Voltage-dependent anion channels: Key players in viral infection.

Viruses control the host cell by exploiting its molecular machinery to facilitate viral replication and propagation. Understanding different viral mechanisms and biochemical pathways is crucial for finding promising therapeutic solutions to viral infections. The mitochondrion is a vital organelle targeted by various types of viruses. More specifically, viruses interact with the voltage-dependent anion channel (VDAC), a porin protein found in the outer mitochondrial membrane. VDAC controls metabolite flux, regulates reactive oxygen species production, and promotes mitochondrial-mediated apoptosis by releasing pro-apoptotic proteins. Hence, a common pathogenic strategy used by many viruses seems to exploit natural pathways that VDAC regulates. This review aims to address the inhibition and enhancement roles of VDAC in viral pathogenesis and outlines multiple links and interactions between VDAC and viral proteins as potential antiviral targets.

Baculovirus displaying SARS-CoV-2 spike RBD promotes neutralizing antibody production in a mouse model.

BACKGROUND: There is always a need for a safe and efficient vaccine platform, especially when facing a pandemic such as COVID-19. Most of the SARS-CoV-2-based vaccines are based on the full spike protein, which is presented as a trimerized protein, and many viral vector vaccines express the spike protein into the host cells and do not display it on virus surfaces. However, the spike receptor-binding domain (RBD)-based vaccines are efficient and are currently under investigation and clinical trials. METHODOLOGY: In this study, we are testing the efficacy of the RBD displayed on a baculovirus as a mean to formulate a safe and stable carrier to induce the immune system against SARS-CoV-2. Therefore, two pseudotyped baculoviruses were constructed to display the RBD, AcRBD-sfGFP-64, and AcRBD-sfGFP-V, using two different displaying strategies based on gp64 and VSV-G envelope glycoproteins, from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and vesicular stomatitis virus (VSV), respectively. BALB/C mice were immunized with the pseudotyped baculoviruses in a dose-optimized manner. Dot blot and Western blot were used to screen and validate the polyclonal antibodies' specificity to the SARS-CoV-2 RBD. A plaque reduction neutralization test (PRNT) was used to measure the sera neutralization capacity against a SARS-CoV-2 wild-type isolate from Egypt. ELISA was used to quantify certain cytokines for the assessment of the immune response. RESULT: The outcome of our investigation showed that the monomeric RBD proteins were properly displayed on baculovirus and efficiently triggered the mouse immune system. The produced sera efficiently neutralized about 50% of SARS-CoV-2 in more than 100-fold serum dilution. The immunized mice showed a significant increase (p<0.01) in the levels of IL-2 and IFN-γ and a significant decrease (p<0.01) and (p<0.001) in the levels of IL-4 and IL-10, respectively, which suggest that AcRBD-sfGFP-64 and AcRBD-sfGFP-V induce Th1 cellular immune response. CONCLUSION: The produced recombinant viruses can induce the immune response without adjuvant, which needs dose optimization and further stability tests. Neutralizing antibodies were induced without affecting the health of immunized mice. Th1 response can be attainable through the system, which is of great benefit in SARS CoV-2 infection and the system can be tested for future applications including vaccine development and polyclonal antibody production.

Impact of Enniatin B and Beauvericin on Lysosomal Cathepsin B Secretion and Apoptosis Induction.

Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from Fusarium and Beauveria bassiana, respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived cell line KB-3-1 cells were treated with different concentrations of these compounds. The extracellular secretion of cathepsin B increased in a concentration-dependent manner, and the lysosomal staining by lysotracker red was reduced upon the treatment with any of the compounds. However, the extracellular secretion of cathepsin L and cathepsin D were not affected. Inhibition of cathepsin B with specific inhibitor CA074 significantly reduced the cytotoxic effect of both compounds, while inhibition of cathepsin D or cathepsin L did not influence the cytotoxic activities of both compounds. In vitro labelling of lysosomal cysteine cathepsins with Ethyl (2S, 3S)-epoxysuccinate-Leu-Tyr-Acp-Lys (Biotin)-NH2 (DCG04) was not affected in case of cathepsin L upon the treatment with both compounds, while it was significantly reduced in case of cathepsin B. In conclusion, ENN B and BEA increase lysosomal Ph, which inhibits delivery of cathepsin B from Golgi to lysosomes, thereby inducing cathepsin B release in cytosol, which activates caspases and hence the apoptotic pathway.

The regulatory role of long non- coding RNAs as a novel controller of immune response against cancer cells.

Immunotherapy has been established as a promising therapy for different cancer types. However, many patients experience primary or secondary resistance to treatment. Immune cells and anti-inflammatory factors are regulated by long noncoding RNAs (lncRNAs). In addition, lncRNAs have a role in immune resistance through antigen presentation loss or attenuation, PD-L1 upregulation, loss of T-cell activities, and activation of G-MDSCs and Tregs in the tumor environment. LncRNAs can also influence the interaction between cancer stem cells and immune cells in the tumor microenvironment, potentially resulting in cancer stem cell resistance to immunotherapy. Immunological-related lncRNAs can influence immune responses either directly by affecting neighboring protein-coding genes or indirectly by sponging miRNAs through various mechanisms. We have emphasized the role and levels of expression of lncRNAs that have been linked to immune cell formation, differentiation, and activation, which may have an influence on immunotherapy efficacy.

The Transgene Expression of the Immature Form of the HCV Core Protein (C191) and the LncRNA MEG3 Increases Apoptosis in HepG2 Cells.

Long non-coding RNAs (lncRNAs) are regulated in cancer cells, including lncRNA MEG3, which is downregulated in Hepatocellular Carcinoma (HCC). In addition, hepatitis C virus (HCV) core proteins are known to dysregulate important cellular pathways that are linked to HCC development. In this study, we were interested in evaluating the overexpression of lncRNA MEG3, either alone or in combination with two forms of HCV core protein (C173 and C191) in HepG2 cells. Cell viability was assessed by MTT assay. Transcripts' levels of key genes known to be regulated in HCC, such as p53, DNMT1, miRNA152, TGF-b, and BCL-2, were measured by qRT-PCR. Protein expression levels of caspase-3 and MKI67 were determined by immunocytochemistry and apoptosis assays. The co-expression of lncRNA MEG3 and C191 resulted in a marked increase and accumulation of dead cells and a reduction in cell viability. In addition, a marked increase in the expression of tumor suppressor genes (p53 and miRNA152), as well as a marked decrease in the expression of oncogenes (DNMT1, BCL2, and TGF-b), were detected. Moreover, apoptosis assay results revealed a significant increase in total apoptosis (early and late). Finally, immunocytochemistry results detected a significant increase in apoptotic marker caspase-3 and a decrease in tumor marker MKI67. In this study, transgene expression of C191 and lncRNA MEG3 showed induction in apoptosis in HepG2 cells greater than the expression of each one alone. These results suggest potential anticancer characteristics.

Genetically Engineered Hepatitis C Virus-like Particles (HCV-LPs) Tagged with SP94 Peptide to Acquire Selectivity to Liver Cancer Cells via Grp78.

Targeted cancer therapy is a challenging area that includes multiple chemical and biological vehicles. Virus-like particles (VLPs) combine safety and efficacy in their roles as potential vaccines and drug delivery vehicles. In this study, we propose a novel drug delivery system based on HCV-LPs engineered with SP94 and RGD peptides mediated by a specific molecular chaperone (Grp78) associated with cancer drug resistance. The PCR primers were designed for engineering two constructs, SP94-EGFP-CORE-HIS and RGD-EGFP-CORE-HIS, by sequential PCR reactions. The two fragments were cloned into pFastBac Dual under the polyhedrin promoter and then used to produce two recombinant baculoviruses (AcSP94 and AcRGD). The VLP's expression was optimized by recombinant virus infection with different MOIs, ranging from 1 to 20 MOI. Recombinant VLP2 were purified by Ni-NTA and their sizes and shapes were confirmed with TEM. They were incubated with different types of cells prior to examination using the fluorescence microscope to test the binding specificity. The effect of the overexpression of the Grp78 on the binding affinity of the engineered VLPs was tested in HepG2 and HeLa cells. The protocol optimization revealed that MOI 10 produced the highest fluorescence intensities after 72 h for the two recombinant proteins (SP94-core and RGD-core). Moreover, the binding assay tested on different types of mammalian cells (HeLa, HEK-293T, and HepG2 cells) showed green fluorescence on the periphery of all tested cell lines when using the RGD-core protein; while, the SP94-core protein showed green fluorescence only with the liver cancer cells, HepG2 and HuH7. Overexpression of Grp78 in HepG2 and HeLa cells enhanced the binding efficiency of the engineered VLPs. We confirmed that the SP94 peptide can be specifically used to target liver cancer cells, while the RGD peptide is sufficiently functional for most types of cancer cells. The overexpression of the Grp78 improved the binding capacity of both SP94 and RGD peptides. It is worth noting that the SP94 peptide can function properly as a recombinant peptide, and not only as a chemically conjugated peptide, as heretofore commonly used.

COVID-19: Risk Factors Associated with Infectivity and Severity.

COVID-19 is highly transmissible; however, its severity varies from one individual to another. Variability among different isolates of the virus and among its receptor (ACE2) may contribute to this severity, but comorbidity plays a major role on disease prognosis. Many comorbidities have been reported to be associated with severe COVID-19 patients. We have collected data from retrospective studies which include clinical and epidemiological features of patients and categorize them into severe/mild, ICU/non-ICU and survivors/dead patients. In this review, we give an update about SARS-CoV-2 structure with emphasis on the possible reasons for the severity of the virus in patients. We also collected information and patients' data to highlight the relation between COVID-19 patients and comorbidities.

In Silico Characterization of Toxin-Antitoxin Systems in Campylobacter Isolates Recovered from Food Sources and Sporadic Human Illness.

Campylobacter spp. represents the most common cause of gastroenteritis worldwide with the potential to cause serious sequelae. The ability of Campylobacter to survive stressful environmental conditions has been directly linked with food-borne illness. Toxin-antitoxin (TA) modules play an important role as defense systems against antimicrobial agents and are considered an invaluable strategy harnessed by bacterial pathogens to survive in stressful environments. Although TA modules have been extensively studied in model organisms such as Escherichia coli K12, the TA landscape in Campylobacter remains largely unexplored. Therefore, in this study, a comprehensive in silico screen of 111 Campylobacter (90 C.jejuni and 21 C.coli) isolates recovered from different food and clinical sources was performed. We identified 10 type II TA systems belonging to four TA families predicted in Campylobacter genomes. Furthermore, there was a significant association between the clonal population structure and distribution of TA modules; more specifically, most (12/13) of the Campylobacter isolates belonging to ST-21 isolates possess HicB-HicA TA modules. Finally, we observed a high degree of shared synteny among isolates bearing certain TA systems or even coexisting pairs of TA systems. Collectively, these findings provide useful insights about the distribution of TA modules in a heterogeneous pool of Campylobacter isolates from different sources, thus developing a better understanding regarding the mechanisms by which these pathogens survive stressful environmental conditions, which will further aid in the future designing of more targeted antimicrobials.

Hepatitis C genotype 4: A report on resistance-associated substitutions in NS3, NS5A, and NS5B genes.

AUTHOR CONTRIBUTION: FN performed the literature review and wrote the manuscript; STZ coauthored, edited, and reviewed the manuscript. ABSTRACT: Treatment response in Hepatitis C virus (HCV) has generated varied effects in patients. Recently, nonresponsive and relapse patients related to host and genotype variabilities have been reported in clinical trials. However, these trials included minimal sample sizes of patients with genotype 4, the most prevalent genotype in Egypt and the Middle East, compared with genotypes 1 and 2. The genetic variabilities that have been detected within the HCV genes, especially the ones associated with genotype 4, and are linked to treatment response, will be the focus of this review with emphasis on direct acting antiviral agents. In addition, the major studies and clinical trials performed globally and their inclusivity of genotype 4 are reported. This review also delineates future study areas and missing data that need further investigation when it comes to genotype 4.

Effect of radiotherapy on the gut microbiome in pediatric cancer patients: a pilot study.

The incidence of pediatric cancer is lower than that of adult cancer worldwide. However, the former has detrimental side effects on the health of individuals, even after the cancer is cured, due to the impact of treatment on development. Recently, correlations have been made between the gut microbiome and cancer in several studies but only on adult participants. There is always a complication of dealing with pediatric cancer treatment protocols because they usually include a combination of chemotherapy, radiotherapy, and intensive prophylactic antibiotics. In the current study, a pilot study was conducted to analyze ten fecal samples from three pediatric cancer patients, suffering from rhabdomyosarcoma near their pelvic region, and two healthy individuals. A correlation between microbial composition and response to treatment was reported, in which the responders had generally a lower microbial diversity compared to non-responders. In addition, nucleotide changes and deletions in the tested 16S rRNA sequences post radiotherapy were detected. Despite the small sample size used in the experiments due to the uncommon rhabdomyosarcoma in children, the results can help in understanding the influence of radiotherapy on the gut microbiome in pediatric cancer patients. More work with larger sample size and different cancer types need to be conducted to understand the influence of radiotherapy on gut microbiome to mitigate the deleterious impact of radiation on treated children.

Back to the Light Side: The Role of Mechanotransduction in the Paradoxical Response to Checkpoint Inhibitors in Cancer Patients.

A growing number of studies and case series point to a dark side of immune checkpoint inhibitors, as they may cause rapid tumor growth with potentially deleterious effects. The pathophysiological mechanism of hyper-progressive disease (HPD) is still unknown; in addition, there are no reliable predictive biomarkers that facilitate the process of patient selection for immunomodulatory antibody-based therapy. The proposed model attempts to reveal the mechanism of such paradoxical response, in a subset of patients receiving anti-PD1/PD-L1 immunotherapy, depending on the biomechanistic properties of the crystal structure of PD-1 protein. PD-1 can exhibit a signaling pattern depending on mechanotransduction upon the formation of PD-1/monoclonal antibody (mAb)/Fc-gamma receptor (Fc-γR) axes resulting from the interaction between PD-1/mAb complex, on the surface of tumor-specific T-cells, and Fc-γR-bearing tumor-associated macrophages (TAMs) within the tumor microenvironment. The generated mechanical force activates ITIM and ITSM on the cytoplasmic endodomain of the PD-1 receptor, leading to suppression of the effector function of tumor-specific T-cells which effectively unleashes cancer cells from the cytotoxic barrier and causes HPD in affected patients. This model provides clues about why patients receiving anti-PD1/PD-L1 mAbs are more prone to develop HPD as well as the variability of the ICIs response among treated patients. Additionally, it features the effect of specific immunophenotypic dynamics, such as TAM infiltration, on the final outcome of antibody-based immunotherapy and gives new insights for designing next-generation immunomodulatory interventions.

Ongoing Transmission of HCV: Should Cesarean Section be Justified? Data Mining Discovery.

BACKGROUND AND OBJECTIVES: Over the past few decades, cesarean section (CS) rates are steadily increasing in most of the middle- and high-income countries. However, most of the pregnant women (particularly undergoing CS) are not screened for hepatitis C virus (HCV); hence, neonates born to HCV-positive mother could be a source of future HCV infection. In this study, the role of the CS and other surgical interventions in HCV transmission in Egypt, the highest endemic country of HCV-4, was investigated. METHODS: From January to June 2016, a prospective cohort study was conducted among 3,836 pregnant women in both urban and rural areas across Egypt for HCV screening in both mothers and neonates born to HCV-positive mother. All pregnant women were screened during third trimester or just before delivery, neonates born to HCV-positive mothers were evaluated within 24-h postdelivery to record vertical transmission cases. Data mining (DM)-driven computational analysis was used to quantify the findings. RESULTS: Among 3,836 randomized pregnant women, HCV genotype 4 was identified in 80 women (2.08%). Out of 80 HCV-infected women, 18 have experienced surgical intervention (22.5%) and 62 CS (77.5%). HCV vertical transmission was identified in 10 neonates, 10/80 (12.5%). CONCLUSION: Screening women who had experienced surgical intervention or CS during child bearing period and before pregnancy might prevent HCV mother-to-child transmission (MTCT). CS should be ethically justified to decrease global HCV transmission.

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).

Verifying the stability of selected genes for normalization in Q PCR experiments of Spodoptera frugiperda cells during AcMNPV infection.

It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. Autographa californica nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of Spodoptera frugiperda cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.

Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression.

Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

AcMNPV enhances infection by ThorNPV in Sf21 cells and SeMNPV in Hi5 cells.

An expression cassette containing the DsRed2 gene, which encodes the red fluorescent protein (RFP), was inserted into the wide-host-range Autographa californica M nucleopolyhedrovirus (AcMNPV) at the polyhedrin locus (vAcDsRed2). An expression cassette containing the enhanced green fluorescent protein (EGFP) gene was inserted at the gp37 locus of the narrow-host-range Thysanoplusia orichalcea MNPV (ThorMNPV) and the p10 locus of Spodoptera exigua MNPV (SeMNPV) to produce vThGFP and vSeGFP, respectively. vThGFP and vSeGFP are poor at infecting Sf21 and Hi5 cells, respectively, whereas vAcDsRed2 is highly infectious to both cell lines. During co-infection, vAcDsRed2 enhanced vThGFP infection in Sf21 cells by approximately 20-fold, and it enhanced vSeGFP infection in Hi5 cells by more than 300-fold, as detected by fluorescence measurements. In contrast, vThGFP reduced vAcDsRed2 infection by 5.4-fold in Sf21 cells, while vSeGFP reduced vAcDsRed2 by 3.2-fold in Hi5 cells. Plaque assay data did not suggest viral recombination, but vThGFP plaques surrounded by vAcDsRed2 plaques were observed. A viral DNA replication assay performed by real-time quantitative PCR suggested that the detected fluorescence correlated with virus replication. Sf21 cells infected with vAcDsRed2 were resistant to superinfection by viruses of the same type expressing EGFP (vAcGFP). These results demonstrated that AcMNPV could enhance replication of ThorMNPV and SeMNPV in non-permissive cells without recombination.

Comprehensive analysis of host gene expression in Autographa californica nucleopolyhedrovirus-infected Spodoptera frugiperda cells.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus and most commonly used virus vector for baculovirus expression vector systems. The effect of AcMNPV infection on host cells is incompletely understood. A microarray based on Spodoptera frugiperda ESTs was used to investigate the impact of AcMNPV on host gene expression in cultured S. frugiperda, Sf21 cells. Most host genes were down-regulated over the time course of infection, although a small number were up-regulated. The most highly up-regulated genes encoded heat shock protein 70s and several poorly characterized proteins. Regulated genes with the highest score identified by functional annotation clustering included primarily products required for protein expression and trafficking in the ER and golgi. All were significantly down-regulated by approximately 12h post-infection. Microarray data were validated by qRT-PCR. This study provides the first comprehensive host transcriptome overview of Sf21 cells during AcMNPV infection.

Tissue tropism of the Musca domestica salivary gland hypertrophy virus.

The tissue tropism of Musca domestica salivary gland hypertrophy virus (MdSGHV) infecting adult house flies was examined by transmission electron microscopy (TEM) and quantitative real-time PCR. TEM demonstrated that characteristic MdSGHV-induced nuclear and cellular hypertrophy was restricted to the salivary glands. Both nucleocapsids and enveloped virions were present in salivary gland cells. In contrast, thin sections of midguts, ovaries, abdominal fat body, crops, air sacs and brains showed the presence of enveloped virions in vacuoles of tracheal cells associated with these tissues. However, no sites of viral morphogenesis were detected in the tracheal cells. Quantitative analysis of MdSGHV DNA and transcript titers revealed that viral DNA was present in all hemolymph and tissue samples collected from MdSGHV-infected flies. Average numbers of MdSGHV genome copies per 50 ng of DNA varied significantly between examined tissues and ranged from 3.83 × 10(8) (±3.75 × 10(7)) in salivary gland samples to 7.98 × 10(5) (±2.91 × 10(5)) in hemolymph samples. High levels of viral genome copies were detected in midgut, fat body and brain samples. Viral transcripts were present in all examined samples, and transcript abundance was also at the highest level in salivary glands and at the lowest level in hemolymph. However, over the range of different tissues that were analyzed, there was no correlation between estimated quantities of genome copies and viral transcripts. The function of viral transcripts in host tissues that do not show sites of viral morphogenesis remains to be elucidated.

Universal primers for rapid detection of hytrosaviruses.

Hytrosaviridae is a proposed virus family encompassing viruses that cause salivary gland hypertrophy (SGH) syndrome in infected insects and reduce the fertility in their dipteran insect hosts. They contain a large, double stranded DNA genome of 120-190 kbp. To date, these viruses have been detected only in adult Diptera. These include hytrosaviruses detected in various tsetse fly species (Glossina spp.), the narcissus bulb fly Merodon equestris and the house fly Musca domestica. The limited number of hytrosaviruses reported to date may be a reflection of the frequent absence of external symptoms in infected adult flies and the fact that the virus does not cause rapid mortality. Based on the complete genome sequence of Glossinia pallidipes (GpSGHV) and Musca domestica (MdSGHV) salivary gland hypertrophy viruses, a PCR based methodology was developed to detect the viruses in these species. To be able to detect hytrosaviruses in other Diptera, five degenerate primer pairs were designed and tested on GpSGHV and MdSGHV DNA using gradient PCR with annealing temperatures from 37 to 61°C. Two pairs of primers were selected from p74, two pairs from PIF-1 and one pair from ODV-e66 homologous proteins. Four primer pairs generated a virus specific PCR product on both MdSGHV and GpSGHV at all tested annealing temperatures, while the ODV-e66 based primers did not generate a virus specific product with annealing temperatures higher that 47°C. No non-specific PCR product was found when using genomic DNA of infected flies as template DNA. These results offer new sets of primers that could be used to detect hytrosaviruses in other insects.

Strategy of the use of 28S rRNA as a housekeeping gene in real-time quantitative PCR analysis of gene transcription in insect cells infected by viruses.

Quantitative real-time reverse transcription-PCR (qRT-PCR) has been used widely to measure gene transcription regulation in cells. qRT-PCR must include one or more internal housekeeping genes to normalize data collection. A strategy to use the host cell 28S rRNA as a housekeeping gene in qRT-PCR analysis of gene transcription of insect cells infected by baculovirus and ascovirus was developed. It has been found that the 28S rRNA reverse primer can be incorporated in the oligo-dT-primed cDNA synthesis reaction. In such a way, amplification of 28S cDNA showed lower and less variable cycle thresholds in cells infected by viruses than by using only oligo-dT and other published housekeeping genes such as the TATA box binding protein (TBP) gene, the peptidyl prolyl isomerase A (PPI) gene and the ribosomal protein 13 (L13) gene. Incorporation of the 28S reverse primer in oligo-dT-primed cDNA synthesis also does not interfere with the detection of other polymerase II transcribed genes.