The insect order Blattodea (cockroaches and termites) has drawn substantial research attention for their dietary habits and lifestyle of living with or around humans. In the present study, we focused on the discovery of RNA viruses hidden in Blattodea insects using the publicly available RNA sequencing datasets. Overall, 136 distinctive RNA viruses were identified from 36 Blattodea species, of which more than 70 % were most closely related to the invertebrate-associated viral groups within Picornavirales, Sobelivirales, Bunyaviricetes, Jingchuvirales, Durnavirales, Lispiviridae, Orthomyxoviridae, Permutotetraviridae, Flaviviridae and Muvirales. Several viruses were associated with pathogens of vertebrates (Paramyxoviridae), plants (Tymovirales), protozoa (Totiviridae), fungi (Narnaviridae) and bacteria (Norzivirales). Collectively, 93 complete or near-complete viral genomes were retrieved from the datasets, and several viruses appeared to have remarkable temporal and spatial distributions. Interestingly, the newly identified Periplaneta americana dicistrovirus displayed a remarkable distinct bicistronic genome arrangement from the well-recognized dicistroviruses with the translocated structural and non-structural polyprotein encoding open reading frames over the genome. These results significantly enhance our knowledge of RNA virosphere in Blattodea insects, and the novel genome architectures in dicistroviruses and other RNA viruses may break our stereotypes in the understanding of the genomic evolution and the emergence of potential novel viral species.

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 significance of gut microbiota in regulating animal immune response to viral infection is increasingly recognized. However, how chronic bee paralysis virus (CBPV) exploits host immune to disturb microbiota for its proliferation remains elusive. Through histopathological examination, we discovered that the hindgut harbored the highest level of CBPV, and displayed visible signs of damages. The metagenomic analysis showed that a notable reduction in the levels of Snodgrassella alvi and Lactobacillus apis, and a significant increase in the abundance of the opportunistic pathogens such as Enterobacter hormaechei and Enterobacter cloacae following CBPV infection. Subsequent co-inoculation experiments showed that these opportunistic pathogens facilitated the CBPV proliferation, leading to accelerated mortality in bees and exacerbation of bloated abdomen symptoms after CBPV infection. The expression level of antimicrobial peptide (AMP) was found to be significantly up-regulated by over 1000 times in response to CBPV infection, as demonstrated by subsequent transcriptome and quantitative real-time PCR investigations. In particular, through correlation analysis and a bacteriostatic test revealed that the AMPs did not exhibit any inhibitory effect against the two opportunistic pathogens. However, they did demonstrate inhibitory activity against S. alvi and L. apis. Our findings provide different evidence that the virus infection may stimulate and utilize the host\'s AMPs to eradicate probiotic species and facilitate the proliferation of opportunistic bacteria. This process weakens the intestinal barrier and ultimately resulting in the typical bloated abdomen.

UNASSIGNED: Asian citrus psyllid (ACP, Diaphorina citri) is an important transmission vector of \"Candidatus Liberibacter asiaticus\" (CLas), the causal agent of Huanglongbing (HLB), the most destructive citrus disease in the world. As there are currently no HLB-resistant rootstocks or varieties, the control of ACP is an important way to prevent HLB. Some viruses of insect vectors can be used as genetically engineered materials to control insect vectors.
UNASSIGNED: To gain knowledge on viruses in ACP in China, the prevalence of five RNA and DNA viruses was successfully determined by optimizing reverse transcription polymerase chain reaction (RT-PCR) in individual adult ACPs. The five ACP-associated viruses were identified as follows: diaphorina citri bunyavirus 2, which was newly identified by high-throughput sequencing in our lab, diaphorina citri reovirus (DcRV), diaphorina citri picorna-like virus (DcPLV), diaphorina citri bunyavirus (DcBV), and diaphorina citri densovirus-like virus (DcDV).
UNASSIGNED: DcPLV was the most prevalent and widespread ACP-associated virus, followed by DcBV, and it was detected in more than 50% of all samples tested. DcPLV was also demonstrated to propagate vertically and found more in salivary glands among different tissues. Approximately 60% of all adult insect samples were co-infected with more than one insect pathogen, including the five ACP-associated viruses and CLas.
UNASSIGNED: This is the first time these viruses, including the newly identified ACP-associated virus, have been detected in individual adult ACPs from natural populations in China\'s five major citrus-producing provinces. These results provide valuable information about the prevalence of ACP-associated viruses in China, some of which have the potential to be used as biocontrol agents. In addition, analysis of the change in prevalence of pathogens in a single insect vector is the basis for understanding the interactions between CLas, ACP, and insect viruses.

Bombyx mori bidensovirus (BmBDV) is one of the most important pathogens of silkworm. It mainly infects midgut cells of silkworm and causes losses to the sericulture industry. Long noncoding RNAs (lncRNAs) have been reported to play an important role in the regulation of antiviral immune response in silkworm. To explore whether lncRNAs are involved in BmBDV infection and immune response of silkworm, we performed a comparative transcriptome analysis to identify the lncRNAs and mRNAs between the BmBDV infected and noninfected silkworm larvae at the early stage. A total of 16,069 genes and 974 candidate lncRNAs were identified, among which 142 messenger RNA (mRNAs) and four lncRNAs were differentially expressed (DE). Target gene prediction revealed that 142 DEmRNAs were coexpressed with four DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans-regulation activities. A regulatory network of DElncRNAs and DEmRNAs was constructed, showing that many genes targeted by different DElncRNAs are involved in metabolism and immunity, which implies that these genes and lncRNAs play an important role in the replication of BmBDV. Our results will help us to improve our understanding of lncRNA-mediated regulatory roles in BmBDV infection, providing a new perspective for further exploring the interaction between host and BmBDV.

The potential of insect viruses in the biological control of agricultural pests is well-recognized, yet their practical application faces obstacles such as host specificity, variable virulence, and resource scarcity. High-throughput sequencing (HTS) technologies have significantly advanced our capabilities in discovering and identifying new insect viruses, thereby enriching the arsenal for pest management. Concurrently, progress in reverse genetics has facilitated the development of versatile viral expression vectors. These vectors have enhanced the specificity and effectiveness of insect viruses in targeting specific pests, offering a more precise approach to pest control. This review provides a comprehensive examination of the methodologies employed in the identification of insect viruses using HTS. Additionally, it explores the domain of genetically modified insect viruses and their associated challenges in pest management. The adoption of these cutting-edge approaches holds great promise for developing environmentally sustainable and effective pest control solutions. © 2023 Society of Chemical Industry.

Invertebrate species are a natural reservoir of viral genetic diversity, and invertebrate pests are widely distributed in crop fields. However, information on viruses infecting invertebrate pests of crops is limited. In this report, we describe the deep metatranscriptomic sequencing of 88 invertebrate samples covering all major invertebrate pests in rice fields. We identified 296 new RNA viruses and 13 known RNA viruses. These viruses clustered within 31 families, with many highly divergent viruses constituting potentially new families and genera. Of the identified viruses, 13 RNA viruses clustered within the Fiersviridae family of bacteriophages, and 48 RNA viruses clustered within families and genera of mycoviruses. We detected known rice viruses in novel invertebrate hosts at high abundances. Furthermore, some novel RNA viruses have genome structures closely matching to known plant viruses and clustered within genera of several plant virus species. Forty-five potential insect pathogenic RNA viruses were detected in invertebrate species. Our analysis revealed that host taxonomy plays a major role and geographical location plays an important role in structuring viral diversity. Cross-species transmission of RNA viruses was detected between invertebrate hosts. Newly identified viral genomes showed extensive variation for invertebrate viral families or genera. Together, the large-scale metatranscriptomic analysis greatly expands our understanding of RNA viruses in rice invertebrate species, the results provide valuable information for developing efficient strategies to manage insect pests and virus-mediated crop diseases.

BACKGROUND: Cell fusing agent virus (CFAV) was the first insect-specific virus to be characterized, and has been reported to negatively influence the growth of arboviruses such as dengue, Zika, and La Cross, making it a promising biocontrol agent for mosquito-borne disease prevention. Aedes aegypti Aag2 cells were naturally infected with CFAV. However, the ability of this virus to stably colonize an Ae. aegypti population via artificial infection and how it influences the vector competence of this mosquito have yet to be demonstrated.
METHODS: CFAV used in this study was harvested from Aag2 cells and its complete genome sequence was obtained by polymerase chain reaction and rapid amplification of complementary DNA ends, followed by Sanger sequencing. Phylogenetic analysis of newly identified CFAV sequences and other sequences retrieved from GenBank was performed. CFAV stock was inoculated into Ae. aegypti by intrathoracic injection, the survival of parental mosquitoes was monitored and CFAV copies in the whole bodies, ovaries, and carcasses of the injected F0 generation and in the whole bodies of the F1 generation on different days were examined by reverse transcription-quantitative polymerase chain reaction.
RESULTS: The virus harvested from Aag2 cells comprised a mixture of three CFAV strains. All genome sequences of CFAV derived from Aag2 cells clustered into one clade but were far from those isolated or identified from Ae. aegypti. Aag2-derived CFAV efficiently replicated in the mosquito body and did not attenuate the survival of Ae. aegypti. However, the viral load in the ovarian tissues was much lower than that in other tissues and the virus could not passage to the offspring by vertical transmission.
CONCLUSIONS: The results of this study demonstrate that Aag2-derived CFAV was not vertically transmitted in Ae. aegypti and provide valuable information on the colonization of mosquitoes by this virus.

Fungi are highly widespread and commonly colonize multicellular organisms that live in natural environments. Notably, studies on viruses infecting plant-associated fungi have revealed the interesting phenomenon of the cross-kingdom transmission of viruses and viroids from plants to fungi. This implies that fungi, in addition to absorbing water, nutrients, and other molecules from the host, can acquire intracellular parasites that reside in the host. These findings further suggest that fungi can serve as suitable alternative hosts for certain plant viruses and viroids. Given the frequent coinfection of fungi and viruses in humans/animals, the question of whether fungi can also acquire animal viruses and serve as their hosts is very intriguing. In fact, the transmission of viruses from insects to fungi has been observed. Furthermore, the common release of animal viruses into the extracellular space (viral shedding) could potentially facilitate their acquisition by fungi. Investigations of the cross-infection of animal viruses in fungi may provide new insights into the epidemiology of viral diseases in humans and animals.

Insects are the host or vector of diverse viruses including those that infect vertebrates, plants, and fungi. Insect viruses reside inside their insect hosts and are vertically transmitted from parent to offspring. The insect virus-host relationship is intricate, as these viruses can impact various aspects of insect biology, such as development, reproduction, sex ratios, and immunity. Arthropod-borne viruses (arboviruses) that cause substantial global health or agricultural problems can also be vertically transmitted to insect vector progeny. Multiple infections with insect viruses and arboviruses are common in nature. Such coinfections involve complex interactions, including synergism, dependence, and antagonism. Recent studies have shed light on the influence of insect viruses on the competence of insect vectors for arboviruses. In this review, we focus on the biological effects of insect viruses on the transmission of arboviruses by insects. We also discuss the potential mechanisms by which insect viruses affect the ability of hosts to transmit arboviruses, as well as potential strategies for disease control through manipulation of insect viruses. Analyses of the interactions among insect vectors, insect viruses and arboviruses will provide new opportunities for development of innovative strategies to control arbovirus transmission.