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Historical presence of IAPV in the U.S.
by Yanping Chen and Jay D. Evans
USDA-ARS, Bee Research Laboratory, Beltsville, MD 20705
Submitted to American Bee Journal 10/29/07
Abstract:
High bee colony losses in the U.S. this past year can be
attributed in part to an unresolved syndrome termed CCD.
An extensive genetic survey found one virus, IAPV, to be
strongly associated with CCD. Using DNA sequencing and
phylogenetic analyses, we provide evidence that IAPV was
present in U.S. bees collected several years prior to
CCD, and prior to the recent importation into the U.S.
of bees from Australia and New Zealand. While downplaying
the importance of bee importation for the appearance of
CCD, these results indicate an urgent need to test
specific strains of IAPV for their disease impacts. Bees
are of great agricultural importance in the U.S. and
worldwide (Morse and Calderone, 2000), and are continu-
ally threatened by parasites and pathogens. During the
winter of 2006-2007, a rare and extreme syndrome of bee
losses was observed. This syndrome, labeled CCD, is
defined by a rapid depopulation of adult bees in colonies,
often leaving a substantial standing brood of healthy
larvae. Survey evidence suggests that roughly 25% of bee-
keepers have suffered the effects of CCD, as defined by
characteristic traits and colony losses of >50% (van
Engelsdorp et al., 2007). Many beekeepers lost sub-
stantially more than 50% of their operations. While
events similar to CCD have occurred in past decades
(Wilson and Menapace, 1979), the severity of this event
has caused appropriate concern nationally and inter-
nationally. Recently, an unprecedented ‘metagenomic’
approach was used to detect parasites and pathogens in
bees associated with CCD and controls (Cox-Foster et
al., 2007). This study described numerous microbes from
bees, some known as pathogens and others that had not
been seen prior in bees. One striking result was the
tight correlation between IAPV, an unclassified
Dicistroviridae virus, and CCD. IAPV was detected in 25
of 30 (83 %) CCD-affected colonies but only once in 21
healthy colonies (Cox-Foster et al., 2007). This virus
was also found in package bees imported from Australia
and isolates of royal jelly imported from China. The
identification of IAPV as a newly described virus for
the U.S., it’s association with an important disease,
and implications for both bee management and trade
issues, have all led to intensive efforts to study this
virus. These efforts are focused on past and present
worldwide distributions IAPV, on determining
mechanisms by which this and related viruses can cause
disease, and on determining whether IAPV strains differ
substantially in their impacts on bees. To help address
these questions, we screened bee samples collected in
California, Maryland and Pennsylvania from 2002 to
2007 for the presence of IAPV. The genetic relation-
ships of different IAPV strains were studied by
sequencing one section (the 5’UTR) from the IAPV
genome for isolates collected from 33 U.S. bees (out of
several hundred screened bees). The results from this
survey indicate that IAPV has been circulating in U.S.
bee populations since at least 2002, and forms a world-
wide species that is greatly diverged from the related
Kashmir Bee Virus (KBV). Specifically, IAPV
isolates from this study can be split into four distinct
clusters supported with bootstrap statistical values
> 55%. These clusters reflect collections from
California, Maryland, Pennsylvania, and Israel. CA,
and PA isolates each formed separate lineages with
strong bootstrap support, while the Maryland and
Israeli lineages were less well defined. Israeli samples,
including the strain first named as IAPV, are not
distinct from the U.S. isolates as a group. We also
sequenced the entire genomes of IAPV isolates from
California, Maryland and a Pennsylvania apiary with
a history of Australian importation and CCD
symptoms, using a combination of long-template
RT-PCR, primer walking, and Rapid Amplification of
cDNA Ends (RACE) methods (protocols available
from Y. Chen). These three complete genomes, when
compared to the definitive (Israeli) IAPV genome
sequence, show 4.2 – 4.7% divergence at the RNA
level, while all IAPV strains showed >25%
divergence from KBV. Genetic heterogeneity across
the studied 5’ region is interesting in that this region
is involved in the initiation of protein translation, and
genetic variability of this region may lead to different
pathogenicities. Further analyses are needed to explore
the implications of these and other genome sequences
for virulence traits of IAPV. Our results show that
IAPV in the U.S. predates both the latest incarnation
of CCD and the importation of Australian packages.
Nevertheless, we caution that much work is still
needed to absolve or implicate this virus, or specific
imports, in CCD. Most importantly, experimental
studies are ongoing to determine the relative virulence
of imported or domestic IAPV strains, and such
studies will provide the best evidence for making
importation and management choices. Viruses with
minimal genome sequence differences can show
greatly different levels of virulence, and all isolates
of IAPV we studied showed at least some sequence
variation. Given its observed association with CCD,
this virus remains an important candidate for bee
disease.
Figure Legend. Phylogenetic tree based on 450 nt of
the 5’-UTR region of 33 IAPV isolates from CA, MD,
PA, and Israel. Sequences were aligned with MegAlign
(DNASTAR Lasergene) and the tree was generated
using a heuristic Maximum Parsimony algorithm
(PAUP 4.03; Sinauer Associates, Sunderland, MA).
The strength of branch relationships was assessed by
bootstrap replication (N=1000 replicates). Sequences
deposited in Genbank by the senior author.
Acknowledgements:
We gratefully acknowledge Jeff Pettis for guidance,
Haim Efrat for Israeli bee samples, Michele Hamilton
and Dawn Lopez for laboratory assistance.
References:
Cox-Foster, D.L., Conlan, S., Holmes, E., Palacios,
G., Evans, J.D., Moran, N.A., Quan, P.L., Briese, T.,
Hornig, M., Geiser, D.M., Martinson, V., van
Engelsdorp, D., Kalkstein, A.L., Drysdale, A., Hui,
J., Zhai, J., Cui, L., Hutchison, S.K., Simons, J. F.,
Egholm, M., Pettis, J. S., Lipkin W. I. (2007).
A metagenomic survey of microbes in CCD.
Science. 318(5848): 283-287. Morse, R. A. and
Calderone, N. W. (2000) The value of bee polli-
nation in the U.S. Bee Culture 128: 1-15. van
Engelsdorp, D., Underwood, R., Caron, D., Hayes,
Jr., J (2007) An estimate of managed colony losses
in the winter of 2006 - 2007: A report commissioned
by the Apiary Inspectors of America, American Bee
Journal 147: 599-603. Wilson, W.T., and D.M.
Menapace, 1979. Disappearing disease of bees: a
survey of the U.S. American Bee Journal 119:
118-119, 184-186, 217.]
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