Microbiology

Mal de Río Cuarto disease, caused by a Fijivirus, is a major constraint for maize production in Argentina. The traditional evaluation of resistant hybrids is limited by the low efficiency of natural virus transmission and the lack of standardized field inoculation methods. We developed a protocol that combines laboratory mass-rearing of the planthopper vector Delphacodes kuscheli with a controlled field transmission system. The method involves the synchronized production of large insect populations, acquisition of viruliferous vectors under controlled conditions, and their safe transport to the field using specialized containers. Transmission is achieved through individual cages placed on maize seedlings, ensuring high inoculation pressure under field-like conditions. This protocol enables reliable and reproducible virus transmission, facilitating large-scale screening of maize hybrids and other cereals. Its main advantages are the high throughput of vector production, improved transmission efficiency, and adaptability to diverse experimental designs.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for exploring cellular heterogeneity and host–pathogen interactions. This protocol details the Zika virus (ZIKV)-targeted scRNA-seq workflow for preparing high-quality single-cell suspensions from the whole brain tissues of neonatal mice, high-quality single-cell sorting, cDNA reverse transcription, amplification, ZIKV enrichment and host transcriptome library preparation, and sequencing dataset integration in downstream analysis to complete the quantification of ZIKV RNA in individual cells.

During herpesvirus replication, capsids are assembled inside the nucleus and translocated into the cytosol by a non-canonical nucleocytoplasmic export process termed nuclear egress. Traditional methods of measuring nuclear egress rely on imaging-based technologies such as confocal and electron microscopy. These techniques are labor-intensive, limited by the number of cells that can be examined, and may not accurately represent the entire population, generating a potential bias during data interpretation. To overcome these problems, we have developed a flow cytometry–based method to measure HSV-1 nuclear egress that we termed FLARE (FLow cytometry–based Assay of nucleaR Egress). This assay uses a double fluorescent reporter system, utilizing HSV-1-tdTomato to identify infected cells and an Alexa Fluor-488-conjugated, capsid-specific antibody to detect cytosolic capsids, thereby distinguishing infected cells with nuclear egress from those without it. This assay provides more quantitative results than traditional methods, enables large-scale high throughput, and can be adapted for use with other herpesviruses.

Human coronavirus OC43 (HCoV-OC43) is an endemic “common cold” coronavirus widely used to study fundamental aspects of coronavirus biology and to test therapeutic interventions. Recently, we used a yeast-based reverse genetics strategy to create recombinant HCoV-OC43 and fluorescent reporter viruses. We assembled a DNA copy of the HCoV-OC43 genome from six linear dsDNA fragments and a linearized yeast centromeric plasmid/bacterial artificial chromosome (YCpBAC) vector in Saccharomyces cerevisiae using transformation-associated recombination (TAR). Reporter genes encoding mCardinal fluorescent protein or histone H2B fused to mClover3 (mClover-H2B) or mRuby3 (mRuby-H2B) were inserted into an intergenic region between the HCoV-OC43 M and N genes. Assembled full-length HCoV-OC43-encoding plasmids were delivered into permissive mammalian cells to initiate viral gene expression, genome replication, and production of infectious progeny. This technique allows for the precise mutagenesis of any area of the HCoV-OC43 genome using homologous recombination, yielding genetically defined reference plasmids for the future generation of HCoV-OC43 virus stocks.

Rice (Oryza sativa), a staple crop sustaining half of humanity’s caloric intake, is threatened by numerous insect-vector-transmitted diseases, such as rice stripe disease, caused by the rice stripe virus (RSV). Most genetic studies on plant antiviral defense mechanisms rely on natural or artificial infection and transgenic approaches, which require months of plant transformation. Here, we present a streamlined protocol that enables rapid analysis of RSV–host interactions within three days. The method encompasses three key phases: (1) polyethylene glycol (PEG)-based precipitation of RSV virions from infected plant tissues, (2) sequential purification through differential ultracentrifugation with glycerol cushion optimization, and (3) high-efficiency transfection of purified virions into rice protoplasts via PEG-mediated delivery. Viral replication is quantitatively assessed using RT-qPCR targeting viral RNA and immunoblotting with RSV nucleocapsid protein-specific monoclonal antibodies. This approach eliminates dependency on stable transgenic lines, allowing the simultaneous introduction of exogenous plasmids for functional studies. Compared with conventional methods requiring several months for transgenic plant generation, our protocol delivers analyzable results within three days, significantly accelerating the exploration of antiviral mechanisms and resistance gene screening.

The HIV-1 reservoir, consisting of transcriptionally silent integrated HIV-1 proviruses, is a major barrier to a cure, as it persists during effective antiretroviral therapy (ART) and is the source of viral rebound upon treatment interruption. Some of the strategies explored for HIV cure focus on the identification of compounds to either reactivate and eliminate the HIV reservoir (“shock and kill”) or to prevent HIV reservoir reactivation and induce deep proviral latency (“block and lock”). Paramount in developing these HIV-1 cure strategies is determining the effect of the compounds on the size of the inducible HIV-1 reservoir in blood from people living with HIV-1 (PWH). Traditionally, viral outgrowth assays have been the primary method to determine the inducible HIV-1 reservoir in CD4+ T cells from PWH. However, these assays are labor-intensive, time-consuming, and often have low sensitivity. We have recently developed the inducible HIV-1 reservoir reduction assay (HIVRRA), a rapid, cost-effective, and sensitive method to measure the impact of compounds on the inducible replication-competent HIV-1 reservoir in total peripheral blood mononuclear cells (PBMCs) from PWH ex vivo. The HIVRRA simultaneously evaluates the effect of test conditions on the size of the inducible replication-competent HIV-1 reservoir as well as the specificity and toxicity of the test strategy. Using total PBMCs instead of purified CD4+ T cells reduces processing time and resource requirements. This makes the HIVRRA a more practical, scalable tool for evaluating potential HIV-1 cure strategies.

Since the establishment of the iSLK-BAC16 cell culture system, iSLK-BAC16 cells and their derivatives have been widely used for Kaposi’s sarcoma-associated herpesvirus (KSHV) studies. However, iSLK-BAC16 cells can be difficult to work with, in part due to the lack of standardized protocols and conflicting troubleshooting suggestions. Here, we describe the protocol for general iSLK-BAC16 cell culture and reactivation, which induces lytic KSHV replication and virion production. This protocol achieves robust levels of KSHV reactivation in our hands and can be readily used for studies of KSHV lytic infection mechanisms.

Human astroviruses pose a significant public health threat, especially to children, the elderly, and immunocompromised individuals. Nevertheless, these viruses remain largely understudied, with no approved antivirals or vaccines. This protocol focuses on leveraging reverse genetics (RG) and replicon systems to unravel the biology of MLB genotypes, a key group of neurotropic astroviruses. Using reverse genetics and replicon systems, we identified critical genetic deletions linked to viral attenuation and neurotropism, pushing forward vaccine development. We also uncovered novel replication mechanisms involving ER membrane interactions, opening doors to new antiviral targets. Reverse genetics and replicon systems are essential for advancing our understanding of astrovirus biology, identifying virulence factors, and developing effective treatments and vaccines to combat their growing public health impact.

Cricket paralysis virus (CrPV), a member of the family Dicistroviridae, is a single-stranded positive-sense RNA virus that primarily infects arthropods. Some members of the dicistrovirus family, including the honey bee viruses Israeli acute paralysis virus and Acute bee paralysis virus and the shrimp-infecting Taura syndrome virus, pose significant threats to agricultural ecosystems and economies worldwide. Dicistrovirus infection in Drosophila is used as a model system to study fundamental insect–virus–host interactions. The availability of a CrPV infectious clone allows controlled manipulation of the viral genome at a molecular level. Effective viral propagation and titration techniques are crucial for understanding the pathogenesis and epidemiology of dicistrovirus infections. Traditional methods for assessing viral titers, such as plaque assays, are unsuitable for CrPV, since Drosophila tissue culture cells like Schneider 2 cells cannot readily form adherent plaques. Here, we present a streamlined protocol for generating a recombinant virus from a CrPV infectious clone, propagating the virus in S2 cells and titering the virus by an immunofluorescence-based focus-forming assay (FFA). This protocol offers a rapid and reliable approach for generating recombinant viruses, viral amplification, and determining CrPV titers, enabling efficient investigation into viral biology and facilitating the development of antiviral strategies.

The bacterial membrane vesicles (MVs) are non-replicative, nanoscale structures that carry specific cargos and play multiple roles in microbe–host interactions. An appropriate MV isolation method that mimics complex pathogen infections in vivo is needed. After bacterial MVs extraction, flagella or pili can be frequently observed along with MVs by transmission electron microscope (TEM). Recently, MVs from Pseudomonas aeruginosa were found to coexist with Pf4 phages, and this MV–phages complex exhibited a different impact on host cell innate immunity compared with MVs or phages solely. The presence of this MVs–phages complex simulates the real condition of complex pathogen infections within the host. This protocol outlines the extraction of the MVs and Pf4 phages complex of P. aeruginosa PAO1, including the respective isolation and qualification approaches. Our step-by-step bacterial MVs–phages complex extraction protocol provides valuable insights for further studying microbe–host cell interactions and the development of novel phage therapies.