Metal-binding proteins and proteases in RNA viruses: unravelling functional diversity and expanding therapeutic horizons.

IMPORTANCE: Metal-binding proteins are pivotal components with diverse functions in organisms, including viruses. Despite their significance, many metalloproteins in viruses remain uncharacterized, posing challenges to understanding viral systems. This study addresses this knowledge gap by identifying and analyzing metal-binding proteins and proteases in RNA viruses. The findings emphasize the prevalence of these proteins as essential functional classes within viruses and shed light on the role of metal ions and metalloproteins in viral replication and pathogenesis. Moreover, this research serves as a crucial foundation for further investigations in this field, offering the potential for developing innovative antiviral strategies. Additionally, the study enhances our understanding of the distribution and evolutionary patterns of metal-binding proteases in major human viruses. Continually exploring metal-binding proteomes across diverse viruses will deepen our knowledge of metal-dependent biological processes and provide valuable insights for combating viral infections, including respiratory viruses and other life-threatening diseases.

Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway.

IMPORTANCE: Type I interferon (IFN-I), produced by the innate immune system, plays an essential role in host antiviral responses. Proper regulation of IFN-I production is required for the host to balance immune responses and prevent superfluous inflammation. IFN regulatory factor 3 (IRF3) and subsequent sensors are activated by RNA virus infection to induce IFN-I production. Therefore, proper regulation of IRF3 serves as an important way to control innate immunity and viral replication. Here, we first identified Prohibitin1 (PHB1) as a negative regulator of host IFN-I innate immune responses. Mechanistically, PHB1 inhibited the nucleus import of IRF3 by impairing its binding with importin subunit alpha-1 and importin subunit alpha-5. Our study demonstrates the mechanism by which PHB1 facilitates the replication of multiple RNA viruses and provides insights into the negative regulation of host immune responses.

Roadblocks and fast tracks: How RNA binding proteins affect the viral RNA journey in the cell.

As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats.

Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome.

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

Simulated vector transmission differentially influences dynamics of two viral variants of deformed wing virus in honey bees (Apis mellifera).

Understanding how vectors alter the interactions between viruses and their hosts is a fundamental question in virology and disease ecology. In honey bees, transmission of deformed wing virus (DWV) by parasitic Varroa mites has been associated with elevated disease and host mortality, and Varroa transmission has been hypothesized to lead to increased viral titres or select for more virulent variants. Here, we mimicked Varroa transmission by serially passaging a mixed population of two DWV variants, A and B, by injection through in vitro reared honey bee pupae and tracking these viral populations through five passages. The DWV-A and DWV-B variant proportions shifted dynamically through passaging, with DWV-B outcompeting DWV-A after one passage, but levels of both variants becoming equivalent by Passage 5. Sequencing analysis revealed a dominant, recombinant DWV-B strain (DWV-A derived 5' IRES region with the rest of the genome DWV-B), with low nucleotide diversity that decreased through passaging. DWV-A populations had higher nucleotide diversity compared to DWV-B, but this also decreased through passaging. Selection signatures were found across functional regions of the DWV-A and DWV-B genomes, including amino acid mutations in the putative capsid protein region. Simulated vector transmission differentially impacted two closely related viral variants which could influence viral interactions with the host, demonstrating surprising plasticity in vector-host-viral dynamics.

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.

Characterization of an infectious clone of pepper ringspot virus and its use as a viral vector.

The genus Tobravirus comprises three species: Tobacco rattle virus, Pea early-browning virus and Pepper ringspot virus. The genomes of tobraviruses consist of two positive-sense single-stranded RNA segments (RNA1 and RNA2). Infectious clones of TRV are extensively used as virus-induced gene-silencing (VIGS) vectors for studies of virus-host interactions and functions of plant genes. Complete infectious clones of pepper ringspot virus (PepRSV), the only tobravirus present in Brazil, however, have not yet been reported. Infectious clones will help to identify unique features of PepRSV RNA2 and provide another option for development of VIGS vectors. We constructed infectious clones based on two PepRSV isolates, CAM (RNA1 and RNA2) and LAV (RNA2). The cDNA constructs for both homologous (RNA1 and RNA2 of the CAM isolate) and heterologous (RNA1/CAM and RNA2/LAV) combinations were infectious in Nicotiana benthamiana plants. VIGS vector constructs with green fluorescent protein or phytoene desaturase genes inserted in RNA2 silenced the target genes. The systemic translocation of the PepRSV RNA1 construct alone (nonmultiple infection) was also confirmed in an N. benthamiana plant. These results are similar to those reported for tobacco rattle virus.

5'-Nor-3-Deaza-1',6'-Isoneplanocin, the Synthesis and Antiviral Study.

The arbocyclic nucleosides aristeromycin and neplanocin have been studied as a source for new antiviral agents. A convenient synthesis of C-5'-truncated 3-deaza-1',6'-isoneplanocin, which combines the features of antiviral candidates 5'-noraristeromycin and 3-deaza-1',6'-isoneplanocin is reported from (-)-cyclopentenone to give the two C-4' epimers of 5'-nor-3-deaza isoneplanocin. Antiviral assays showed activity against the JC virus (EC50 = 1.12 µM for (4'R)-8; EC50 = 59.14 µM for (4'S)-7) and inactivity of both compounds against several DNA and RNA viruses. Both compounds lacked cytotoxicity.

Virus Size Analysis by Gas-Phase Mobility Measurements: Resolution Limits.

Electrospraying (ES) dissolved viral particles, followed by charge reduction and size analysis with a differential mobility analyzer (DMA), offers a flexible size-analysis tool for small particles in solution. The technique relies on pioneering work by Kaufman and colleagues, commercialized by TSI, and often referred to as GEMMA. However, viral studies with TSI's GEMMA have suffered from limited resolving power, possibly because of imperfections in either the instrument (DMA or charge reduction) or the sample solution preparation. Here, we explore the limits of the resolution achievable by GEMMA, taking advantage of (i) cleaner charge reduction methods and (ii) DMAs of higher resolving power. Analysis of the literature provides indications that mobility peak widths (fwhm) of 2% or less may be achieved by combining careful sample preparation with improved instrumentation. Working with purified PP7 bacteriophage particles small enough to be classifiable by existing high-resolution DMAs, we confirm that fairly narrow viral mobility peaks may be obtained (relative full width at half-maximum fwhm <5%). Comparison of spectra of a given apian virus sample obtained with TSI's GEMMA and our improved instrumentation confirms that one critical limitation is the DMA. This is further verified by narrow peaks from murine parvovirus, norovirus, and encephalomyelitis virus samples, obtained in our improved GEMMA with little sample preparation, directly from infected cell cultures. Classification of purified large (60 nm) coliphage PR772 particles leads to broad peaks, due to both viral degradation and limited intrinsic resolution of the DMAs used to cover the range of such large particles. We conclude that improved DMAs suitable for high-resolution analysis of particles larger than 30 nm need to be developed to determine the intrinsic mobility width of viral particles.

Partial Inactivation of the Chromatin Remodelers SMARCA2 and SMARCA4 in Virus-Infected Cells by Caspase-Mediated Cleavage.

The BAF-chromatin remodeling complex, with its mutually exclusive ATPases SMARCA2 and SMARCA4, is essential for the transcriptional activation of numerous genes, including a subset of interferon-stimulated genes (ISGs). Here, we show that C-terminally truncated forms of both SMARCA2 and SMARCA4 accumulate in cells infected with different RNA or DNA viruses. The levels of truncated SMARCA2 or SMARCA4 strongly correlate with the degree of cell damage and death observed after virus infection. The use of a pan-caspase inhibitor and genetically modified cell lines unable to undergo apoptosis revealed that the truncated forms result from the activity of caspases downstream of the activated intrinsic apoptotic pathway. C-terminally cleaved SMARCA2 and SMARCA4 lack potential nuclear localization signals as well as the bromo- and SnAC domain, with the latter two domains believed to be essential for chromatin association and remodeling. Consistent with this belief, C-terminally truncated SMARCA2 was partially relocated to the cytoplasm. However, the remaining nuclear protein was sufficient to induce ISG expression and inhibit the replication of vesicular stomatitis virus and influenza A virus. This suggests that virus-induced apoptosis does not occur at the expense of an intact interferon-mediated antiviral response pathway.IMPORTANCE Efficient induction of interferon-stimulated genes (ISGs) prior to infection is known to effectively convert a cell into an antiviral state, blocking viral replication. Additionally, cells can undergo caspase-mediated apoptosis to control viral infection. Here, we identify SMARCA2 and SMARCA4 to be essential for the efficient induction of ISGs but also to be targeted by cellular caspases downstream of the intrinsic apoptotic pathway. We find that C-terminally cleaved SMARCA2 and SMARCA4 accumulate at late stages of infection, when cell damage already had occurred. Cleavage of the C terminus removes domains important for nuclear localization and chromatin binding of SMARCA2 and SMARCA4. Consequently, the cleaved forms are unable to efficiently accumulate in the cell nucleus. Intriguingly, the remaining nuclear C-terminally truncated SMARCA2 still induced ISG expression, although to lower levels. These data suggest that in virus-infected cells caspase-mediated cell death does not completely inactivate the SMARCA2- and SMARCA4-dependent interferon signaling pathway.

Pollen reverses decreased lifespan, altered nutritional metabolism and suppressed immunity in honey bees (Apis mellifera) treated with antibiotics.

Nutrition is involved in regulating multiple aspects of honey bee biology such as caste, immunity, lifespan, growth and behavioral development. Deformed wing virus (DWV) is a major pathogenic factor which threatens honey bee populations, and its replication is regulated by the nutrition status and immune response of honey bees. The alimentary canal of the honey bee is home to a diverse microbial community that provides essential nutrients and serves to bolster immune responses. However, to what extent gut bacteria affect honey bee nutrition metabolism and immunity with respect to DWV has not been investigated fully. In this study, newly emerged worker bees were subjected to four diets that contained (1) pollen, (2) pollen and antibiotics, (3) neither pollen nor antibiotics or (4) antibiotics alone. The expression level of two nutrition genes target of rapamycin (tor) and insulin like peptide (ilp1), one nutritional marker gene vitellogenin (vg), five major royal jellyprotein genes (mrjp1-5), one antimicrobial peptide regulating gene relish (rel), and DWV virus titer and its replication intermediate, negative RNA strand, were determined by qRT-PCR from the honey bees at 7 days post-antibiotic treatment. Additionally, honey bee head mass and survival rate were measured. We observed that antibiotics decreased the expression of tor and rel, and increased DWV titer and its replication activity. Expression of ilp1, mrjp1-5 and vg, and honey bee head mass were also reduced compared with bees on a pollen diet. Antibiotics also caused a significant drop in survivorship, which could be rescued by addition of pollen to the diet. Of importance, pollen could partially rescue the loss of vg and mrjp2 while also increasing the head mass of antibiotic-treated bees. Our results illuminate the roles of bacteria in honey bee nutrition, metabolism and immunity, which confer the ability to inhibit virus replication, extend honey bee lifespan and improve overall health.

Temperature-driven changes in viral loads in the honey bee Apis mellifera.

Many of the physiological traits in insects are shaped by environmental temperatures, which can influence their interactions with pathogens. Therefore, quantifying the thermal responses of the host-pathogen system is crucial for better understanding and predicting their dynamics due to environmental changes. This is particularly important in honey bees, which are experiencing severe colony losses around the world, notably due to infection with the Deformed wing virus (DWV). To investigate the influence of temperature on the honey bee/DWV relationship we exposed adult bees to low or high temperatures and determined the effects on viral titers and bee survival. Emerging bees naturally infected with DWV were reared in vitro at different temperatures ranging from 15 °C to 37 °C. In addition, some bees reared at 37 °C were exposed daily to acute heat treatments (40 and 43 °C). High temperatures significantly decreased DWV titers close to the initial viral load at emergence but increased bee mortality. The lowest temperature resulted in higher mortality, but virus load was not significantly impacted. In conclusion, our results indicate that temperature could contribute to seasonal variations in viral loads but do not suggest temperature to be used as a tool to eliminate viruses, even given that high temperatures limit viral multiplication.

Mechanisms and consequences of positive-strand RNA virus recombination.

Genetic recombination in positive-strand RNA viruses is a significant evolutionary mechanism that drives the creation of viral diversity by the formation of novel chimaeric genomes. The process and its consequences, for example the generation of viruses with novel phenotypes, has historically been studied by analysis of the end products. More recently, with an appreciation that there are both replicative and non-replicative mechanisms at work, and with new approaches and techniques to analyse intermediate products, the viral and cellular factors that influence the process are becoming understood. The major influence on replicative recombination is the fidelity of viral polymerase, although RNA structures and sequences may also have an impact. In replicative recombination the viral polymerase is necessary and sufficient, although roles for other viral or cellular proteins may exist. In contrast, non-replicative recombination appears to be mediated solely by cellular components. Despite these insights, the relative importance of replicative and non-replicative mechanisms is not clear. Using single-stranded positive-sense RNA viruses as exemplars, we review the current state of understanding of the processes and consequences of recombination.

Detection of novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico using metagenomics and characterization of their in vitro host ranges.

A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes, Anopheles, Culex, Mansonia and Psorophora) were sorted into 37 pools, homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses, designated Uxmal virus and Mayapan virus, were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae), with one and two isolates being recovered, respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian, human, monkey, hamster, murine, lepidopteran and mosquito (Aedes, Anopheles and Culex) origin, revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion, we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico, and add to the growing plethora of viruses discovered recently through the use of metagenomics.

Ubiquitin-conjugating enzyme UBE2J1 negatively modulates interferon pathway and promotes RNA virus infection.

BACKGROUND: Viral infection activates innate immune pathways and interferons (IFNs) play a pivotal role in the outcome of a viral infection. Ubiquitin modifications of host and viral proteins significantly influence the progress of virus infection. Ubiquitin-conjugating enzyme E2s (UBE2) have the capacity to determine ubiquitin chain topology and emerge as key mediators of chain assembly. METHODS: In this study, we screened the functions of 34 E2 genes using an RNAi library during Dengue virus (DENV) infection. RNAi and gene overexpression approaches were used to study the gene function in viral infection and interferon signaling. RESULTS: We found that silencing UBE2J1 significantly impaired DENV infection, while overexpression of UBE2J1 enhanced DENV infection. Further studies suggested that type I IFN expression was significantly increased in UBE2J1 silenced cells and decreased in UBE2J1 overexpressed cells. Reporter assay suggested that overexpression of UBE2J1 dramatically suppressed RIG-I directed IFNß promoter activation. Finally, we have confirmed that UBE2J1 can facilitate the ubiquitination and degradation of transcription factor IFN regulatory factor 3 (IRF3). CONCLUSION: These results suggest that UBE2 family member UBE2J1 can negatively regulate type I IFN expression, thereby promote RNA virus infection.

Broad-spectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response.

Our previous screening of 50 240 structurally diverse compounds led to the identification of 39 influenza A virus infection inhibitors (Kao R.Y., Yang D., Lau L.S., Tsui W.H., Hu L. et al. Nat Biotechnol 2010;28:600-605). Further screening of these compounds against common respiratory viruses led to the discovery of compound FA-613. This inhibitor exhibited low micromolar antiviral activity against various influenza A and B virus strains, including the highly pathogenic influenza A strains H5N1 and H7N9, enterovirus A71, respiratory syncytial virus, human rhinovirus A, SARS- and MERS-coronavirus. No significant cellular toxicity was observed at the effective concentrations. Animal studies showed an improved survival rate in BALB/c mice that received intranasal FA-613 treatments against a lethal dose infection of A/HK/415742Md/2009 (H1N1). Further cell-based assays indicated that FA-613 interfer with the de novo pyrimidine biosynthesis pathway by targeting the dihydroorotate dehydrogenase. Surprisingly, FA-613 lost its antiviral potency in the interferon-deficient Vero cell line, while maintaining its inhibitory activity in an interferon-competent cell line which showed elevated expression of host antiviral genes when infected in the presence of FA-613. Further investigation of the specific connection between pyrimidine synthesis inhibition and the induction of host innate immunity might aid clinical development of this type of drug in antiviral therapies. Therefore, in acute cases of respiratory tract infections, when rapid diagnostics of the causative agent are not readily available, an antiviral drug with properties like FA-613 could prove to be very valuable.

Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection.

BACKGROUND: Tomato zonate spot virus (TZSV), a dominant species of thrips-transmitted orthotospoviruses in Yunnan and Guangxi provinces in China, causes significant loss of yield in lots of crops and is a major threat to incomes of rural families. However, the detailed molecular mechanism of crop disease caused by TZSV remains obscure. METHODS: Next-generation sequencing (NGS)-based transcriptome analysis (RNA-seq) was performed to investigate and compare the gene expression changes in systemic leaves of tobacco upon infection with TZSV and mock-inoculated plants as a control. RESULTS: De novo assembly and analysis of tobacco transcriptome data by RNA-Seq identified 135,395 unigenes. 2102 differentially expressed genes (DEGs) were obtained in tobacco with TZSV infection, among which 1518 DEGs were induced and 584 were repressed. Gene Ontology enrichment analysis revealed that these DEGs were associated with multiple biological functions, including metabolic process, oxidation-reduction process, photosynthesis process, protein kinase activity. The KEGG pathway analysis of these DEGs indicated that pathogenesis caused by TZSV may affect multiple processes including primary and secondary metabolism, photosynthesis and plant-pathogen interactions. CONCLUSION: Our global survey of transcriptional changes in TZSV infected tobacco provides crucial information into the precise molecular mechanisms underlying pathogenesis and symptom development. This is the first report on the relationships in the TZSV-plant interaction using transcriptome analysis. Findings of present study will significantly help enhance our understanding of the complicated mechanisms of plant responses to orthotospoviral infection.

Functional identification of two minor capsid proteins from Chinese wheat mosaic virus using its infectious full-length cDNA clones.

Full-length cDNA clones of Chinese wheat mosaic virus (CWMV) RNA1 and RNA2 were produced from single reverse transcription PCR reactions and transcripts were shown to be infectious in both wheat and Nicotiana benthamiana. An efficient and reliable agro-infiltration method was then developed for reverse genetic assays in N. benthamiana. Inoculation of infectious cDNA clones resulted in obvious chlorotic symptoms, and CWMV viral genomic RNAs, capsid protein (CP)-related proteins, and typical rod-shaped particles were detectable on the inoculated and upper leaves, similar to those of WT virus. The optimal temperature for virus multiplication was 12 °C, but the optimum for systematic infection in plants was 17 °C. Mutant clones that abolished the N- or C-terminal extensions of the major CP did not inhibit systemic infection or the formation of rod-shaped particles but sometimes modified the symptoms in inoculated plants. These results suggest that the two minor CP-related proteins of CWMV are dispensable for viral infection, replication, systemic movement and virion assembly in plants.

Quantitative modeling of virus evolutionary dynamics and adaptation in serial passages using empirically inferred fitness landscapes.

We describe a stochastic virus evolution model representing genomic diversification and within-host selection during experimental serial passages under cell culture or live-host conditions. The model incorporates realistic descriptions of the virus genotypes in nucleotide and amino acid sequence spaces, as well as their diversification from error-prone replications. It quantitatively considers factors such as target cell number, bottleneck size, passage period, infection and cell death rates, and the replication rate of different genotypes, allowing for systematic examinations of how their changes affect the evolutionary dynamics of viruses during passages. The relative probability for a viral population to achieve adaptation under a new host environment, quantified by the rate with which a target sequence frequency rises above 50%, was found to be most sensitive to factors related to sequence structure (distance from the wild type to the target) and selection strength (host cell number and bottleneck size). For parameter values representative of RNA viruses, the likelihood of observing adaptations during passages became negligible as the required number of mutations rose above two amino acid sites. We modeled the specific adaptation process of influenza A H5N1 viruses in mammalian hosts by simulating the evolutionary dynamics of H5 strains under the fitness landscape inferred from multiple sequence alignments of H3 proteins. In light of comparisons with experimental findings, we observed that the evolutionary dynamics of adaptation is strongly affected not only by the tendency toward increasing fitness values but also by the accessibility of pathways between genotypes constrained by the genetic code.

Replication of Chinese sacbrood virus in primary cell cultures of Asian honeybee (Apis cerana).

A primary cell culture system was established for the first time from embryonic tissues of Asian honeybee, Apis cerana, and used to trace the early infection process of Chinese sacbrood virus (CSBV), an iflavirus in the family Iflaviridae. A monolayer of epithelium-like cells of A. cerana, approximately 8-10 µm in diameter, was grown in Kimura's insect medium at 28 °C within 3-4 days of setting up the cultures. Such cultured cells were inoculated with CSBV purified from infected larvae or pupae for 2 h. In electron and confocal micrographs, viral particles accumulated as filamentous or vesicular inclusions in the cytoplasm of infected cultured cells at 36 h post-inoculation (hpi). Real-time quantitative RT-PCR assay showed that the expression levels of four cistrons of CSBV in the cultured cells increased rapidly between 12 and 48 hpi. This newly established primary cell culture derived from A. cerana will be useful for further studies of infection caused by CSBV.