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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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