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Prevalence of bee viruses in Apis cerana cerana populations from different locations in the Fujian Province of China

Abstract Prevalence of honeybee viral diseases has recently been causing major problems in the beekeeping industry, causing economic losses worldwide. Honeybees are susceptible to a variety of diseases and various pathogens. Among these pathogens, prevalence viruses, along with other factors, are seriously threatening the health of bee species. In the present study, samples were collected from 80 Apis cerana cerana (A. c. cerana) colonies from three different locations, Cangshan, Fuan, and Yongtai, in the Fujian Province of China. All samples were screened using the reverse transcription polymerase chain reaction (RT‐PCR) method for detection of seven honeybee viruses, namely, Chinese sacbrood virus (CSBV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV), and Kashmir bee virus (KBV). Our results showed that CSBV was the most prevalent as it was detected in (90%), of the samples, DWV was detected in (81.25%), and IAPV was detected in (26.25%). In contrast, insignificant prevalence results were obtained from all apiaries for BQCV, CBPV, APBV, and KBV, which were not detected in any sample. Here, we are providing the first report on the molecular detection of honeybee viruses, especially the prevalence of IAPV, from different regions in the Fujian Province of China with a high prevalence of bee viruses, on A. c. cerana, and there is great concern for the presence of honeybee viruses in the population of the native honeybee (A. c. cerana) in China.

globe, causing economic losses worldwide (Gisder & Genersch, 2015Martinello et al., 2017). The number of RNA and DNA viruses that have impacted bee species as investigated by metagenomics and applied next-generation sequencing technologies are increasing; at present, 27 honeybee viruses have been identified (Galbraith et al., 2018;McMenamin & Flenniken, 2018;Schoonvaere, Smagghe, Francis, & de Graaf, 2018). The seven most common and destructive bee viruses are sacbrood virus (SBV), deformed wing virus (DWV), black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV), and Kashmir bee virus (KBV) (Ai, Yan, & Han, 2012). Chinese sacbrood virus (CSBV) and DWV are the most widespread and distributed viruses among A. c. cerana colonies in China (Ai et al., 2012;Zhang et al., 2013). Meanwhile, in recent years, DWV, IAPV, and BQCV have also created a major problem in the beekeeping industry (Ward et al., 2007). China has more than 2 million A. c. cerana colonies, but recently, the number of colonies is declining because of the presence of bee viruses, along with some other threatening factors (Huang et al., 2017;Shan et al., 2017;Shi et al., 2013;Wang et al., 2012). The devastation of bees by viruses of the native honeybee (A. c. cerana), which are widely maintained in southern China, causes colony losses, and most of the studies focus on the European honeybee (Ai et al., 2012). Investigation and molecular research on the Chinese honeybee A. c. cerana are scarce (Shi et al., 2013).
RT-PCR was conducted to study the prevalence and distribution of seven bee viruses in A. c. cerana colonies. Among these seven viruses, three viruses (CSBV, DWV, and IAPV) were detected with high prevalence. However, BQCV, ABPV, CBPV, and KBV were not detected in any sample. This study has provided a recent report on the distribution of bee viruses in the different geographic regions of Fujian Province and evidence of IAPV infection in A. c. cerana colonies, which has not been reported before in this species from Fujian Province, and it may be helpful for the possible control of viral epidemic diseases.  (Figure 1). A total of 1,200 live samples were randomly collected (Ten 4 th instar larvae and five capped pupae) from every colony separately. Each apiary was randomly selected, and samples were collected from 6 colonies with an obvious symptom of a sacbrood virus infection from Fuan apiary and from 74 colonies without any exhibited symptoms of any virus infection. Samples were collected from three combs of each hive based on the frames. We selected the combs from both the side and the center of the hives. After collection from the frames, all samples were immediately transferred into a sterilized 2 ml centrifuge tube and were kept in 700 μl RNA later (Solarbio ® Life Science) solution according to the manufacturer's instructions. Later, larvae and pupae samples were grounded in a single tube, and 30 μl was taken from a pool of ground samples for further molecular analysis.

| Sample collection
F I G U R E 1 Sample collection sites from three different locations, namely, Fuan, Cangshan and Yongtai (n = 26, 14 and 40 colonies, respectively), and virus detection from larvae and pupae using RT-PCR, gel electrophoresis and percentage of infected A. c. cerana colonies

| RNA extraction and cDNA synthesis
Total RNA was extracted from collected samples using a LabServ Universal RNA kit (Thermo Scientific KingFisher Flex) with magnetic beads according to the manufacturer's instructions. The concentration of RNA was determined by spectrometer using a NanoDrop ND-2000 (Thermo Scientific). Extracted RNA was used immediately for generation of first strand cDNA to eliminate genomic DNA, certifying that only RNA existed in the final preparation. Total RNA (4 μg for a 20 μl reaction) was used to obtain cDNA. The RNA template was primed using an oligonucleotide-deoxythymidine (oligo-dT) primer with Transcript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Beijing) according to the manufacturer's instructions. Reactions were incubated at 65°C for 5 min, 4°C for 2 min, 25°C for 10 min, 42°C for 90 min, and 85°C for 10 s, and cDNA was kept at −20°C for later use.

| RT-PCR assays for virus detection
An RT-PCR was developed for better diagnosis of viral diseases by the following standard method for the accurate detection of several honeybee viruses (Yoo, Thi et al., 2012). RT-PCR was conducted to detect seven common bee viruses (CSBV, DWV, BQCV, KBV, ABPV, IAPV, and CBPV). We screened all the samples using specific primer pairs ( Table 1). Amplification of cDNA was carried out using 2X EasyTaq ® PCR SuperMix (TransGen Biotech Beijing). We used a total reaction volume of 15 μl in the PCR for diagnosis of each virus. For CSBV detection, PCR amplification was conducted via initial denaturation at 95°C for 1:30 min, followed by 39 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 25 s, and finally 72°C for 1 min. For the detection of DWV, PCR amplification was conducted via an initial denaturation at 95°C for 1:30 min, followed by 39 cycles at 95°C for 15 s, 57°C for 30 s, and 72°C for 30 s, and finally 72°C for 1 min.
Likewise, for IAPV detection, PCR amplification was conducted through an initial denaturation at 95°C for 1:30 min, followed by 39 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and finally 72°C for 2 min. CBPV, ABPV, BQCV, and KBV were not detected from any samples, so we did not mention the PCR amplification protocol here. The PCR products were analyzed by electrophoresis in a 2% 50X TBE buffer agarose gel and stained with ethidium bromide.
Amplified products were visualized under UV light, and their length was compared to a standard 100-bp molecular weight DNA ladder (Invitrogen, USA). To ensure that the PCRs were running correctly, we used an apis-β-actin primer pair, size: 181 bp (Table 1), for an internal positive control in all PCR virus detection assays, and for the negative control, we used double-distilled water (ddH 2 O). We also used previously identified positive sample cDNA as a positive control for CSBV and DWV in every PCR reaction.

| Statistical analysis
Statistical analysis was carried out using SPSS version 21.0 (Inc.,

Chicago, Illinois, USA).
To compare the means distribution of each detected virus between different locations. Data were analyzed using one-way ANOVA, Post Hoc Test; Tukey HSD, with p-value of ≤0.05 considered significant difference. GraphPad Prism version 7.00 for Windows (GraphPad Software, California, USA) was used to draw the graphs. was virus free, and this result is very interesting (Figure 3). Likewise, most of the colonies presented multiple-virus infections, whereas a few colonies showed a single-virus infection (Figure 4)

Infected colonies in (%)
Cangshan apiaries, most of the colonies did not present any disease

| D ISCUSS I ON
China has the largest managed honeybee population in the world, with more than two million A. c. cerana and 8.77 million A. mellifera colonies. However, the presence of bee viruses is becoming a major problem, causing concern among beekeepers and economic losses.
The prevalence of viral disease in various honeybee species has been investigated by several researchers, and it was observed that the trend of infection is worldwide (Shi et al., 2013;Wang et al., 2012;Xia, Mao, Wang, Zhou, & Wei, 2014 (Ai et al., 2012).
These results are similar to our findings that the prevalence of CSBV was (90%), followed by DWV at (81.25%), but our results for IAPV showed increasing infections at (26.25%) in A. c. cerana colonies. A few colonies were infected with a single virus, whereas most of the colonies were infected with multiple viruses. CSBV, IAPV, and DWV were detected in all apiaries. CSBV was the most prevalent in Fuan and Cangshan, DWV was the most prevalent in Yongtai and IAPV showed the same prevalence in Fuan and Cangshan, but IAPV was the most prevalent in Yongtai. However, BQCV, CBPV, ABPV, and KBV were not detected in any geographic locations (Figures 2 and 4).

The surveys of domestic A. c. cerana populations in China have
suggested that CSBV is the primary disease involved in the declining population of colonies (Ai et al., 2012). The prevalence of CSBV in Taiwan increased from (41.1% to 69.6%), over 4 months in 2015.
Additionally, (37.5%) of A. mellifera colonies were infected with CSBV, which showed that the western honeybee is also susceptible to CSBV (Nai et al., 2018). ABPV infection rate of (22%) in A. c. cerana was reported in Guangdong, while there was no infection of this virus in Fujian until now (Ding et al., 2016). IAPV detection was reported in Fujian Province with a prevalence of (11%) in A. mellifera, and the prevalence of IAPV in A. c. cerana was also reported in Hainan at (60%) and in Beijing at (30%-35%) (Antunez et al., 2012).

There are two serotypes, a European serotype that infects
A. mellifera throughout the world and an Asian serotype that can infect A. cerana, and multiple complete genomes and genetic variation of both serotypes have been described, including distinct strains in South Africa and Russia (Choe, Nguyen, Hyun et al., 2012;Choe, Nguyen, Jin et al., 2012;Roberts & Anderson, 2014). CSBV can infect honeybees at all stages from eggs to adults (Shan et al., 2017).
DWV is also the primary concern among the honeybee viruses that belongs to the Iflaviridae, (family) threatening honeybee colonies worldwide (Gisder & Genersch, 2017). KBV has not been detected in China in both species of honeybee until now (Ai et al., 2012). A survey of the honeybee A. mellifera in China from 2010 to 2013 showed losses of (10%) of colonies and overwintering losses of (770,000) colonies per year (Liu et al., 2016). While in 6 decades the number of honeybee colonies increased by (45%) globally, in recent decades, colony losses have increased, and overwintering losses have been reported frequently in different countries worldwide (Gisder & Genersch, 2015).
In conclusion, many studies have been conducted on the presence of honeybee viruses in western honeybees (A. mellifera) and

CO N FLI C T O F I NTE R E S T S
None declared.

E TH I C S S TATEM ENT
None required.

DATA ACCE S I B I LIT Y
All data are provided in the results section of this paper.