them from finding broad usage in the field. Hemagglutination
(HA) test, viral isolation, enzyme-linked immune sorbent assay (ELISA; SNAP Parvo, Idexx USA),
and polymerase chain reaction are the diagnostic procedures often used for the identification
of CPV-2 (PCR).[1-4] For quick detection, a variety of commercial immune chromatographic (IC)
strip-based assays are available. For quick detection, a variety of commercial immune
chromatographic (IC) strip-based assays are available under field circumstances, of CPV-2. The
majority of these kits are not made in India and must be imported, which raises the price each
test.[4] These strip tests utilize either a single monoclonal antibody (mAb) or two distinct CPV-
specific mAbs as the detector and capture antibodies.[2] They are based on the sandwich
concept. The use of the same mAb for both capture and detection in these IC tests may reduce
the sensitivity of detection in situations with low faecal CPV load since the capture and detector
mAbs would compete for the same epitope, even though this has not been discussed in the
literature.[3] This could be the cause of the tests' low sensitivity, especially in the latter stages
of illness when faecal shedding is significantly decreased. A more expensive alternative would
be to use two different mAbs in these strip-based assays, which would restrict their usefulness
on a larger scale.[2] Additionally, when one mAb (detector) binds to an epitope, it has the
ability to change the binding of another mAb by potentially affecting some other,
geographically unrelated epitopes. As utilizing mAbs as detector antibodies may result in
altering just part of the original epitopes, leaving the remainder of the epitopes untouched,
using a polyclonal antibody (pAb) in place of a mAb as the capture antibody in the IC test may
hold promise. Bovine parvovirus and canine minute virus are two distinct parvoviruses that
have been discovered for many years and placed in the genus Bocavirus.[1-4] Human faecal
samples occasionally included tiny, spherical viruses of unknown relevance that could only be
identified using electron microscopy; nevertheless, limited sequencing of the DNA extracted
from such samples revealed a B19 parvovirus-like resemblance. Human bocavirus (HBoV1) was
discovered in children's respiratory secretions in Sweden in 2005, while HBoV2 and HBoV3 were
discovered in children's stool samples in the US and Australia in 2009. In stool samples more
recently, a fourth member, HBoV4, was also discovered. Non-structural protein 1 is one of 4
proteins that the human boca virus genome encodes (NS1). A nuclear phosphor protein NP-1 of
unknown function, the capsid proteins VP1 and VP2, and a DNA-binding protein involved in
gene transcription and replication. It appears that HBoV1 is largely a respiratory pathogen, and
infection is common.[5] It is not known if it can be a main cause of disease, whether it can lead
to more serious disease, or whether it is often a harmless passenger because it is frequently
discovered in respiratory samples from unwell children, with or without other recognized
pathogens.[5] In one research, HBoV2 and HBoV3 were the third most frequent agents
discovered after rotavirus and astrovirus. They are typically identified in stool samples,
especially from instances of severe gastroenteritis. However, in order to clearly establish the
real pathogenic function of these, several meticulous case control studies are necessary.[4] The
Boca parvovirus genus is a subfamily of the Parvoviridae family that includes the bovine
parvovirus (BPV). The major effects of BPV infection include respiratory and gastrointestinal
illnesses in newborn calves as well as reproductive abnormalities in pregnant cows. Three open
reading frames (ORFs) make up the BPV genome: ORF1 encodes the non-structural protein NS1,
ORF2 the phosphorylated protein NP1, and ORF3 the structural proteins VP1, VP2, and VP3.[1-
4] The majority (80%) of the structural proteins in BPV are found in VP2, which is the principal
structural protein. The well-known disease known as the bovine viral diarrhoea virus (BVDV)
affects cows and causes significant financial losses. BVDV is a member of the Flaviviridae family
and the Pestivirus genus. Intrauterine infection with BVDV can result in a chronic infection,
which leads to a condition of immune tolerance in addition to producing respiratory, gastro
enteric, and reproductive problems.[5] The 50 UTR region is highly conserved in different BVDV
strains and is frequently employed as a marker for BVDV categorization or diagnosis. Bovine
rotavirus, bovine parvovirus, and bovine viral diarrhoea virus are all responsible for intestinal
illnesses in cattle. Mixed infections frequently happen as a result of the closeness of their
clinical presentations and infectious pathways. A technique that concurrently detects the three
pathogens is required since it can reduce time and labour and has a significant benefit in clinical
detection. There are a few diagnostic methods that may be used to identify CPV-2 in biological
material. Molecular methods, like qPCR, are used for the gold standard diagnosis. Similar to
CPV-1, CPV-2 may be found via hemagglutination assays, transmission electron microscopy,
ELISA, and immunochromatographic testing (IC). They can, however, be costly and time-
consuming methods.[2]
MATERIALS AND METHODS:
Shortened CPV VP2 proteins (CPV/VP2) were cloned, expressed in
bacteria, and purified:
DNA from faecal samples was used in a polymerase chain reaction, with recombinant plasmid
DNA (KJ364525) serving as the positive control and no template DNA serving as the negative
control. Using the standardized conditions, a PCR reaction was set up in a standard 25 l reaction
using the following chemicals.[2] All of the PCR chemicals, which are listed below, were
purchased from Thermoscientific in the USA.Utilizing 1% agarose gel electrophoresis, the PCR
result was seen. CPV-NR (5-ATCTAGATTCTCGAGTGTTCC TGTAGCAAATTCATC-3), while the
partial C-terminal region was amplified using CPV-CF (5-ACT
GCAGCGAATTCAGGTGATGAATTTGCTACAG-3). The partial C-terminal region was amplified using
CPV-CR (5-GGGCTCGAGTGGATTCCAAGTATGAGA G-3).[1] Using genomic DNA from cell-culture-
adapted CPV-2a BE-1 (NCBI Gen Bank accession number KJ364524), two overlapping segments
corresponding to the N-terminal end and the majority of the C-terminal end of the VP2 gene
were independently targeted for amplifcation. The N-terminal region was amplified using the
primers CPVNF (5-ACACAGAATTCAGGAGCAGTTCAACCAGA C-3). After the membrane had been
probed with a recognized anti-CPV dog serum (1:100 dilution) obtained from healthy
vaccinated dogs, the proteins on the membrane were discovered using an enzymatic reaction.
[1-3]

Conjugation of mAb with colloidal gold nanoparticle:

The Turkevich and Frens synthesis technique was used to create gold nanoparticles with an
average diameter of 10 nm (Emmanuel et al., 2018). Following a three-minute boil of 100 mL of
0.01% gold chloride, 5 mL of 1% trisodium citrate solution was quickly added while being stirred
with a glass rod. It took the solution another 5-8 minutes of boiling before it turned wine red.
The GNPs were cooled to room temperature, kept at 4 °C, and seen using a transmission
electron microscope.[2-3]
To remove unbound antibodies from the solution, centrifugation was performed on the mixture
at 8000 rpm for 30 min at 4 °C. The soft pellet was then rinsed with 0.01 M PBS, pH 7.2. The
pellet was then placed in a tenth of its original volume of 0.01 M PBS, pH 7.2, containing 1%
BSA, and kept at 4 °C until it was needed.[4] Briefly, 0.1 N K2 CO3 was used to adjust 1 mL of
colloidal gold solution to the ideal pH, and the right quantity of anti-CPV mAb was then added
while stirring continuously. For 30 min. at room temperature, with steady stirring, mAbs were
allowed to bind to the surface of the GNPs.[1] By adding 0.01 M PBS, pH 7.2, containing 10%
bovine serum albumin (BSA), at room temperature, with constant stirring for 15 minutes,
unreactive sites on GNPs were blocked.
The CPV-2 detection process:
There were examined 60 positive and 5 negative stool samples for CPV-2. The samples were
generously given through collaborations with the Laboratory of Veterinary Molecular Diagnosis
(LDMVET). Until processing, the samples were kept in 15 mL conical centrifuge tubes at 20 °C.
The gold standard diagnostic method, quantitative polymerase chain reaction (qPCR), was used
to confirm the presence of CPV-2 in the feaces. Adapted magnetic beads (Sera-MagTM
Magnetic Speed Beads TM Carboxylate-Modified) were used for DNA extraction.10 L of the
qPCR Master Mix, 0.8 L of each unique primer14 (10 pmol L1), and 4.4 L nuclease-free water
were used in the reaction.[5] The cycling included 40 cycles of 95 °C every 15 seconds and 60 °C
every minute, followed by a melting curve, after five minutes of 95 °C denaturation. The AriaMX
real-time PCR System (Agilent, Santa Clara, CA, EUA) was used to carry out the reaction.
Nuclease-free water was used as the negative control in all reactions, and an earlier positive
control.

Making a lateral flow immune chromatographic test strip based on

mAb/pAb:
The membrane was coated, air dried at 37 °C for 2 hours, and then put together with additional
pads. The test and control lines were spaced apart by about 5 mm. Other factors that could
influence how well the IC test performed were also optimized, including. By manually pipetting
(10 µl of conjugate per strip), colloidal gold conjugate was applied to the conjugation pad and
let to air dry. Using an Easy Printer or a hand pipette, test and control antibodies were added to
a nitrocellulose membrane. Anti-CPV-tVP2 rabbit polyclonal IgG and goat anti-mouse pAbs were
coated on the test and control lines, respectively, at different doses (1, 2 and 5 mg/ml).