The Consequence of a Single Nucleotide Substitution on the Molecular Diagnosis of the Chicken Anemia Virus

June 9, 2015 — admin
AttachmentSize
single_nucleotide_substitution.pdf357.39 KB
Embedded Scribd iPaper - Requires Javascript and Flash Player

Israel Journal of Veterinary Medicine  Vol. 70 (2)  June 2015 Davidson, I. 30
Te Consequence of a Single Nucleotide Substitution on the
Molecular Diagnosis of the Chicken Anemia Virus
Davidson, I.,
1
* Raibshtein, I.,
1
Al Tori, A.
1
and Elrom, K.
2
1
Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan P.O. Box 12, Israel 50250.
2
Private Poultry Veterinarian, Kiryat Tivon, Israel 79330.
*
Corresponding Author: Dr. Irit Davidson, Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan, P.O.Box 12, Israel 50250.
Email: davidsoni@int.gov.il
ABSTRACT
While genomic variations, including single nucleotide polymorphism (SNP) are expected and common for
RNA viruses, their occurrence is anticipated at a fairy low frequency for Chicken Anemia Virus (CAV), as
it contains a conserved DNA genome. Te present report demonstrate that in 4/80 CAV feld isolates one
nucleotide substitution, from G to A, located in the middle of the real-time probe was responsible for false-
negative real-time PCR amplifcation results. Tis fnding emphasizes the need of awareness to harmful
mismatches that occur even in conserved genomes, and the need for periodical verifcation of amplifcation
primers and probes according to the clinical picture in the feld.
Keywords: Chickens; Chicken Anemia Virus; Molecular Diagnosis; SNP
INTRODUCTION
Chicken anemia virus (CAV) is ubiquitous with a worldwide
distribution having a considerable economic impact due to
its ability to cause clinical morbidity, increased mortality,
but also sub-clinical infections and immune-suppression
(1). CAV belongs to the family Circoviridae, the Gyrovirus
genus and is a non-enveloped virus with a negative sense
single-stranded circular DNA genome and is considered a
conserved virus of one serotype.
Te viral genome consists of 2.3 kb with three partially or
completely overlapping ORFs (open reading frames). ORF3
(1,347 bp) encodes the major viral structural protein VP1 (52
kDA) and partially overlaps with ORF1 (648 bp), VP2 which
encodes the 24 kDa protein VP2, a scafolding protein.ORF3
(VP3) encodes a non-structural protein named apoptin (13.6
kDa) for its ability to cause apoptosis. VP1 is the target of
neutralizing antibodies, while VP2 is important to fold VP1
in the proper format. CAV sequence variability consisted of
less than 6% at the amino acid level, especially at the VP1
hypervariable region, amino acids 139-151 of VP1 (2, 3)
and also at the carboxy-terminus of VP2 and VP3 (4). Te
present phylogenetic classifcation of sequenced worldwide
CAV isolates is not supported by any biological distinction;
therefore, the signifcance of the phylogenetic grouping is
still rather vague (5).
Te VP2 protein was demonstrated in several Bangladesh
and American CAV isolates to be the most conserved among
the three CAV proteins, showing only 1.4% diversity, compared
to about 4% and 2.2% for VP1 and VP3, respectively (6, 7). To
reduce possible genomic mismatches due to genetic variability
between feld strains and to consolidate the diagnostic assay,
the VP2 gene was adopted at our laboratory for molecular
diagnosis by end-point PCR according to Imai et al. (8) and by
real-time amplifcation based on a plasmid in which the end-
point PCR amplicon was inserted, which served as the positive
control DNA and as the standard for quantifcation (9).
While the PCR-based detection of CAV is the most
widely applied diagnostic test, due to its straightforward
advantages in relative easiness of fast performance and sen-
sitivity, minor genomic changes might impede virus detec-
Israel Journal of Veterinary Medicine  Vol. 70 (2)  June 2015 31 Chicken Anemia Virus
tion. While genomic variations, including single nucleotide
polymorphisms (SNP) are expected and common for RNA
viruses, their occurrence and detrimental infuence on di-
agnosis is unanticipated for CAV, as the virus is considered
genetically conserved DNA genome. Te evolutionary rate of
DNA genomes is 10
-3
lower than RNA genomes, 10
-6
to 10
-8
/
base/generation compared to 10
-3
to 10
-5
/base/generation,
for DNA and RNA viruses, respectively (10). Strain-specifc
primers which discriminate between CAV strains have been
reported previously (11).
In this communication we describe an uncommon SNP
that occurred in the genomic area encompassing the probe
used in the real-time PCR (2).
MATERIALS AND METHODS
Clinical samples
Te study included 80 commercial chicken focks of various
ages and type that were submitted for molecular diagnosis,
between January 2013 to March 2014 and was positive for
CAV by end-point PCR (12). Basically, DNA was purifed
from samples of spleen and liver as a pool and from 10 pieces
of 5 feather shafts, as a second pool. Similar organs from 3
chickens were pooled for DNA purifcation and used as one
DNA sample.
DNA purifcation and end-point PCR
DNA was purifed using Maxwell 16 Tissue DNA Cat.
# 1030 kit, (Promega, Mad. WI, U.S.A.), according to the
manufacturer instruction, and amplifed for CAV (1). Te
PCR cloned amplicon, was used as positive control.
Primers and probes of real-time PCR
Te cloned CAV amplicon, obtained from a local CAV isolate
was the basis of the following original frst set of real-time
PCR primers and probes:
Forward, 5' – CCGCGCTAAGATCTGCAACTG,
Reverse, 5' – GAGGGAGGCTTGGGTTGATC
Probe 5' FAM/3' BHQ-1 CGGACAATTCCGAAA
GCACTGGTTTCA.
Following the identifcation of the SNP, a second pair of
primers and probe was designed, matching with the Cux-1
CAV prototype (Acc. No. M55918):
Forward, 5' – CCA CGCTAAGATCTGCAACTG,
Reverse, 5' – GAGGGAGGCTTGGGTTGATC
Probe 5' FAM/3' BHQ-1 CGGACAATTCAGAAAG/
ACACTGGTTTCA.
A sample of 2 µl of the extracted DNA, according to
the kit instructions, was used in the real-time PCR assay, as
described (9).
RESULTS AND DISCUSSION
In November 2013 three broiler focks derived from the same
broiler breeding fock that showed typical clinical signs of
CAV and immunosuppression-related morbidity, were sub-
mitted for CAV diagnosis. About 4% mortality was recorded
on days 13-26 of age, accompanied by severe hypocellularity
of the bone marrow, thymus and spleen atrophy with lym-
phocytic depletion, bone marrow lesions, foot infammatory
lesions, necrosis, and perihepatitis/pericarditis in about 95%
of the birds that were examined post-mortem.
Te real-time amplifcation of these samples was nega-
tive for CAV, therefore the samples were amplifed by the
end-point PCR assay. As suspected, the presence of CAV
was demonstrated. After elimination of technical factors, a
mismatch between the CAV genome and the real-time PCR
primers and probes was suspected. To evaluate whether ad-
ditional false negative diagnostic cases occurred since January
2013, all CAV-negative cases (n=77) by real-time PCR,
amplifed with the frst primer and probe set, were retested
by end-point PCR. Only one out of 77 focks was found
false-negative by real-time PCR. In total, the false-negative
samples were 5% out of 80 focks, that is 4 focks.
We then compared the end-point PCR amplicon se-
quences of the 4 focks that were negative by the real-time
PCR (Katriel #128, Katriel #131, Katriel #137 and #109), 4
reactive focks (#77, #78, #82 and #96), as well as the end-point
PCR amplicon of the local isolate (positive control), the CAV
Cux-1 prototype amplicon and the real-time PCR frst set
of primers and probe (Fig. 1). As refected in the sequence
alignment, the non-reactive 4 focks difered by one nucleo-
tide substitution, from G to A, located in the middle of the
real-time probe. Te presence of an additional SNP, derived
from the local isolate sequence that was present on the probe
sequence allowed amplifcation, however, the addition of a
second SNP present on the forward primer, was detrimental to
amplifcation. Furthermore, to ascertain the SNP contribution,
a second set of CAV primers and probes were synthesized
according to the Cux-1 sequence, i.e., without the local isolate
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (2)  June 2015 Davidson, I. 32
SNP. By employing the second set of primers all samples were
amplifed by real-time PCR, indicating the importance of the
novel G to A substitution in the probe region.
Te present fndings confrm many previous studies report-
ing the efect of various sequence mismatches, demonstrating
more pronounced efects on amplifcation caused by mismatch-
modifed probe than those of mismatch-modifed primers.
Specifcally, Süss et al. (13) showed that probe mismatches
have a double weighted efect compared to primer mismatches.
In summary, the present case emphasizes the need of
awareness to detrimental mismatches that occur even in con-
served genomes, leading to the need of periodical verifcation
of amplifcation primers and probes, according to the clinical
picture in the feld.
REFERENCES
1. Schat, K.A. and van Santen, V.L.: Chicken infectious anemia.
In: Swayne, D.E., Glisson, J.R., McDougald, L.R., Nolan, L.K.,
Suarez, D.L. and Nair, V. eds. Diseases of Poultry, 13th edition,
John Wiley & Sons, Inc. p 248-264, 2013.
2. Ducatez, M.F., Owoade, A.A., Abiolaand, J.O. and Muller, C.P.:
Molecular epidemiology of chicken anemia virus in Nigeria. Arch.
Virol. 151: 97-111, 2006.
3. Ducatez, M.F., Chen, H., Guan, Y. and Muller, C.P.: Molecular
epidemiology of chicken anemia virus (CAV) in South Eastern
Chinese live bird markets. Avian Dis. 52: 68-73, 2008.
4. Renshow, R.W., Soine, C., Weinkle, T., O'Connell, P.H., Ohashi,
K., Watson, S., Lucio, B., Harrington S. and Schat, K.A.: A
hypervariable region in VP1 of chicken infectious anemia virus
mediates rate of spread and cell tropism in tissue culture. J. Virol.
70: 8872-8878, 1996.
5. Schat, K.A.: Chicken anemia virus Curr. Top. Microbiol. Immunol.
331: 151-183, 2009.
6. Islam, M.R., Johne, R., Raue, R., Todd, D. and Muller, H.:
Sequence analysis of the full-length cloned DNA of a chicken
anaemia virus (CAV) strain from Bangladesh: evidence for genetic
grouping of CAV strains based on the deduced VP1 amino acid
sequences. J. Vet. Med. B 49, 332-337, 2002.
7. Van Santen, V.L., Li, L., Hoerr F.L. and Lauerman, L.H.: Genetic
characterization of chicken anemia virus from commercial broiler
chickens in Alabama. Avian Dis. 45: 373-388, 2001.
8. Imai, K., Mase, M., Tsukamoto, K., Hihara, H. and Yuasa, N.:
Persistent infection with chicken anemia virus and some efects
of highly virulent infectious bursal disease virus infection on its
persistency. Res. Vet. Sci. 67: 233-238, 1999.
9. Davidson, I., Reibshtein, I. and Altori, A.: Quantitation of Marek's
disease and chicken anemia viruses in organs of experimentally-
infected and of commercial chickens by multiplex real-time PCR.
Avian Dis. 57, Suppl. 532-538, 2013.
10. Lauring, A.S., Frydman, J. and Andino, R.: Te role of mutational
robustness in RNA virus evolution Nat. Rev. Microbiol. 11: 327-
336, 2013.
11. Markowski-Grimsrud, C.J., Miller, M.M. and Schat, K.A.: Develop-
ment of strain-specifc real-time PCR and RT-PCR assays for quan-
titation of chicken anemia virus. J. Virol. Meth. 101: 135-147, 2002.
12. Davidson, I., Kedem, M., Borochovitz, H., Kass, N., Ayali, G.,
Hamzani, E., Perelman, B., Smith, B. and Perk, S.: Chicken infec-
tious anemia virus infection in Israeli commercial focks; virus
amplifcation, clinical signs, performance and antibody status.
Avian Dis. 48: 108-118, 2004.
13. Süss, B., Flekna, G., Wagner, M. and Hein, I.: Studying the ef-
fect of single mismatches in primer and probe binding regions
on amplifcation curves and quantifcation on real-time PCR. J.
Microbiol. Methods. 76: 316-319, 2009.
Figure 1. Sequence alignment of 4 reactive and 4 non-reactive focks by CAV real-time PCR with the frst set of primers and probe, performed
with Mega program ver. 5.0.
Research Articles

Published under a Creative Commons License By attribution, non-commercial