An investigation of honey bee virus prevalence in managed honey bees (Apis mellifera and Apis cerana) undergone colony losses: a case study in China

Objective: In the absence of known clinical symptoms, viruses were considered to be the most probable key pathogens of honey bee. Therefore, the aim of this study was to investigate the prevalence and distribution of honey bee viruses in managed Apis mellifera and Apis cerana in China. Results: We conducted a screening of 9 honey bee viruses on A. mellifera and A. cerana samples collected from 54 apiaries from 13 provinces in China. We found that the types and numbers of viruses signicantly differed between A. mellifera and A. cerana. Black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Apis mellifera lamentous virus (AmFV) and Kakugo virus (DWV-C/KV) were the primary viruses found in A. mellifera colonies, whereas Chinese sacbrood bee virus (CSBV) and sacbrood bee virus (SBV) were the primary viruses found in A. cerana. The percentage infection of BQCV and CSBV were 84.6% and 61.6% in all detected samples. We rst detected the occurrences of Varroa destructor virus-1(VDV-1, DWV-B) and DWV-C/KV in China but not ABPV in both A. mellifera and A. cerana.


Introduction
Viruses, bacteria, fungi and parasites were all con rmed to be capable of inducing reductions in colony populations [1][2][3][4]. Among these pathogens, more than 20 viruses were identi ed to infect honey bee, most of them belonged to the family Picornaviridae and possessed a single-strand positive-sense RNA genome. These viruses were demonstrated to not only have severe impacts on individual bees by damaging their physiology [2], interfering with their productivity [5] and contributing to behavioral disorders [6] but also on the colony growth rate and colony tness (i.e., decrease of foraging activity at the colony level [7]).
However, most honey bee viruses cause covert infections without signi cant clinical symptoms, which is a barrier to their study. A few viruses can be recognized during the late stage of infection by their appearance, such as chronic bee paralysis virus (CBPV), black queen cell virus (BQCV), deformed wing virus (DWV), sacbrood virus (SBV) and Israeli acute paralysis virus (IAPV). Nevertheless, little is known about DNA viruses of honey bees compared to RNA viruses. Recently, the DNA virus Apis mellifera lamentous virus (AmFV) was reported to be associated with crawling bees. The AmFV viral loads were reported to be higher in the bees in spring in Switzerland and demonstrated a signi cant correlation with RNA viruses in infected honey bees [8]. Owing to the constant discovery of emerging viruses, many investigations have reported the occurrence and prevalence of honey bee viruses as well as other viruses that may infect honey bees, especially since IAPV was found to be associated with colony collapse disorder (CCD) [9]. Ai et al. [10] investigated the occurrence of 7 honey bee viruses in 2011 in China, and found that DWV was the most prevalent of the viruses. Subsequently, a report that demonstrated the prevalence of 6 viruses in A. cerana found that BQCV had an infection rate of 98% in A. cerana [11]. In contrast, Yang et al. [12] found that DWV was the most prevalent among the 7 viruses in China regardless of whether A. mellifera or A. cerana was investigated.
Despite the prevalence of honey bee viruses that have been extensively investigated around the world, several neglected and new honey bee viruses have not been described in China. To obtain a broader spectrum of information about honey bee viruses, we investigated the presence of viruses in A. mellifera and A. cerana bee samples from different provinces that reported large bees crawling in front of the hives and undergone colony losses. We screened bee samples from 56 apiaries from 13 provinces to identify the presence and distribution of 9 honey bee viruses in China. We found that BQCV and CSBV were the most prevalent in A. mellifera and A. cerana, respectively. Additionally, we discovered that a high percentage of colonies were infected by AmFV and DWV-C/KV in many regions in China for the rst time.

Bee samples
Honey bees were sampled from 56 apiaries in 13 provinces from March to November, 2015. Worker samples of A. mellifera were collected from 38 apiaries in 8 provinces and samples of A. cerana were obtained from 18 apiaries in 10 provinces (Table S1). Ten colonies were randomly selected in each apiary per province. Clinical signs of virus infection were not found in these colonies; thus, these colonies were thought to be capable of honey production, although the colonies were reported to have undergone large losses or were identi ed with crawling bees. All of the above colonies were maintained consistent with guidelines for beekeeping practice manipulation and regularly monitored and treated for mites (only for A. mellifera).

PCR detection
The primer sequences, orientation and references are provided in Table S2. For the 8 RNA viruses, the initial cycle for reverse transcription was 80 °C for 30 min, followed by the PCR cycles. The PCR reaction for AmFV and the other 10 RNA viruses consisted of a total 20 µl volume containing 2 × GoTaq reaction buffer (Promega, USA), 1 µM of the sense and antisense primers, 1 µL of cDNA or DNA, and nuclease-free water. The cycling conditions were as follows: 1 min at 95 °C; 33 cycles of 30 s at 94 °C, 30 s at 55 °C and 72 °C for 1 min; a nal extension of 10 min at 72 °C; and cooling to 4 °C. The PCR ampli cation products were separated in a 2% agarose gel stained with GV II (Biomec, China) and photographed with a FR-200A luminescent and uorescent biological image analysis system (Furi, China). The product size was determined using a 100-bp molecular size ladder.

Statistical analysis
The results were expressed as the presence or absence of virus in an apiary. We considered virus to be present in an apiary if samples from any colony were positive for any virus. Differences in the viral infection percentages of different provinces and virus co-infection were estimated using the Chi-square test with the SPSS 21 software.

Virus prevalence and occurrence
As shown in Table 1, different types of viruses were detected in all provinces with the exception of Jiangsu province where no virus was detected. All of the viruses were present in the different provinces with the exception of KBV and ABPV, which were not detected. BQCV was the most prevalent virus and was present in 18 % of the A. mellifera apiaries and in seven provinces. CSBV was found in 38.9 % of the A. cerana apiaries and in seven provinces. In contrast, BQCV was detected in 22 % of the A. cerana apiaries and CSBV was found in 2.6 % of the A. mellifera apiaries. The following frequencies were obtained for the remainder of the viruses: IAPV was detected in 7.9 % of the A. mellifera apiaries and 5.6 % of the A. cerana apiaries; CBPV was found in 15.8 % of the A. mellifera apiaries and was not detected in A. cerana; SBV was identi ed in 5.3 % of the A. mellifera apiaries and 16.7 % of the A. cerana apiaries; DWV-A was found in 13.2 % of the A. mellifera apiaries and 5.6 % of the A. cerana apiaries; DWV-B was found in 10.5 % of the A. mellifera apiaries and was not detected in the A. cerana apiaries; AmFV was found in 13.2 % of the A. mellifera apiaries and 11.1 % of the A. cerana apiaries; and DWV-C/KV was found in 10.5 % of the A. mellifera apiaries and 11.1 % of the A. cerana apiaries.
BQCV was the most common virus and accounted for 84.6 % among the tested samples in each province ( Figure 1A). The second highest frequency virus was CSBV (61.6%), followed by another 3 viruses at equal frequency (53.8 %; DWV-A, AmFV and DWV-C/KV). The least frequent viruses were IAPV and DWV-B with 30.8 %. The number of honey bee viruses in A. mellifera was higher than that in A.cerana in the same province ( Figure 1B) with the exception of Chongqing and Yunnan, which gave priority to keeping A. cerana. The number of viruses in A. mellifera in Zhejiang was the same as that of A. cerana. Liaoning province had the highest number of viruses, with 9 viruses in A. mellifera and only one in A. cerana. Similarly, there were 7 viruses in A. mellifera in Inner Mongolia and Heilongjiang and no viruses were detected in A. cerana.

Co-infection of viruses in each province
The co-occurrence of more than one virus in one province was fairly common in both the A. mellifera and A. cerana apiaries (Table S3 and Table S4). In A. mellifera, virus co-infection varied from 2 to 6 viruses. Co-infection with 2 viruses represented 28.9 % of the occurrences, 3 viruses represented 31. 6 (Table S5), whereas in A. cerana co-infection with 2 viruses was signi cantly more common than the other co-infection types (e.g., BQCV and CSBV; χ 2 =0.0009, P=0.02, df=1) (Table S6).

Discussion
The results showed that several viruses with a high prevalence and wide distribution in China were not previously reported, such as DWV-B, DWV-C/KV and AmFV. Our surveys indicated that DWV-C/KV and AmFV were widely distributed in China in both A. mellifera and A. cerana. Additionally, neither ABPV nor KBV was found in any province. This primary investigation helps to narrow future efforts to identify the cause of colony decline.
In addition, we found multiple virus co-infections in honey bee colonies of both A. mellifera and A. cerana. The widespread distribution of honey bee viruses in the two honey bee species suggests a stable interaction between the viruses and their hosts. Simultaneous infection with more than 3 viruses (65.78%) occurred in more than 12 apiaries of A. mellifera, which was more than occurred in A. cerana, suggesting that multiple infections were very common in A. mellifera; in contrast, most cases of virus coinfection in A. cerana involved 2 viruses. Data from experiment studies indicated that multiple virus infections will increase the virulence [13]. A recent report showed that IAPV could be elevated to a higher level when several viruses were mixed to infect honey bees, although the initial IAPV infection level was not the highest [14]. Thus, colony losses might be due to a combination of multiple viruses.

Conclusion
We demonstrated for the rst time the presence of DWV-B and DWV-C/KV. The most prevalent viruses were BQCV, CBPV, DWV-C/KV and AmFV in A. mellifera and CSBV and SBV in A. cerana. The results of this study suggest that future studies should focus on these viruses to narrow our objectives and help us understand the differences in different areas and hosts.

Limitations
A main limitation of current study is that we did not investigate the honey bee samples from all provinces. Therefore, we cannot draw conclusions regarding the prevalence of honey bee viruses in China. In addition, the differences in the distributions and levels of different viruses among the different tissues of honey bees also require further study, although multiple virus co-infections were found.

Declarations
Availability of data and materials All data generated or analysed during this study are included in this published article and Additional le.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
Authors have no potential con ict or competing interests to declare.