Introduction:

Worldwide, parasites and pathogens present serious threats to the health of the European honey bee

(Apis mellifera). In Asia, A. mellifera is commonly infested with two different genera of parasitic mites

(Varroa spp. and Tropilaelaps spp.) (Anderson & Morgan, 2007; Anderson & Roberts, 2013; Anderson

& Trueman, 2000). Although these mites have similar life cycles (Kapil & Aggarwal, 1987, 1989), the

populations of Tropilaelaps spp. are generally higher than populations of Varroa in A. mellifera

colonies (Anderson & Morgan, 2007; De Jong, De Jong, & Goncalves 1982; Oldroyd & Wongsiri,

2006). Hence, in Asia, Tropilaelaps mites inflict more serious damage to A. mellifera colonies than

varroa (Burgett, Akratanakul, & Morse, 1983; Burgett, Rossignol, & Kitprasert, 1990; Koeniger &

Musaffar, 1988; Laigo & Morse, 1968; Otis & Kralj, 2001). Moreover, Tropilaelaps mites are the

major health threat to Apis mellifera colonies in Asia because of their widespread occurrence, rapid

population growth and potential ability to transfer bee viruses. Furthermore, viruses, particularly RNA

viruses, constantly change and adapt to new host species and vectors, presenting a potential threat of

new and reemerging infectious diseases. Acute bee paralysis virus (ABPV) and Deformed wing virus

(DWV) are two of the most common honey bee viruses found in European honey bees Apis mellifera.

Furthermore, this study provides the first evidence that ABPV occurs in both A. cerana and T.

mercedesae in northern Thailand. The geographical proximity of host species likely played an

important role in the initial exposure and the subsequent cross-species transmission of these viruses.

The results from this study indicate that viral populations will continue to evolve and increase the need

for effective surveillance and control of virus infections in honey bee populations.

Summary:

Honey bees play a vital role in our global agricultural economy by pollinating a wide variety of

food crops and flowers and producing honey, beeswax, pollen, propolis, royal jelly, and other hive

products (Chen & Siede, 2007). The European honey bee (Apis mellifera) was first introduced into
Thailand in the 1980's joining other Asian honey bee species including Apis cerana that have been

commercially kept for pollination.

In recent decades, beekeepers around the world have encountered serious honey bee colony losses.

During the winter of 2006-2007, a phenomenon called Colony Collapse Disorder (CCD) mysteriously

wiped out honey bee colonies from most parts of the United States (Cox-Foster et al., 2007) but the

cause of CCD remains unknown. Several factors such as emergence of new parasites and pathogens,

poor nutrition due to loss of forage land and monocropping, excessive and inappropriate use of

pesticides and other environmental threats have been suggested to be involved in the widespread bee

population declines (Lever et al., 2014; Potts et al., 2010; Henry et al., 2012; Doublet et al., 2014;

Goulson et al., 2015). Of the potential factors reported so far, viruses have been suggested to be one of

the major risk factors that negatively affect bee health (Tencheva et al., 2004; Tantillo et al., 2015).

Honey bee viruses are widely distributed throughout the world. Among 22 viruses reported so far

(Tantillo et al., 2015), seven are highly prevalent in honey bee colonies: Acute bee paralysis virus

(ABPV), Deformed wing virus (DWV), Sacbrood virus (SBV), Black queen cell virus (BQCV), Israeli

acute paralysis virus (IAPV), Chronic bee paralysis virus (CBPV), and Kashmir bee virus (KBV). The

viruses infect different life stages of the honey bee including eggs, larvae, pupae, and adults and can

significantly affect honey bee health and shorten their life span (Chen & Siede, 2007). Furthermore, the

RNA viruses are especially prone to mutation which comes from the lack of proofreading ability in

RNA polymerases (Elena & Sanjuán, 2005). The higher mutation rate of RNA viruses increases the

adaptability of the viruses (Ferris et al., 2007), which allows the viruses to evolve and adapt to

changing environmental conditions during host expansion (Chen & Siede, 2007; Longdon et al., 2014).

A. mellifera and A. cerana are the only honey bee species that have been truly domesticated.

Populations of these species share food sources and habitats and interract with each other in East Asia

(Yang et al., 2011; Chantawannakul et al., 2016), creating opportunities for the transmission of

pathogens and parasites between the two species (Kojima et al., 2011). Virus transmission can also
occur during foraging through virus contaminated pollen and nectar (Singh et al., 2010) or through

vectors during infestation of parasitic mites such as Varroa destructor (Anderson & Trueman, 2000)

and Tropilaelaps mercedesae (Anderson & Morgan, 2007; Khongphinitbunjong et al., 2015). The

parasitic mite Varroa and intracellular microsporidian parasite Nosema ceranae were first described in

A. cerana (Fries et al., 1996) and jumped from this original host into the new host A. mellifera

(Anderson & Trueman, 2000; Higes et al., 2006). An earlier study showed that BQCV and DWV have

spread from A. mellifera to A. florea and A. dorsata in China (Zhang et al., 2012). Another study by

scientists from Japan also displayed inter-species transmission of IAPV infection from A. mellifera to

A. cerana (Kojima et al., 2011).

Earlier reports on virus prevalence in Asia did not provide evidence that ABPV infection occurs in

Asian honey bees (Choe et al., 2012; Ai et al., 2012; Yañez et al., 2015). In a regional survey for

parasites and pathogens including eight common bee viruses; DWV, BQCV, SBV, ABPV, KBV, IAPV,

CBPV, and Slow bee paralysis virus (SBPV) in South Korea, China and Vietnam showed that, except

for DWV, no clear evidence of unique interspecific transmission of virus infections between the two

honey bee species (Forsgren et al., 2015), was detected.

Moreover, Tropilaelaps mites are native to Asia and feed on the hemolymph of developing brood

of the giant Asian honey bee, A. dorsata (Chantawannakul et al., 2016). DWV was found in

Tropilaelaps mites that were screened for the presence of bee viruses during the previous Asian

regional survey for pathogens and parasites. DWV was found to be the most predominant honey bee

virus in Thailand (Sanpa & Chantawannakul, 2009) and to be tightly associated with infestations of

Varroa mites (Chantawannakul et al., 2006) and Tropilaelaps mites (Khongphinitbunjong et al., 2015).

In the present study, the researchers conducted an investigation into the occurrence of highly

predominant A. mellifera viruses in both A. cerana and the parasitic mite T. mercedesae collected from

A. mellifera to further explore and understand cross-species infections of viruses in honey bees.
 Moreover, samples of Asian honey bee adult workers were collected from three colonies kept in

the wild and located in Chiang Rai province, Thailand. Samples were randomly collected into an ice

box and immediately sent to the Bee laboratory, Chiang Mai University, Chiang Mai, Thailand. T.

mercedesae, which has only been found parasitizing in A. mellifera (Anderson & Morgan, 2007), were

collected from a controlled A. mellifera beehive in Chiang Mai University area.

Figure 1

RT-PCR detection of ABPV in A. cerana and T. mercedesae. The 100 bp size ladder,

742 bp ABPV specific PCR fragments detected in A. cerana and T. mercedesae and

negative control (NC) are marked.

This study showed that ABPV was detected in both A. cerana and T. mercedesae (Figure 1).

Sequence analyses indicated that RT-PCR bands corresponding ABPV were virus specific. Detection of

ABPV in A. cerana were 6 (15%), 14 (35%) and 4 (10%) from 40 samples collected from each

colonies, respectively. Of 21 mite samples tested, ABPV was detected in 4 samples.

Furthermore, multiplex RT-PCR assay was performed to check for the presence of seven viruses

including ABPV, BQCV, CBPV, DWV, IAPV, KBV and SBV. The reaction mixture consisted of 1X

AMV/Tfl reaction buffer, 4 mM MgSO4, 0.6 mM dNTP, 0.4 units AMV reverse transcriptase, 0.4 units

Tfl DNA polymerase, 2 ug total RNA, 0.6 uM of each specific primer in a total volume of 50 μl. The
cycling conditions consisted of one cycle at 48°C for 45 min for reverse transcription followed by one

cycle at 94°C for 2 minutes, 40 cycles of 94°C for 30 sec, 55°C for 1 min, and 68°C for 2 minutes

followed by a final elongation step at 68°C for 7 minutes. To ensure the absence of contamination, a

negative control was included in each reaction run. The PCR products were electrophoresed in a 1%

UltraPure™ Low Melting Point Agarose gel (Life Technologies, USA), stained with ethidium bromide

and photographed under UV light. Fragment sizes were determined with reference to a 100-bp ladder

(Invitrogen, USA).

Figure 2

RT-PCR detection of DWV in A. cerana and T. mercedesae. The 100 bp size ladder,

302 bp DWV specific PCR fragments detected in A. cerana, T. mercedesae, and

negative control (NC) are marked.

The researchers' RT-PCR detection and sequence analysis confirmed the previous finding that

DWV could be detected in both A. cerana and T. mercedesae (Figure 2). Among 71 adult A. cerana

bees randomly selected from three colonies and 14 T. mercedesae mite samples tested, DWV was

detected in 26 adults and 3 mites, representing about 36.62% and 21.42%, respectively.

Furthermore, to understand the phylogenetic relationship of ABPV isolates from A. cerana, A.

mellifera, and T. mercedesae, the researchers compared the partial capsid protein coding region (ORF2)
of Acute bee paralysis virus (ABPV) from 18 Thai A. cerana (Accession number KY451673-

KY451690) and four Thai T. mercedesae (Accession number KY451691-KY451694), as well as the

previously reported A. mellifera genotypes from GenBank.

The DWV phylogenetic tree was inferred by using DWV data from 12 Thai A. cerana (Accession

number KY451695-KY451706), three Thai Tropilaelaps mites (Accession number KY451707-

KY451709) and seven A. mellifera retrieved from GenBank. The comparison of the partial polyprotein

coding region of DWV revealed that A. cerana and A. mellifera shared 96- 99% sequence homology

while A. cerana and T. mercedesae shared 92-93% sequence homology. All sequences are formed into

three groups. Similar to the ABPV tree, the DWV tree exhibited short genetic distance due to a high

level of genetic similarity. However, Tropilaelaps mite-infected DWV sequences were significantly

separated from the other groups. DWV from A. mellifera were grouped in the middle of the tree while

all A. cerana infected DWV sequences formed another distinct clade. There was no significance in

geographic separation between DWV isolated from A. mellifera in different geographical origins.

This study provides the first evidence of ABPV occurrence in Asian honey bees A. cerana and the

parasitic mite T. mercedesae that also infect A. mellifera. ABPV was first described in honey bees in

1963 without causing apparent disease symptom in the hosts (Bailey et al., 1963). Since then, ABPV

has become widely spread throughout the world in such places as the United Kingdom (Baker &

Schroeder, 2008), United States(Evans, 2001), Austria (Berenyi et al., 2006), Hungary (Forgách et al.,

2008), Argentina (Reynaldi et al., 2010), Brazil (Teixeira et al., 2008), France (Tentcheva et al., 2004),

China (Ai et al., 2012; Forsgren et al., 2015) and Thailand (Sanpa & Chantawannakul, 2009). ABPV

appears to be the most common honey bee virus in Europe and South America (de Miranda et al.,

2010). ABPV has been reported as a major cause of honey bee mortality in association with Varroa

mite infestation (Antúnez et al., 2006; Bakonyi et al., 2002).

Since the 1980’s, many European honey bees have been introduced to Thailand (especially the

Northern part) for commercial purposes (Sanpa & Chantawannakul, 2009). Following its introduction,
A. mellifera has been exposed to a broad range of parasites and pathogens infecting the native Asian

honey bee A. cerana. In Asia, A. mellifera is commonly infested with two parasitic mites; Varroa spp.

and Tropilaelaps spp. but the populations of Tropilaelaps mites are normally higher than populations of

Varroa (Khongphinitbunjong et al., 2015). Tropilaelaps remain currently confined to Asia and

bordering areas (Anderson & Roberts, 2013). As a result, whatever viruses exist in Varroa could be

possibly transmitted to A. mellifera or Tropilaelaps, or in reverse direction.

Previous reports indicated that ABPV was not found in A. cerana samples from Japan, Korea,

China, and Vietnam (Ai et al., 2012; Choe at al., 2012; Kojima et al., 2011; Li et al., 2012; Thu et al.,

2016; Yañez et al., 2015) and Tropilaelaps mites(Khongphinitbunjong et al., 2015). A previous study in

Thailand, however, detected the presence of ABPV in Varroa mites (Chantawannakul et al., 2006). The

positive detection of ABPV in feral colonies of A. cerana and T. mercedesae mites provides an update

on the ABPV infection in Asia and warrants in-depth future studies to elucidate the virus epidemiology

and transmission pathways in host populations. The results of this study support the pattern of

evolutionary direction that ABPV was primarily evolved in T. mercedesae, and then infected A.

mellifera which is comparatively more susceptible to the effects of parasitism than native Asian honey

bee. Sanpa and Chantawannakul (2009) have observed that ABPV was the second most prevalent virus

infection in A. mellifera in northern Thailand. Because of the sufficient infection rate of ABPV in A.

mellifera, it is possible that an originally European honey bee infecting virus has adapted recently to A.

cerana. The inter-species viral transmission from A. mellifera to A. cerana could possibly be occurring

with help of both mites as vectors or sharing of the range of food and nectar sources or other interaction

such as with robber bees. A previous study also demonstrated the presence of several viruses in both

honey and pollen samples (Chen et al., 2006).

DWV is the most prevalent virus infection in honey bees (Ai et al., 2012; Yañez et al., 2015;

Kojima et al., 2011), also as A. mellifera in Thailand (Sanpa & Chantawannakul, 2009). In contrast to

ABPV, DWV seems to have an opposite evolutionary pathway. DWV was first detected in Japan from
adult bees (Bailey & Ball, 1991). Studies have shown that DWV is known to be associated with

parasitic mite infestation, V. destructor(Chen & Siede, 2007) and T. mercedesae (Forsgren et al., 2009).

Tropilaelaps-infested A. mellifera pupae revealed higher levels of DWV compared to uninfested pupae

(Khongphinitbunjong et al., 2015). The natural host of Varroa is A. cerana, from which the mite moved

onto A. mellifera. V. destructor has only been reported in A. cerana and A. mellifera colonies

(Anderson & Trueman, 2000). The widespread appearance of V. destructor during recent decades has

resulted in the occurrence of DWV around the world. The prevalence of DWV was found to be higher

in frequency in A. mellifera compared to A. cerana (Yañez et al., 2015; Li et al., 2012). It is likely that

DWV has lower rates of infection in A. cerana because of the strong ability of this bee to resist both

viruses and mites. With the high potential of Varroa carrying the virus and other transmission routes,

DWV has been transmitted from A. cerana to A. mellifera and then may have transferred inter-species

to Tropilaelaps via A. mellifera.

Additionally, the immune response of A. mellifera towards Tropilaelaps parasitism and to the

different honey bee viruses this mite may vector remains unknown. Moreover, there is little knowledge

on the host specificity of honey bee viruses and on their species of origin. Viruses can only be detected

with methods that postdate the human mediated sympatry of honey bees and the potential exchange of

viruses. To improve understanding of population dynamics of honey bee viruses, the researchers

focused on the transmission of viruses between neighboring colonies at the apiary and regional scale,

where robbing, drifting (Moritz & Neumann, 1996), and usage of the same foraging range constitute

potential pathways for interspecific pathogen transmission.

Viruses can adapt to new environments and exploit multiple host species to expand their ecological

niche. When viruses jump over the species barrier to spread to new hosts, the interspecies transmission

may result in more virulent strains of viruses or emergence of new diseases. Through several attempts

to detect ABPV in A. cerana in Asia, it was eventually found in Thailand. The high level of genetic

homology of the viruses among A. cerana, A. mellifera and T. mercedesae reflects the similarly
ecological environment that these three species host share together. These results suggest further

evidence that ABPV is a multiple host virus and can expand its host range to A. cerana and T.

mercedesae. Further studies on genetic variability of honey bee viruses and whether factors involved in

host specificity will shed more light on the co-evolution between pathogens and hosts and on the

pathogenesis of honey bee virus infections.