Varroa destructor shapes the unique viral landscapes of the honey bee populations of the Azores archipelago.

The worldwide dispersal of the ectoparasitic mite Varroa destructor from its Asian origins has fundamentally transformed the relationship of the honey bee (Apis mellifera) with several of its viruses, via changes in transmission and/or host immunosuppression. The extent to which honey bee-virus relationships change after Varroa invasion is poorly understood for most viruses, in part because there are few places in the world with several geographically close but completely isolated honey bee populations that either have, or have not, been exposed long-term to Varroa, allowing for separate ecological, epidemiological, and adaptive relationships to develop between honey bees and their viruses, in relation to the mite's presence or absence. The Azores is one such place, as it contains islands with and without the mite. Here, we combined qPCR with meta-amplicon deep sequencing to uncover the relationship between Varroa presence, and the prevalence, load, diversity, and phylogeographic structure of eight honey bee viruses screened across the archipelago. Four viruses were not detected on any island (ABPV-Acute bee paralysis virus, KBV-Kashmir bee virus, IAPV-Israeli acute bee paralysis virus, BeeMLV-Bee macula-like virus); one (SBV-Sacbrood virus) was detected only on mite-infested islands; one (CBPV-Chronic bee paralysis virus) occurred on some islands, and two (BQCV-Black queen cell virus, LSV-Lake Sinai virus,) were present on every single island. This multi-virus screening builds upon a parallel survey of Deformed wing virus (DWV) strains that uncovered a remarkably heterogeneous viral landscape featuring Varroa-infested islands dominated by DWV-A and -B, Varroa-free islands naïve to DWV, and a refuge of the rare DWV-C dominating the easternmost Varroa-free islands. While all four detected viruses investigated here were affected by Varroa for one or two parameters (usually prevalence and/or the Richness component of ASV diversity), the strongest effect was observed for the multi-strain LSV. Varroa unambiguously led to elevated prevalence, load, and diversity (Richness and Shannon Index) of LSV, with these results largely shaped by LSV-2, a major LSV strain. Unprecedented insights into the mite-virus relationship were further gained from implementing a phylogeographic approach. In addition to enabling the identification of a novel LSV strain that dominated the unique viral landscape of the easternmost islands, this approach, in combination with the recovered diversity patterns, strongly suggests that Varroa is driving the evolutionary change of LSV in the Azores. This study greatly advances the current understanding of the effect of Varroa on the epidemiology and adaptive evolution of these less-studied viruses, whose relationship with Varroa has thus far been poorly defined.

Highly divergent and diverse viral community infecting sylvatic mosquitoes from Northeast Brazil.

Mosquitoes can transmit several pathogenic viruses to humans, but their natural viral community is also composed of a myriad of other viruses such as insect-specific viruses (ISVs) and those that infect symbiotic microorganisms. Besides a growing number of studies investigating the mosquito virome, the majority are focused on few urban species, and relatively little is known about the virome of sylvatic mosquitoes, particularly in high biodiverse biomes such as the Brazilian biomes. Here, we characterized the RNA virome of 10 sylvatic mosquito species from Atlantic forest remains at a sylvatic-urban interface in Northeast Brazil employing a metatranscriptomic approach. A total of 16 viral families were detected. The phylogenetic reconstructions of 14 viral families revealed that the majority of the sequences are putative ISVs. The phylogenetic positioning and, in most cases, the association with a high RNA-dependent RNA polymerase amino acid divergence from other known viruses suggests that the viruses characterized here represent at least 34 new viral species. Therefore, the sylvatic mosquito viral community is predominantly composed of highly divergent viruses highlighting the limited knowledge we still have about the natural virome of mosquitoes in general. Moreover, we found that none of the viruses recovered were shared between the species investigated, and only one showed high identity to a virus detected in a mosquito sampled in Peru, South America. These findings add further in-depth understanding about the interactions and coevolution between mosquitoes and viruses in natural environments. IMPORTANCE: Mosquitoes are medically important insects as they transmit pathogenic viruses to humans and animals during blood feeding. However, their natural microbiota is also composed of a diverse set of viruses that cause no harm to the insect and other hosts, such as insect-specific viruses. In this study, we characterized the RNA virome of sylvatic mosquitoes from Northeast Brazil using unbiased metatranscriptomic sequencing and in-depth bioinformatic approaches. Our analysis revealed that these mosquitoes species harbor a diverse set of highly divergent viruses, and the majority comprises new viral species. Our findings revealed many new virus lineages characterized for the first time broadening our understanding about the natural interaction between mosquitoes and viruses. Finally, it also provided several complete genomes that warrant further assessment for mosquito and vertebrate host pathogenicity and their potential interference with pathogenic arboviruses.

Leveraging transcriptome sequence read archives for virus detection in wild and colony populations of triatomines (Hemiptera: Reduviidae: Triatominae).

Triatomines are infamous as vectors of the parasite Trypanosoma cruzi, the causative agent of Chagas disease. However, climate-driven range expansion and urbanization adaptation of triatomine populations, coupled with their highly diverse feeding strategies (vertebrate haematophagy, kleptohaematophagy, and coprophagy), and has elevated interest in triatomines as potential arboviral vectors. Information on the triatomine virome is scant, with prior records including only eight insect-specific viruses: Triatoma virus (TrV) and Rhodnius prolixus viruses 1-7. Here, we leverage publicly available transcriptome datasets to assess viral diversity in 122 wild and colony kissing bugs representing eight species from six countries. In total, six viruses were detected (including Rhodnius prolixus viruses 4-6), and TrV was detected in almost half of all screened triatomines. This is the first report of TrV in Triatoma brasiliensis and in members of the genus Mepraia (M. gajardoi, M. spinolai, and M. parapatrica), and this effort has vastly expanded the publicly available genomic resources of TrV, adding 39 genome sequences to the single genome sequence currently available in the GenBank database. Furthermore, two additional viruses-Meccus longipennis virus 1 and Drosophila melanogaster Nora virus-are herein reported for the first time from kissing bugs. Meccus longipennis virus 1 was detected in Triatoma infestans from Argentina, Brazil, Chile, and Peru, and Drosophila melanogaster Nora virus was found in T. infestans from Argentina. Our results illustrate the advantage and utility of low-cost transcriptome data mining for the discovery of known and novel arboviruses in triatomines and other potential insect vectors.

Prevalence and molecular analysis of some important viruses in honey bee colonies in Türkiye: the status of multiple infections.

In this study, seven bee viruses of significant importance for bee health in Türkiye were investigated using one-step RT-PCR. For this purpose, larvae from 1183 hives and adult bees from 1196 hives were sampled from 400 apiaries in 40 provinces. The prevalence of viral infections in hives was as follows: acute bee paralysis virus (ABPV), 6.4%; black queen cell virus (BQCV), 77%; chronic bee paralysis virus (CBPV), 3.2%; deformed wing virus (DWV), 63.8%; Israel acute bee paralysis virus (IAPV), 7%; Kashmir bee virus (KBV), 2.7%; sacbrood virus (SBV), 49.7%. Moreover, 50 different combinations of viral infections were identified in the hives. While dual infections (36.1%) were the most common in hives, triple infections with BQCV, DWV, and SBV were found to have the highest prevalence (22.1%). At least one viral infection was detected in all of the apiaries tested. Phylogenetic analysis showed that the isolates from this study generally exhibited the highest similarity to previously reported Turkish isolates. When similarity ratios and the locations and types of amino acid mutations were analyzed, it was observed that the isolates from our study exhibited high similarity to isolates from various countries, including China, the United Kingdom, Syria, and Germany.

First metagenomic analysis of virome in Uzbekistan honey bee (Apis mellifera): Investigating basic information on honey bee viruses.

Honey bees are economically important insects. However, they face multiple biotic and abiotic stresses, such as diseases, pesticides, climate change, and pests, which cause the loss of honey bee colonies worldwide. Among these factors, viruses have been identified as the major cause of colony loss. Research on honey bee viruses in Uzbekistan is limited. This study investigated the viruses affecting honey bees in Uzbekistan. Virome analysis was conducted for each sample using high-throughput sequencing and bioinformatics. Nine honey bee viruses have been identified: the acute bee paralysis virus, aphid lethal paralysis virus, Apis rhabdovirus 1 and 2, black queen cell virus, deformed wing virus, Lake Sinai virus 10, sacbrood virus, and Hubei partiti-like virus 34. Additionally, 15 plant viruses were identified, 7 of which were novel. This study is the first virome analysis of Uzbekistan honey bees and provides a foundation for understanding the viruses affecting honey bees and plants in Uzbekistan.

Identification and Genome Characterization of a Novel Nege-like Virus Isolated from Aphids (Aphis gossypii) in Yunnan Province.

Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named Aphis gossypii nege-like virus (AGNLV, GenBank: OR880429.1), was isolated from aphids (Aphis gossypii) in Lijiang City, Yunnan, China. AGNLV has a genome sequence of 9258 nt (excluding the polyA tail) encoding three open reading frames (ORFs). ORF1 (7149 nt) encodes a viral methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase. ORF2 (1422 nt) encodes a DiSB-ORF2_chro domain and ORF3 encodes an SP24 domain. The genome sequence of AGNLV shares the highest nucleotide identity of 60.0% and 59.5% with Wuhan house centipede virus 1 (WHCV1) and Astegopteryx formosana nege-like virus (AFNLV), respectively. Phylogenetic analysis based on the RNA-dependent RNA polymerase shows that AGNLV is clustered with other negeviruses and nege-like viruses discovered in aphids, forming a distinct "unclassified clade". Interestingly, AGNLV only encodes three ORFs, whereas AFNLV and WHCV1 have four ORFs. Structure and transmembrane domain predictions show the presence of eight alpha helices and five transmembrane helices in the AGNLV ORF3. Translational enhancement of the AGNLV 5' UTR was similar to that of the 5' UTR of plant viruses. Our findings provide evidence of the diversity and structure of nege-like viruses and are the first record of such a virus from a member of the genus Aphis.

The first complete genome sequence of an iflavirus from the endoparasitoid wasp Tetrastichus brontispae.

The complete genome sequence of a novel iflavirus isolated from the gregarious and koinobiont endoparasitoid Tetrastichus brontispae, tentatively named "Tetrastichus brontispae RNA virus 3" (TbRV-3), was determined by total RNA and Sanger sequencing. The complete genome is 9998 nucleotides in length, 8934 nt of which encodes a putative polyprotein of 2978 amino acids. TbRV-3 was found to have a similar genome organization and to contain conserved domains and motifs found in other iflaviruses, with some variations. Phylogenetic analysis based on deduced amino acid sequences of the RdRp domain showed that TbRV-3 clustered with Dinocampus coccinellae paralysis virus (DcPV). However, the percent amino acid sequence identity of the putative capsid proteins of TbRV-3 and DcPV determined using BLASTp was below the species demarcation threshold (90%), suggesting that TbRV-3 is a new iflavirus. This is the first virus of the family Iflaviridae to be isolated from a wasp of the family Eulophidae.

Effect of Wolbachia wAlbB on a positive-sense RNA negev-like virus: a novel virus persistently infecting Aedes albopictus mosquitoes and cells.

The Aedes aegypti mosquito is the primary vector of several medically important arboviruses. The endosymbiotic bacterium, Wolbachia pipientis, has emerged as a means of blocking transmission of arboviruses such as dengue and Zika viruses. One Wolbachia strain that has shown potential in field trials is wAlbB, a naturally occurring Wolbachia strain of the Asian tiger mosquito Aedes albopictus. When transinfected into Ae. aegypti, wAlbB exhibits strong virus inhibition. In addition to modulating arboviruses, Wolbachia also modulates some insect-specific viruses. Here, we explored the effect of Wolbachia on the virome of the Ae. albopictus cell line Aa23 naturally infected with wAlbB and also a stably transinfected recipient Ae. aegypti cell line (Aag2.wAlbB). RNA sequencing and bioinformatic analysis on both cell lines revealed an 11 kb genome of a single-stranded positive-sense RNA negev-like virus related to the recently proposed negevirus taxon. We denoted this novel virus as Aedes albopictus negev-like virus (AalNLV). Tetracycline clearance of Wolbachia from Aa23 cells did not significantly affect AalNLV levels, while in Aag2.wAlbB cells, a significant increase in virus genome RNA copies was observed. We further investigated the inhibitory effect of wAlbB on AalNLV and another positive-sense RNA virus, cell fusing agent virus, which is present in Aag2 cells and known to be suppressed by Wolbachia. wAlbB suppressed both viruses, with the effect on AalNLV being more striking. The findings from this study further supplement our understanding of the complex interaction between Wolbachia, host and virome.

Novel monoclonal antibodies against Australian strains of negeviruses and insights into virus structure, replication and host -restriction.

We report the isolation of Australian strains of Bustos virus and Ngewotan virus, two insect-specific viruses in the newly identified taxon Negevirus, originally isolated from Southeast Asian mosquitoes. Consistent with the expected insect-specific tropism of negeviruses, these isolates of Ngewotan and Bustos viruses, alongside the Australian negevirus Castlerea virus, replicated exclusively in mosquito cells but not in vertebrate cells, even when their temperature was reduced to 34 °C. Our data confirmed the existence of two structural proteins, putatively one membrane protein forming the majority of the virus particle, and one glycoprotein forming a projection on the apex of the virions. We generated and characterized 71 monoclonal antibodies to both structural proteins of the two viruses, most of which were neutralizing. Overall, these data increase our knowledge of negevirus mechanisms of infection and replication in vitro.

A novel circular Rep-encoding single-stranded DNA virus detected in Agrotis ipsilon (Lepidoptera: Noctuidae) in China.

Long-distance migratory insects carry microorganisms that can potentially play a crucial role in the life cycles of their hosts. Here, we used Illumina and Sanger sequencing to determine the complete genome sequence of a novel circular Rep-encoding single-stranded (ss) DNA virus from an important migratory pest, Agrotis ipsilon (Hufnagel). The full genome of this new virus is about 2, 242 nt in length and shares 55-75% genome-wide pairwise sequence identity with members of the family Genomoviridae but 91% nucleotide sequence identity with finch-associated genomovirus 3 isolate S30P_D, which is tentatively abbreviated "FaGmV-3". Viral infection rates in A. ipsilon from Yantai, Langfang and Xinxiang were 4.5% (n = 88), 11.8% (n = 85) and 0% (n = 35), respectively. Phylogenetic analysis based on the deduced amino acid sequence of Rep indicated that the Agrotis ipsilon-associated virus is closely related to members of the genus Gemykibivirus, and we propose it to be a new member of this genus. Hence, it is tentatively named "Agrotis ipsilon-associated genomovirus" (AiGmV).

A novel negevirus isolated from Aedes vexans mosquitoes in Finland.

Negeviruses are insect-specific enveloped RNA viruses that have been detected in mosquitoes and sandflies from various geographical locations. Here, we describe a new negevirus from Northern Europe, isolated from pool of Aedes vexans mosquitoes collected in Finland, designated as Mekrijärvi negevirus (MEJNV). MEJNV had a typical negevirus genome organization, is 9,740 nucleotides in length, and has a GC content of 47.53%. The MEJNV genome contains three ORFs, each containing the following identified conserved domains: ORF1 (7,068 nt) encodes a viral methyltransferase, an FtsJ-like methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase, ORF2 (1,242 nt) encodes a putative virion glycoprotein, and ORF3 (660 nt) encodes a putative virion membrane protein. A distinctive feature relative to other currently known negeviruses is a 7-nucleotide-long overlap between ORF1 and ORF2. MEJNV shares the highest sequence identity with Ying Kou virus from China, with 67.71% nucleotide and 75.19% and 59.00% amino acid sequence identity in ORF 1 and ORF 2, respectively. ORF3 had the highest amino acid sequence similarity to Daeseongdong virus 1 and negevirus Nona 1, both with 77.61% identity, and to Ying Kou virus, with 71.22% identity. MEJNV is currently the northernmost negevirus described. Our report supports the view that negeviruses are a globally distributed, diverse group of viruses that can be found from mosquitoes in a wide range of terrestrial biomes from tropical to boreal forests.

Chronic bee paralysis virus (CBPV) in South American non-Apis bees.

Chronic bee paralysis virus (CBPV) is a positive single-stranded RNA virus that exhibits a worldwide distribution. Although the effects of this virus on honeybees' health are well known, its presence in other bee species has not been fully studied. In this work, CBPV was detected in several native bees from Argentina, including Bombus pauloensis, Halictillus amplilobus, Peponapis fervens, and members of the genus Xylocopa. Here, we report for the first time the presence of CBPV in native bees from South America.

Complete genome sequence of a novel rhabdo-like virus from the Chinese black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae).

This work identified a novel rhabdo-like virus in a Chinese black cutworm (Agrotis ipsilon), which we tentatively named "Agrotis ipsilon virus" (AIpsV). The complete genome of AIpsV is 15,454 nucleotides in length and contains seven open reading frames, collectively encoding more than 160 amino acids. The AIpsV genome is predicted to encode three structural proteins, nucleoprotein (N), glycoprotein (G), and large polymerase protein (L), and four unknown proteins. Phylogenetic analysis revealed that the AIpsV clusters with Wuhan ant virus and Hubei rhabdo-like virus 1 within the rhabdo-like virus clade. The level of expression of AIpsV genes was found to be higher in the pupal and adult stages than in the egg and larval stages.

Isolation and characterization of a novel cripavirus, the first Dicistroviridae family member infecting the cotton mealybug Phenacoccus solenopsis.

A new virus belonging to the family Dicistroviridae was identified in the hibiscus-infesting cotton mealybug Phenacoccus solenopsis. Using high-throughput sequencing (HTS) on an Illumina HiSeq platform, a single contig of the complete genome sequence was assembled. The authenticity of the sequence obtained by HTS was validated by RT-PCR and Sanger sequencing of the amplicons, which was also employed for the 3' untranslated region (UTR). The 5' UTR was sequenced using a rapid amplification of cDNA ends kit. A large segment encompassing the whole genome was amplified by RT-PCR using viral RNA extracted from mealybugs. A whole-genome nucleotide sequence comparison showed 89% sequence identity to aphid lethal paralysis virus (ALPV), covering a short segment of 44 bp. Pairwise amino acid sequence comparisons of the protein encoded by open reading frame (ORF) 2 with its counterparts in the GenBank database, showed less than 40% identity to several members of the genus Cripavirus, including ALPV. Phylogenetic analysis based on the deduced amino acid sequence of the ORF 2 protein showed that the new virus grouped with members of the genus Cripavirus. The intergenic region (IGR) internal ribosome entry site (IRES) showed the conserved nucleotides of a type I IGR IRES and had two bulge sites, three pseudoknots, and two stem-loops. Virus morphology visualized by transmission electron microscopy demonstrated spherical particles with a diameter of ~30 nm. This virus was the only arthropod virus identified in the sampled mealybugs, and the purified virus was able to infect cotton mealybugs. To the best of our knowledge, this is the first report of a Dicistroviridae family member infecting P. solenopsis, and we have tentatively named this virus Phenacoccus solenopsis virus (PhSoV).

Mosquito-Borne Viruses and Insect-Specific Viruses Revealed in Field-Collected Mosquitoes by a Monitoring Tool Adapted from a Microbial Detection Array.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.IMPORTANCE Viruses associated with mosquitoes have made a large impact on public and veterinary health. In the United States, several viruses, including WNV, DENV, and chikungunya virus (CHIKV), are responsible for human disease. From 2015 to 2018, imported Zika cases were reported in the United States, and in 2016 to 2017, local Zika transmission occurred in the states of Texas and Florida. With globalization and a changing climate, the frequency of outbreaks linked to arboviruses will increase, revealing a need to better detect viruses in vector populations. With the capacity of the LLMDA to detect viruses, bacteria, and fungi, this study highlights its ability to broadly screen field-collected mosquitoes and contribute to the surveillance and management of arboviral diseases.

Complete genome sequence of a novel negevirus isolated from Culex tritaeniorhynchus in China.

A novel negevirus, tentatively named Manglie virus (MaV), was isolated from Culex tritaeniorhynchus from the village of Manglie, Yunnan, China, in August 2011. It was identified by high-throughput sequencing of cell culture supernatants, and the complete genome was sequenced using an Illumina MiSeq sequencer. The complete MaV genome comprised 9,218 nt encoding three hypothetical proteins and had a poly(A) tail. BLASTn analysis showed that the genome had the greatest similarity to Ngewotan virus strain Nepal22, with query coverage of 100% and 79% identity. Genomic and phylogenetic analyses demonstrated that MaV should be considered a novel negevirus.

Discovery of three RNA viruses using ant transcriptomic datasets.

Three novel RNA viruses, named Formica fusca virus 1 (GenBank accession no. MH477287), Lasius neglectus virus 2 (MH477288) and Myrmica scabrinodis virus 2 (MH477289), were discovered in ants collected in Cambridge, UK. The proposed virus names were given based on the hosts in which they were identified. The genome sequences were obtained using de novo transcriptome assembly of high-throughput RNA sequencing reads and confirmed by Sanger sequencing. Phylogenetic analysis showed that Formica fusca virus 1 grouped within the family Nyamiviridae, Lasius neglectus virus 2 grouped within the family Rhabdoviridae and Myrmica scabrinodis virus 2 belongs to the family Dicistroviridae. All three viruses are highly divergent from previously sequenced viruses.

Rapid detection of Bombyx mori bidensovirus by loop-mediated isothermal amplification based lateral flow dipstick assay for field applications.

Bombyx mori bidensovirus (BmBDV), is the only bipartite single-stranded DNA (ssDNA) insect virus reported to date. BmBDV causes fatal flacherie disease in silkworm, resulting in large economic losses to sericulture. We developed a loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD) method that can successfully and rapidly detect BmBDV DNA with a low limit of detection (5 fg, 400 copies of the BmBDV genome). The method was evaluated and improved for direct field diagnosis using silkworm faeces. In the field, the actual limit of detection was ∼50 fg (4000 copies of the BmBDV genome). The results demonstrated that, compared with traditional methods for BmBDV detection, our new LAMP-LFD method was much more rapid, sensitive and cost-effective, with less dependence on equipment, making it easier to use. The method has potential to be translated into a new diagnostic product for field applications in the sericulture industry.

Discovery and characterization of a novel picorna-like RNA virus in the cotton bollworm Helicoverpa armigera.

We characterize a novel picorna-like virus, named Helicoverpa armigera Nora virus (HaNV), with a genome length of 11,200 nts, the sequence of which was isolated from the lepidopteran host cotton bollworm Helicoverpa armigera, using RNA-Seq. Phylogenetic analysis, using the putative amino acid sequence of the conserved RNA-dependent RNA polymerase (RdRp) domain, indicated that HaNV clustered with Spodoptera exigua Nora virus, Drosophila Nora virus and Nasonia vitripennis virus-3 with a high bootstrap value (100%), which might indicate a new viral family within the order Picornavirales. HaNV was efficiently horizontally transmitted between hosts via contaminated food, and transmission was found to be dose-dependent (up to 100% efficiency with 109 viral copy number/µl). HaNV was also found to be transmitted vertically from parent to offspring, mainly through transovum transmission (virus contamination on the surface of the eggs), but having a lower transmission efficiency (around 43%). Infection distribution within the host was also investigated, with HaNV mainly found in only the gut of both adult moths and larvae (>90%). Moreover, our results showed that HaNV appears not to be an overtly pathogenic virus to its host.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.