Diversity and Dynamics: Virome Analysis of Two Honey Bee Species across Pakistan and Bangladesh

INTRODUCTION

The Apis mellifera species is globally cultivated as a pollinator for numerous crops and is also economically significant to produce industrial goods like honey, royal jelly, wax, and propolis (Popovska Stojanov et al., 2021; Zacepins et al., 2021). A. cerana remains primarily managed in Asian countries (Theisen-Jones and Bienefeld, 2016). Both species are under constant threat from diseases, environmental changes, and pesticides, significantly impacting bee health and stability of bee colonies (Atkins, 1992; Allen and Ball, 1996; Le Conte and Navajas, 2008). A prominent issue for A. mellifera is Colony Collapse Disorder (CCD), which has led to considerable population declines worldwide. A. cerana, in contrast, has faced notable mortality from sacbrood virus (SBV) outbreaks in countries such as Korea, Vietnam, Thailand, India, and China (Bailey et al., 1983, Liu et al., 2010, Choe et al., 2012; Nguyen and Le, 2013).

Virological research on honey bees initially lagged behind other fields, with sacbrood virus identified as the first known honey bee virus (White, 1913). Interest in honey bee virology surged following the CCD phenomenon in 2007, with the number of identified viruses rising from just 17 prior to CCD to over 128 by 2023 (Chen and Siede, 2007; Oldroyd, 2007; Beaurepaire et al., 2020; Kwon et al., 2023a). Despite these advances, significant gaps remain, particularly in regions like Pakistan and Bangladesh, where honey bees are extensively managed but relatively under-researched.

Beekeeping in Pakistan mainly involves the commercial beekeeping of two species, A. mellifera and A. cerana, with recent estimates indicating approximately 600,000 colonies managed by 10,000 beekeepers, producing about 12,000 tons of honey annually (Khan and Ghramh, 2023). Alongside virus threats, honey bees in Pakistan face various pests and diseases, including Varroa destructor, Tropilaelaps clareae, American foulbrood (AFB), and chalkbrood (CB) (Waghchoure-Camphor and Martin, 2009; Rehman et al., 2018; Khan and Ghramh, 2023). In Bangladesh, beekeeping encompasses five species, of which four (A. cerana, A. dorsata, A. florea, Trigona sp.) are native, including A. cerana (TBS, 2020). In 2023 survey, commercial apiculture is projected to involve around 70,000 colonies, maintained by 12,000-15,000 beekeepers with an annual honey yield of 5,000 tons (Haque, 2023). However, studies on honey bee pests and pathogens in Bangladesh are still limited.

Advancements in sequencing technologies, from Sanger sequencing to second-generation high-throughput sequencing (HTS), have greatly expanded virological research by reducing analysis time and costs (Collins et al., 2003; Mardis, 2011; Pareek et al., 2011). Additionally, several thirdand fourth-generation analysis platforms are now in use (Feng et al., 2015; Rhoads and Au, 2015; Deamer et al., 2016). These advancements have significantly reduced the extensive time required for analysis by Sanger sequencing and the cost of analysis by more than 100-fold (Schloss, 2008). HTS has catalyzed progress in bioinformatics, especially virome analysis, which comprehensively examines viral communities in a sample, including known, novel, and asymptomatic viruses (Ercolini, 2013; Kapoor and Lipkin, 2014; Conesa et al., 2016; Sekse et al., 2017; Kwon et al., 2023b). In honey bees, virome analysis has illuminated the viral diversity affecting bees, such as the discovery of Lake Sinai virus (LSV) species and the tripartite classification of deformed wing virus (DWV) associated with CCD (Runckel et al., 2011; Roberts et al., 2018a; Bonilla-Rosso et al., 2020; Daughenbaugh et al., 2021; Kadlečková et al., 2022; Li et al., 2022). Since then, several researchers have conducted virome analyses, confirming that LSV group is a virus complex with multiple species (Šimenc et al., 2020; Daughenbaugh et al., 2021; Čukanová et al., 2022; Kadlečková et al., 2022). Research has also demonstrated that DWV, which is associated with CCD, can be classified into three types (Mordecai et al., 2016; Natsopoulou et al., 2017). In some instances, plant viruses have been detected in honey bees (Granberg et al., 2013; Li et al., 2014). Interestingly, virome studies have also identified plant viruses in bees, suggesting honey bees may play a role in plant virus transmission during pollination (Darzi et al., 2018; Roberts et al., 2018b).

This study aims to explore the viral landscape within A. mellifera and A. cerana populations in Pakistan and Bangladesh. By employing virome analysis through HTS, we seek to identify the diversity of viruses present, including novel species, and provide a foundation for future virological research and conservation efforts in these regions.

MATERIALS AND METHODS

1. Honey bee sample collection

Honey bee samples were collected from multiple apiaries across Pakistan and Bangladesh (Fig. 1). In Pakistan, honey bees were obtained from the regions of Fateh Jang (33°33ʹ59.6ʺN 72°36ʹ51.1ʺE; 33°35ʹ28.9ʺN 72°40ʹ48.2ʺ E), Jand (33°25ʹ57.1ʺN 72°00ʹ57.1ʺE; 33°25ʹ44.0ʺN 72° 01ʹ40.2ʺE; 33°26ʹ12.8ʺN 72°00ʹ32.4ʺE), Khunda (33°27ʹ 43.8ʺN 72°23ʹ55.4ʺE; 33°27ʹ31.8ʺN 72°23ʹ49.3ʺE), and PindiGheb (33°14ʹ58.1ʺN 72°16ʹ19.4ʺE; 33°14ʹ26.8ʺN 72°16ʹ20.2ʺE; 33°13ʹ56.5ʺN 72°16ʹ36.0ʺE). In Bangladesh, samples were collected from Tangail (24°14ʹ59.4ʺN 89°54ʹ59.6ʺE). From each location, a minimum of 50 honey bees were collected, preserved in 70% ethanol within 50 mL conical tubes, and stored at 4℃ until RNA extraction.

Fig. 1.

Geographic distribution of viruses identified in honey bee samples from Bangladesh and Pakistan. Red circles indicate sampling locations on the map. The list of detected viral taxa from virome analysis is shown beside each country. Viruses are color-coded based on host specificity: green for plant viruses and yellow for honey bee viruses.

RESULTS

1. High-throughput sequencing overview

The average number of reads generated per sample was 64,236,804. Notably, the Fateh Jang sample had approximately 10 million fewer reads than other samples, though all samples maintained significant Q30 values, ensuring high data quality. Tangail 1 had the highest host reads (55,812,561), while Tangail 2 held the highest viral reads count, with 23,491,534 reads (Fig. 2, Table S1). Thirteen viruses spanning seven families, along with an unclassified DNA virus, were identified (Fig. 3). Among the sixteen identified viruses, nine were honey bee viruses, while seven were plant viruses, including four novel viruses (Table 1). Apis mellifera filamentous virus (AmFV) showed the highest viral read count in Tangail 2, with approximately 20 million reads. Khunda recorded the highest virus diversity, with eight viruses identified. Plant viruses were detected in most samples, except for Fateh Jang and Tangail 2.

Fig. 2.

Distribution of reads following quality trimming for high-throughput sequencing. Black bars represent host-related reads, red bars represent viral reads, and gray bars represent other reads. Samples from Fateh Jang, Jand, Khunda, PindiGheb, and Tangail 1 correspond to Apis mellifera, while the Tangail 1 sample represents A. cerana.

Fig. 3.

Virus read counts by family across detected viral sequences. This analysis identified eight well-known viral families and one unclassified DNA virus family from the honey bee virome samples.

Table 1.

Virus read counts from virome analysis in honey bee samples

2. Honey bee viruses

1) Dicistroviridae

Aphid latent paralysis virus (ALPV) sequences from PindiGheb (PP971556) had 96.98% similarity with MF458892 from Kenya, while sequences from Fateh Jang (PP971553) and Jand (PP971554) showed 98.51% and 97.26% similarity with MW063131 from South Africa (Fig. S1A). Acute bee paralysis virus (ABPV) identified in PindiGheb (PP971549) showed 94.52% similarity with MZ821785 reported in China (Fig. S1B). Black queen cell virus (BQCV) was detected in four samples, with highest homology values of 91.88% (Fateh Jang, PP971562), 93.54% (Jand, PP971563), 93.88% (Tangail 2, PP971589), and 95% (Khunda, PP971564) to MW397638 identified in Israel (Fig. S1D). A partial sequence from the Triatovirus genus was identified in Fateh Jang (PP971572), showing 87% coverage and 80.64% identity to Vespula vulgaris picorna-like virus 2 (VvPLV2, QZZ63316) and was classified as a novel virus named “Apis mellifera associated triato-like virus (AmTLV)” (Table 2).

Table 2.

Summary of BLAST analysis results for novel viruses identified in this study

2) Partitiviridae

The Hubei partiti-like virus 34 (HPLV34) virus detected in PindiGheb (PP971577) showed 95.34% similarity to ON648754 from Slovenia, while sequences from Fateh Jang (PP971576) and Jand (PP971578) showed similarities of 95.48% and 97.63% to MT747982 from Italy (Fig. S1C). The Apis mellifera associated partiti-like virus 2 (AmPLV2), previously reported in South Korea, was detected in Pakistani samples, with sequences from Fateh Jang (PP971557) and PindiGheb (PP971559) showing 96.53% and 96.39% similarity to OR496402, and sequences from Jand (PP971558) and Khunda (PP971560) showing 96.61% and 96.24% similarity to OR496403.

3) Iflaviridae

Deformed wing virus (DWV) was identified in Fateh Jang (PP971573), and Tangail 2 (PP971590), showing 92.76%, 92.85%, and 93.39% similarity with MT940255 from the USA. The sequence in Jand (PP971574) exhibited 96.09% similarity with MN607197 from Viet Nam (Fig. S1E).

4) Sinhaliviridae

Lake Sinai virus 4 (LSV4) was detected in all samples except Tangail 1. The sequences showed similarities of 90.05% (Khunda, PP971580), 90.12% (Jand, PP971579), 89.30% (PindiGheb, PP971581), and 89.22% (Tangail 2, PP971591) with MZ821913 from China. The Fateh Jang (PP971582) sample yielded a partial sequence overlapping the ORF1 and RdRp regions, showing 98% similarity with LSV4 (Fig. S1F).

5) Unclassified DNA virus

The complete genome sequence of Apis mellifera filamentous virus (AmFV) was confirmed in Tangail 2 from Bangladesh (PP971592). This is the fourth country where AmFV has been fully sequenced, following China (OK 392616), Hungary (OR644609-OR644611, OR270109), and Switzerland (NC_027925).

3. Plant viruses

1) Endornaviridae

Bell pepper alphaendornavirus (BPADV) was identified in Khunda (PP971561), showing 97.66% similarity to MH182675 from Capsicum annuum in China (Fig. S1M). Four novel virus sequences were detected, with two classified under the Alphaendornavirus genus. Alphaendornavirus 1 (provisional) showed 65-70% identity and 3-5% coverage with known sequences, while alphaendornavirus 2 (provisional) had 70% identity and 15-18% coverage (Table 2). These novel viruses, with 40% amino acid sequence similarity to previously reported viruses, were named “Apis mellifera associated alphaendornavirus 1 (AmADV1)” and “Apis mellifera associated alphaendornavirus 2 (AmADV2)”, and their sequences were uploaded to NCBI GenBank (AmADV1; PP 971550, PP971551, AmADV2; PP971552) (Fig. S1O).

2) Bromoviridae

Three genome segments of cucumber mosaic virus (CMV) were identified in both the Khunda and PindiGheb samples. For segment RNA1, the sequence identified in Khunda (PP971565) showed a 99.41% similarity to OQ993352, previously reported in Cucumis sativus from Canada, while the sequence from PindiGheb (PP971566) showed a 99.50% similarity to OP722596, identified in Pelargonium peltatum in Germany (Fig. S1G). Segment RNA2 showed similarities of 98.72% and 99.31% with MG882751, reported in Cucurbita pepo from Poland, in Khunda (PP971567) and PindiGheb (PP971568), respectively (Fig. S1H). For segment RNA3, the sequence identified in Khunda (PP971569) had 98.01% similarity to OQ993354 from C. sativus in Canada, and the sequence from PindiGheb (PP971570) had 98.60% similarity to ON13912 identified in Solanum lycopersicum in Greece (Fig. S1I).

Tobacco streak virus (TSV) was found in Khunda, with three RNA segments. RNA1 (PP971583) (Fig. S1J), RNA2 (PP971584) (Fig. S1K), and RNA3 (PP971585) (Fig. S1L) showed similarities of 98.72%, 99.52%, and 99.55% with Indian isolates from Gossypium barbadense, Parthenium hysterophorus, and Gomphrena globosa, respectively.

3) Chrysoviridae

Brassica campestris chrysovirus 1 (BrcCV1), previously reported only in China, was identified in Tangail 1. Complete genome sequences of RNA1 (PP971586), RNA2 (PP971587), and RNA3 (PP971588) showed similarities of 97.3%, 98.08%, and 98.43% with NC_043661, NC_043660, and NC_043659, respectively.

4) Virgaviridae

One of the four novel virus sequences was classified under the Tobamovirus genus. Comparative analysis of the complete genome nucleotide sequence using BLASTn, according to ICTV genus demarcation criteria, showed 80-99% identity with previously reported tobamovirus sequences, though the coverage was limited to 1-2%. Despite the low coverage, this virus sequence contained all five conserved domains characteristic of the Tobamovirus genus (Fig. 4). Each domain showed approximately 50% similarity to known tobamovirus sequences in BLASTp analysis (Table 2). Based on these findings, this novel virus was classified as a new member of the Tobamovirus genus and named “Jand tobamovirus 1 (JTV1).” Its sequence has been deposited in NCBI GenBank (PP 971571) (Fig. S1N).

Fig. 4.

Genomic structure of Jand tobamovirus 1 (JTV1), showing the open reading frames (ORFs) and corresponding functional domains. Domains were annotated based on nucleotide positions, including Mtr (Methyltransferase), Hel (Helicase), RdRp (RNA-dependent RNA polymerase), MP (Movement protein), and CP (Coat protein).

DISCUSSION

Since the emergence of CCD in 2007, interest in honey bee viruses has surged, driving research efforts worldwide (Beaurepaire et al., 2020). HTS-based virome analyses have been conducted in many regions, revealing diverse viral profiles across Europe, North America, South America, Africa, the Middle East, and Asia (Granberg et al., 2013; Tozkar et al., 2015; Remnant et al., 2017; Galbraith et al., 2018; Haddad et al., 2018; Regan et al., 2018; Roberts et al., 2018a; Thaduri et al., 2018; Kraberger et al., 2019; Bonilla-Rosso et al., 2020; Bovo et al., 2020; Deboutte et al., 2020; Gebremedhn et al., 2020; Roberts et al., 2020; Šimenc et al., 2020; Daughenbaugh et al., 2021; da Silva et al., 2023; Kwon et al., 2023b; Lee et al., 2023; Li et al., 2023; Shojaei et al., 2023). Our study represents the first comprehensive virome analysis of two honey bee species, Apis mellifera and A. cerana, from Pakistan and Bangladesh, contributing valuable data on honey bee viruses in these countries on the Indian subcontinent.

Our results revealed the presence of multiple honey bee and plant viruses, including several previously unreported in these countries. Although the sample size is limited and may not capture the full viral diversity across all seasons and regions, this study lays the groundwork for future research. Broader, seasonal, and geographically extensive sampling is expected to reveal a more comprehensive viral landscape, potentially aiding in predicting and managing future honey bee health challenges in these regions.

Notably, we identified Apis mellifera-associated partitivirus 2 (AmPLV2), previously reported only in South Korea (Kwon et al., unpublished data, NCBI GenBank accession OR496401-OR496403), in honey bees from Pakistan. Future research should investigate the routes of transmission of this virus and its potential impact on bee health. Two novel viruses within the Alphaendornavirus genus were also identified: AmADV1 in Jand, Khunda, and PindiGheb, and AmADV2 in Jand. Given that alphaendornaviruses are pollen-transmissible (Moriyama et al., 1996), it is possible that these viruses were introduced through pollen carried by the bees. This finding underscores the potential role of honey bees as vectors for plant viruses, though it remains challenging to determine the exact plant host without further analysis.

We also detected a novel tobamovirus in honey bees from Jand. The limited sequence similarity between this virus and previously reported tobamoviruses suggests an unexplored diversity of viruses affecting plants in Pakistan (Dombrovsky et al., 2017; Zhang et al., 2022; Spiegelman and Dinesh-Kumar, 2023). Expanding virome studies to include plant hosts in Pakistan could yield new insights into viral diversity and potential reservoirs.

The viral profiles of the two bee species from Tangail, Bangladesh, showed notable differences. In A. cerana, only one plant virus was detected, while A. mellifera harbored four honey bee viruses but no plant viruses. This study also marks the first identification of Apis mellifera filamentous virus (AmFV) in A. mellifera samples from Bangladesh. Although AmFV has been linked to DWV, SBV, and BQCV in other studies (Hartmann et al., 2015), our findings provide limited data to establish relationships among these viruses. Further investigation is warranted to explore possible interactions among these pathogens.

In conclusion, this study presents the first virome analysis of honey bees in Pakistan and Bangladesh, identifying both known and novel viruses that may influence honey bee health. These findings provide foundational data for future research on honey bee viruses in these countries.

Acknowledgments

This study was conducted as part of the “Glocal University Project Group in Andong National University-Gyeongbuk Provincial College” supported by the Ministry of Education and National Research Foundation of Korea.

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