184 L. Ward et al.Figure 1. Multipl

184 L. Ward et al.
Figure 1. Multiple alignment of all-available KBV sequences alongside ABPV sequences (for ABPV, if
more than one sequence is available with 100% identity in this region, only one of each sequence ‘type’
is shown), indicating the positions of the primers and probes designed (in boxes). Only polymorphic nucleotides
are shown, (.) indicates an identical nucleotide at that position.
The quantity of KBV virus in the UK and Australian
samples were extrapolated from the standard curve.
The virus concentrations were normalised by dividing
the inverse log of virus quantity by the inverse
log of the bee IPC quantity.
3. RESULTS
3.1. Real-time primer and probe design
Although KBV and ABPV are closely
related it was possible to discriminate between
the two viruses by designing TaqMan

primers and a probe to conserved region of
KBV sequence but where the ABPV sequence
was variable (Fig. 1, Tab. I). The KBV assay
gave a positive PCR result when tested with
3 purified virus preparations, KBV14, KBV16
and KBVNZ. No cross-reaction was observed
(no amplification after 40 PCR cycles) when
RNA was extracted from pure virus or bees
infected with the following viruses ABPV,
BQCV, SBV, CWV, SPV, DWV, CPV and
AIV. To confirm the presence of virus in the
pure virus preparations electron microscopy
was performed, and in each case virus particles
of the correct size were observed (data
not shown). For the viruses where sequence
was available (BQCV, SBV, DWV and AIV)
the presence of virus was confirmed by RTPCR/PCR
(data not shown). For Cloudy wing
virus (CWV) electron microscopy confirmed
the presence of virus particles of the correct
size, whilst the KBV assay did not detect viral
RNA, even though the available sequence
of CWV is identical to KBV. Although AIV
is a DNA virus it could still be detected in
the RNA extractions from infected bees using
PCR (without an RT step), indicating that suf-
ficient DNA was co-purified in the extraction
procedure to enable effective detection.
KBV was successfully detected in different
life stages of Apis mellifera L. including Australian
worker honey bees, worker pupae and
drone pupae. Some of the worker honey bees
from America also tested positive for the virus,
along with one Australian wasp (Vespula germanica).
This further indicates that the assay
developed is able to detect different sources
of KBV in A. mellifera as well as KBV in
different insect species. Detection in other insect
species is further supported by the successful
amplification of KBV sequence from
the virus preparation KBV16, obtained from a
bumblebee. The German bees all tested negative
for KBV, however, the internal control results
show that adequate levels of total RNA
had been extracted (Tab. II).
3.2. Survey of honeybee colonies
for KBV
RNA extracted from 5 bees in each survey
sample was tested with the real-time PCR
First detection of Kashmir bee virus in the UK 185
Table II. Results from testing known infected samples and samples suspected of being KBV-infected,
including the number of positive samples and the average Ct values and standard deviations for the KBV
assay in positive samples and the internal positive control assay in all samples extracted. The instrument
records a Ct value of 40 for the negative samples (-).
Source Sample type Number of positive Average Ct for KBV Average Ct for 18S assay
individuals tested positive samples for all samples
Australia Wasp pupa 1/2 24.36 23.52 ± 0.18
Australia Honey bee worker pupa 6/6 29.09 ± 3.28 14.64 ± 0.13
Australia Honey bee drone pupa 3/4 36.18 ± 1.66 14.48 ± 0.23
Australia Honey bee worker 3/10 35.36 ± 6.54 16.48 ± 1.20
Australia Honey bee worker 6/15 36.50 ± 1.63 23.24 ± 3.49
USA Honey bee worker 2/10 31.85 ± 1.45 16.39 ± 1.55
Germany Honey bee worker 0/10 - 17.59 ± 5.12
Germany Honey bee worker 0/10 - 17.38 ± 0.75
assays for the bee 18S rRNA (IPC). Using the
automated extraction method, RNA was successfully
recovered from all bee samples. A
similar extraction efficiency was observed for
all samples indicating that the method was reproducible.
The average real-time PCR Ct (denoted
by the point at which amplification is
first observed above the base threshold) value
for two replicates from 458 survey samples
was 10.39 ± 1.52.
Following testing using the KBV real-time
PCR assay, of the 458 samples tested, three
tested positive for the presence of KBV. Two
of the positive samples were detected in different
hives from the same apiary. Details of
these colonies are shown in Figure 2. One of
the sampled colonies was infected with European
foulbrood (a statutory notifiable disease)
and was destroyed as part of the national
disease control strategy. PCR products ampli-
fied during the real-time PCR testing were
cloned and sequenced. The sequence of the
cloned PCR product sequence was confirmed
to be KBV following a blast search on the
NCBI database which matched other KBV sequences
(Accessions AY275710, AF263723–
AF263732, AY452696).
3.3. Quantification of virus load in UK
bee samples
The quantity of KBV in the positive UK
samples were compared to Australian bees that
were known to be naturally infected with KBV
at low levels (non-symptomatic) or high levels
(showing clinical symptoms). A standard
curve method was used to quantify virus load
in the bees rather than the ∆Ct method as described
in Chen et al. (2005). When the relative
efficiencies of the bee IPC and KBV were
plotted as ∆Ct [Ct(KBV)−Ct(IPC) ], versus the log of
the RNA dilution, the ∆Ct had a value greater
than 0.1 (the slope was 0.9513), indicating that
the amplification efficiencies of KBV and bee
IPC were not equal. In this instance, the ∆Ct
method was not suitable for quantification. In
addition, as the starting level of virus and bee
IPC RNA in the standard dilutions was unknown,
relative rather than absolute quantifi-
cation was used.
A standard curve was constructed by plotting
Ct values of bee and KBV standard RNA
dilutions against the log of the RNA dilution.
The quantity of KBV and bee IPC RNA
from each bee was extrapolated from the standard
curve. The normalised quantity of KBV
in each bee was determined by dividing the
amount of KBV RNA by the amount of bee
IPC RNA. The results show that the virus levels
in the infected bees from the UK are similar
to those in bees with covert levels of infection
from Australia. In comparison, other bees
(also from Australia) believed to be highly infected,
contained around 400 times more virus
than the bees at the UK Manchester site and
100 times more virus than covertly infected
Australian bees and bees from the UK Hull site
(Fig. 3).
186 L. Ward et al.
Figure 2. A Map showing the locations of the 458 bee colonies sampled in the UK 2004 KBV survey
including the two colonies that tested positive for KBV (indicated by black diamonds).
4. DISCUSSION
This paper reports the first finding of KBV
in the UK and the first large-scale survey for
bee viruses in Europe using TaqMan technology.
This study demonstrates the practical application
of this technology as a support tool
for surveys and contingency management, and
to provide robust surveillance data on the presence
or otherwise of KBV and other honey bee
viruses.
The incorporation of an internal control assay
to detect the bee 18S rRNA gene allowed
the assessment of extraction efficiency and allowed
interpretation of false negative results.
First detection of Kashmir bee virus in the UK 187
Figure 3. Relative quantification of KBV in bee samples. (A) TaqMan Standard curves for KBV virus and
bee IPC. (B) Relative comparison of KBV levels in UK bees in relation to bees known to be naturally
infected with high and covert levels of KBV infection.
Also, the real-time PCR assays allowed the
quantitative assessment of virus levels over
several orders of magnitude. This proved to
be valuable for detection of low-titre infections
within the bees tested. In this study,
quantification of KBV levels in the UK bees
showed they contained similar levels to Australian
bees having covert infections. The bee
colonies from the UK sites showed no obvious
signs of virus infection at the time of collection
suggesting the virus was covert or latent
in these apiaries. It is known that the viruses
can exist in individual bees or entire colonies
for long periods without causing noticeable
clinical symptoms (Dall, 1985; Anderson and
Gibbs, 1988; Hung et al., 1996a, b). Studies
by Anderson and Gibbs (1988) showed that
covert virus infections were common in pupae
with KBV. The status and sites where the
virus may remain in the bee in this state are
unknown, however, it has been suggested the
lining of the gut may be the site of ‘unapparent’
or covert infection (Anderson and Gibbs,
1989). Anderson and Gibbs (1988) have stated
that these infections do possess a latent capacity
to become activated and develop into acute
infections.
Some studies have suggested that viruses
such as KBV and ABPV may exist as natural
infections in a broader range of genera other
than Apis. For example KBV has been detected
in the wasp Vespula germanica (Anderson,
1991) and ABPV in Bombus species (Bailey
and Gibbs, 1964). Our study provides further
support for this, as KBV was detected in the
same species of wasp and one of the KBV
serotypes tested was extracted from a bumblebee
from New Zealand. In terms of trade and
regulation of honeybee viruses between countries,
if these viruses exist in different insect
hosts, it makes such controls and restrictions
difficult to impose or justify. In view of this,
the full host-range of honeybee viruses warrants
further study.
At the time of sampling, all three KBV positive
UK colonies were in good condition, with
healthy numbers of adult bees and brood, however,
the colonies were infested with varroa
and one of the colonies was found to have European
foulbrood (EFB). The association of184 L. Ward et al.
Figure 1. Multiple alignment of all-available KBV sequences alongside ABPV sequences (for ABPV, if

Kết quả (Việt) 1:

184 L. Phường et al.Hình 1. Nhiều liên kết có sẵn tất cả KBV cuỗi cùng với trình tự ABPV (cho ABPV, nếunhiều hơn một trình tự có sẵn với 100% identity trong khu vực này, chỉ là một trong mỗi chuỗi 'loại'được hiển thị), chỉ ra vị trí của chất nền, mồi và đầu dò thiết kế (trong hộp). Chỉ bướu nucleotideĐang hiển thị, (.) cho biết một nucleotide giống hệt nhau tại vị trí đó.Số lượng KBV virus ở Anh và Úcmẫu đã suy luận từ các đường cong tiêu chuẩn.Nồng độ virus đã được normalised bằng cách chiađăng nhập nghịch đảo của vi rút số lượng bởi nghịch đảoNhật ký của số lượng IPC ong.3. KẾT QUẢ3.1. thời gian thực thiết kế mồi và thăm dòMặc dù KBV và ABPV là chặt chẽliên quan đến nó đã có thể phân biệt đối xử giữaCác virus hai bằng cách thiết kế TaqManchất nền, mồi và một tàu vũ trụ tới các khu vực bảo tồn củaKBV chuỗi nhưng nơi dãy ABPVđổi biến (hình 1, Tab. I). The KBV khảo nghiệmđã cho một đảng Cộng sản Romania tích cực kết quả khi thử nghiệm với3 chuẩn bị tinh khiết virus, KBV14, KBV16và KBVNZ. Không có cross-reaction được quan sát thấy(không có khuếch đại sau khi 40 PCR chu kỳ) khiRNA được chiết xuất từ tinh khiết virus hay con ongbị nhiễm virus sau ABPV,BQCV, nhà nước, CWV, SPV, DWV, CPV vàAIV. Để xác nhận sự hiện diện của virus trong cáckính hiển vi điện tử chuẩn bị tinh khiết virusđã được thực hiện, và trong mỗi trường hợp vi rút hạtKích thước chính xác đã được quan sát (dữ liệukhông hiển thị). Các virus nơi trình tựwas available (BQCV, SBV, DWV and AIV)the presence of virus was confirmed by RTPCR/PCR(data not shown). For Cloudy wingvirus (CWV) electron microscopy confirmedthe presence of virus particles of the correctsize, whilst the KBV assay did not detect viralRNA, even though the available sequenceof CWV is identical to KBV. Although AIVis a DNA virus it could still be detected inthe RNA extractions from infected bees usingPCR (without an RT step), indicating that suf-ficient DNA was co-purified in the extractionprocedure to enable effective detection.KBV was successfully detected in differentlife stages of Apis mellifera L. including Australianworker honey bees, worker pupae anddrone pupae. Some of the worker honey beesfrom America also tested positive for the virus,along with one Australian wasp (Vespula germanica).This further indicates that the assaydeveloped is able to detect different sourcesof KBV in A. mellifera as well as KBV indifferent insect species. Detection in other insectspecies is further supported by the successfulamplification of KBV sequence fromthe virus preparation KBV16, obtained from abumblebee. The German bees all tested negativefor KBV, however, the internal control resultsshow that adequate levels of total RNAhad been extracted (Tab. II).3.2. Survey of honeybee coloniesfor KBVRNA extracted from 5 bees in each surveysample was tested with the real-time PCRFirst detection of Kashmir bee virus in the UK 185Table II. Results from testing known infected samples and samples suspected of being KBV-infected,including the number of positive samples and the average Ct values and standard deviations for the KBVassay in positive samples and the internal positive control assay in all samples extracted. The instrumentrecords a Ct value of 40 for the negative samples (-).Source Sample type Number of positive Average Ct for KBV Average Ct for 18S assayindividuals tested positive samples for all samplesAustralia Wasp pupa 1/2 24.36 23.52 ± 0.18Australia Honey bee worker pupa 6/6 29.09 ± 3.28 14.64 ± 0.13Australia Honey bee drone pupa 3/4 36.18 ± 1.66 14.48 ± 0.23Australia Honey bee worker 3/10 35.36 ± 6.54 16.48 ± 1.20Australia Honey bee worker 6/15 36.50 ± 1.63 23.24 ± 3.49USA Honey bee worker 2/10 31.85 ± 1.45 16.39 ± 1.55Germany Honey bee worker 0/10 - 17.59 ± 5.12Germany Honey bee worker 0/10 - 17.38 ± 0.75assays for the bee 18S rRNA (IPC). Using theautomated extraction method, RNA was successfullyrecovered from all bee samples. Asimilar extraction efficiency was observed forall samples indicating that the method was reproducible.The average real-time PCR Ct (denotedby the point at which amplification isfirst observed above the base threshold) valuefor two replicates from 458 survey sampleswas 10.39 ± 1.52.Following testing using the KBV real-timePCR assay, of the 458 samples tested, threetested positive for the presence of KBV. Two
of the positive samples were detected in different
hives from the same apiary. Details of
these colonies are shown in Figure 2. One of
the sampled colonies was infected with European
foulbrood (a statutory notifiable disease)
and was destroyed as part of the national
disease control strategy. PCR products ampli-
fied during the real-time PCR testing were
cloned and sequenced. The sequence of the
cloned PCR product sequence was confirmed
to be KBV following a blast search on the
NCBI database which matched other KBV sequences
(Accessions AY275710, AF263723–
AF263732, AY452696).
3.3. Quantification of virus load in UK
bee samples
The quantity of KBV in the positive UK
samples were compared to Australian bees that
were known to be naturally infected with KBV
at low levels (non-symptomatic) or high levels
(showing clinical symptoms). A standard
curve method was used to quantify virus load
in the bees rather than the ∆Ct method as described
in Chen et al. (2005). When the relative
efficiencies of the bee IPC and KBV were
plotted as ∆Ct [Ct(KBV)−Ct(IPC) ], versus the log of
the RNA dilution, the ∆Ct had a value greater
than 0.1 (the slope was 0.9513), indicating that
the amplification efficiencies of KBV and bee
IPC were not equal. In this instance, the ∆Ct
method was not suitable for quantification. In
addition, as the starting level of virus and bee
IPC RNA in the standard dilutions was unknown,
relative rather than absolute quantifi-
cation was used.
A standard curve was constructed by plotting
Ct values of bee and KBV standard RNA
dilutions against the log of the RNA dilution.
The quantity of KBV and bee IPC RNA
from each bee was extrapolated from the standard
curve. The normalised quantity of KBV
in each bee was determined by dividing the
amount of KBV RNA by the amount of bee
IPC RNA. The results show that the virus levels
in the infected bees from the UK are similar
to those in bees with covert levels of infection
from Australia. In comparison, other bees
(also from Australia) believed to be highly infected,
contained around 400 times more virus
than the bees at the UK Manchester site and
100 times more virus than covertly infected
Australian bees and bees from the UK Hull site
(Fig. 3).
186 L. Ward et al.
Figure 2. A Map showing the locations of the 458 bee colonies sampled in the UK 2004 KBV survey
including the two colonies that tested positive for KBV (indicated by black diamonds).
4. DISCUSSION
This paper reports the first finding of KBV
in the UK and the first large-scale survey for
bee viruses in Europe using TaqMan technology.
This study demonstrates the practical application
of this technology as a support tool
for surveys and contingency management, and
to provide robust surveillance data on the presence
or otherwise of KBV and other honey bee
viruses.
The incorporation of an internal control assay
to detect the bee 18S rRNA gene allowed
the assessment of extraction efficiency and allowed
interpretation of false negative results.
First detection of Kashmir bee virus in the UK 187
Figure 3. Relative quantification of KBV in bee samples. (A) TaqMan Standard curves for KBV virus and
bee IPC. (B) Relative comparison of KBV levels in UK bees in relation to bees known to be naturally
infected with high and covert levels of KBV infection.
Also, the real-time PCR assays allowed the
quantitative assessment of virus levels over
several orders of magnitude. This proved to
be valuable for detection of low-titre infections
within the bees tested. In this study,
quantification of KBV levels in the UK bees
showed they contained similar levels to Australian
bees having covert infections. The bee
colonies from the UK sites showed no obvious
signs of virus infection at the time of collection
suggesting the virus was covert or latent
in these apiaries. It is known that the viruses
can exist in individual bees or entire colonies
for long periods without causing noticeable
clinical symptoms (Dall, 1985; Anderson and
Gibbs, 1988; Hung et al., 1996a, b). Studies
by Anderson and Gibbs (1988) showed that
covert virus infections were common in pupae
with KBV. The status and sites where the
virus may remain in the bee in this state are
unknown, however, it has been suggested the
lining of the gut may be the site of ‘unapparent’
or covert infection (Anderson and Gibbs,
1989). Anderson and Gibbs (1988) have stated
that these infections do possess a latent capacity
to become activated and develop into acute
infections.
Some studies have suggested that viruses
such as KBV and ABPV may exist as natural
infections in a broader range of genera other
than Apis. For example KBV has been detected
in the wasp Vespula germanica (Anderson,
1991) and ABPV in Bombus species (Bailey
and Gibbs, 1964). Our study provides further
support for this, as KBV was detected in the
same species of wasp and one of the KBV
serotypes tested was extracted from a bumblebee
from New Zealand. In terms of trade and
regulation of honeybee viruses between countries,
if these viruses exist in different insect
hosts, it makes such controls and restrictions
difficult to impose or justify. In view of this,
the full host-range of honeybee viruses warrants
further study.
At the time of sampling, all three KBV positive
UK colonies were in good condition, with
healthy numbers of adult bees and brood, however,
the colonies were infested with varroa
and one of the colonies was found to have European
foulbrood (EFB). The association of184 L. Ward et al.
Figure 1. Multiple alignment of all-available KBV sequences alongside ABPV sequences (for ABPV, if