Viral Messenger RNA Research Articles

Evaluation of the hologic gen-probe aptima HPV assay with the panther system

The Royal Prince Alfred Hospital (RPAH) receives gynecological samples from previously clinically screened high risk patients. In February 2012 the Microbiology Department at RPAH evaluated the APTIMA HPV (AHPV) assay on the fully automated PANTHER system. This assay is a target amplification nucleic acid probe test that utilises target capture for the qualitative detection of E6/E7 viral messenger RNA (mRNA) from 14 high-risk HPV types. The E6 and E7 genes from high-risk genotypes are known oncogenes. 201 ThinPrep specimens were tested in parallel using the Digene Hybrid Capture 2 (HC2) HPV DNA assay and AHPV assay. The results from each assay were directly compared to each other. Cytological and/or histological findings were used to aid resolution of discordant results. The overall agreement between the assays was 92.4%. The sensitivity specificity, positive predictive value and negative predictive value were as follows: for the AHPV assay, 97.1%, 96.9%, 94.3% and 98.4%; and for the HC2 assay, 91.2%, 97.7%, 95.4% and 95.5%. In this small sample size study of tertiary referral samples, the AHPV assay demonstrated good clinical performance. The PANTHER, which is a random access instrument capable of performing up to four different assays at one time, has very significant workflow advantages in comparison with the HC2 assay.

Reactivation of Latent HIV-1 by Inhibition of BRD4

HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy. Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell-line models. In multiple instances, JQ1, when used in combination with the NF-κB activators Prostratin or PHA, enhanced the invitro reactivation of latent HIV-1 in primary Tcells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection.

Conservation of a Triple-Helix-Forming RNA Stability Element in Noncoding and Genomic RNAs of Diverse Viruses

Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of four of the ENEs was demonstrated by increased accumulation of an intronless polyadenylated reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2. Our findings demonstrate that searching for structural elements can lead to rapid identification of lncRNAs.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

An equilibrium-dependent retroviral mRNA switch regulates translational recoding

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag-Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5-10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag-Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

Viral-mediated stabilization of AU-rich element containing mRNA contributes to cell transformation

E4orf6 is one of the oncogene products of adenovirus, and it also has an important role for transportation of cellular and viral messenger RNA (mRNA) during the late phase of virus infection. We previously revealed that E4orf6 controls the fate of AU-rich element (ARE) containing mRNA by perturbing the chromosome maintenance region 1-dependent export mechanism. Here, we show that E4orf6 stabilizes ARE-mRNA through the region required for its oncogenic activity and ubiquitin E3 ligase assembly. Cells that failed to stabilize ARE-mRNA after HuR knockdown were unable to produce colonies in soft agar, even when E4orf6 was expressed. Furthermore, the stabilized ARE-mRNA induced the transformation of rodent immortalized cells. These findings indicate that stabilized ARE-mRNA is necessary, if not all, for the oncogenic activity of E4orf6 and has the potential to transform cells, at least under a certain condition.

In vitro inhibition of feline coronavirus replication by small interfering RNAs

Infection with virulent biotypes of feline coronavirus (FCoV) can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. In this study we demonstrate the ability of small interfering RNA (siRNA) mediated RNA interference (RNAi) to inhibit the replication of virulent FCoV strain FIPV WSU 79-1146 in an immortalised feline cell line. A panel of eight synthetic siRNAs targeting four different regions of the FCoV genome were tested for antiviral effects. Efficacy was determined by qRT-PCR of intracellular viral genomic and messenger RNA, TCID50 infectivity assay of extracellular virus, and direct IFA for viral protein expression. All siRNAs demonstrated an inhibitory effect on viral replication in vitro. The two most effective siRNAs, targeting the untranslated 5′ leader sequence (L2) and the nucleocapsid gene (N1), resulted in a >95% reduction in extracellular viral titre. Further characterisation of these two siRNAs demonstrated their efficacy when used at low concentrations and in cells challenged with high viral loads. Taken together these findings provide important information for the potential therapeutic application of RNAi in treating FIP.

In Vitro and in Vivo Anti-Influenza Virus Activity of Diarylheptanoids Isolated from Alpinia Officinarum

Diarylheptanoids (AO-0002 [7-(4''-hydroxy-3''-methoxyphenyl)-1-phenyl-4E-hepten-3-one] and AO-0011 [(5S)-5-hydroxy-7-(4''-hydroxyphenyl)-1-phenyl-3-heptanone]) isolated from Alpinia officinarum have been reported to exhibit anti-influenza virus activity in vitro. Hence, efficacies against influenza virus infection and the mode of antiviral action were evaluated in vivo and in vitro, respectively. In a murine influenza virus infection model, diarylheptanoids were orally administered three times daily to mice infected with influenza A/PR/8/34 virus for 6 days after infection. AO-0002 was examined for its antiviral activity against the wild types of influenza viruses A/PR/8/34 (H1N1), oseltamivir-resistant A/PR/8/34 (H1N1), A/Bangkok/93/03 (H1N1), A/Ishikawa/7/82 (H3N2), A/Fukushima/13/43 (H3N2), B/Singapore/222/79 and B/Fukushima/15/93 in plaque reduction or yield reduction assays. The mode of anti-influenza virus action was assessed by a virus adsorption assay, immunofluorescence assay of viral antigens, and inhibition of viral messenger RNA synthesis using real-time reverse transcriptase PCR. AO-0002 at 100 mg/kg was significantly effective in reducing the body weight loss and prolonging survival times of infected mice without toxicity, but AO-0011 was not. AO-0002 at 30 and 100 mg/kg significantly reduced virus titres in bronchoalveolar lavage fluids of the lungs on days 3 and 6 after infection. AO-0002 exhibited anti-influenza virus activity against all viruses used, including the oseltamivir-resistant strain in vitro. The compound had no effect on virus adsorption or invasion into cells, but dose-dependently suppressed the expression of viral messenger RNA and antigens. AO-0002 was suggested to have a different anti-influenza virus action to that of oseltamivir and was verified to show anti-influenza activity in vitro and in vivo.

Prevalence and Activity of Epstein-Barr Virus and Human Cytomegalovirus in Symptomatic and Asymptomatic Apical Periodontitis Lesions

Introduction Apical periodontitis is a polymicrobial inflammation with a dominant flora of opportunistic Gram-negative bacteria; however, a pathogenic role of human herpesviruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) has been implicated recently. The aims of this study were to determine the prevalence, activity, and disease association of EBV and HCMV in apical periodontitis in an Eastern Hungarian population. Methods Forty samples with apical periodontitis (17 symptomatic and 23 asymptomatic) and 40 healthy pulp controls were collected. EBV and HCMV prevalences were measured by polymerase chain reaction (PCR) detection of the viral DNA and viral activity was tested by reverse-transcription PCR amplification of viral messenger RNA. Results EBV DNA and EBNA-2 messenger RNA were found in apical periodontitis lesions at significantly ( p < 0.0001) higher frequencies (72.5% and 50%, respectively) than in controls (both 2.5%). The occurrence of HCMV infection was rare in both apical lesions (10%) and controls (0%). The presence of EBV DNA in apical lesions was associated significantly with large (≥5 mm) lesion size ( p = 0.02) but not with symtoms ( p = 0.30). Symptomatic manifestation was significantly associated with the co-occurrence (odds ratio [OR], 8.80; 95% confidence interval [CI], 1.69-45.76) but not the sole occurrences of EBNA-2 messenger RNA (OR, 2.29; 95% CI, 0.48-11.06) and large lesion size (OR, 4.02; 95% CI, 0.81-19.89). Conclusion EBV infection is a frequent event in apical periodontitis, whereas the involvement of HCMV still remains to be elucidated. This study showed that symptomatic manifestation was likely to occur if a large-sized apical periodontitis lesion is aggravated with active EBV infection.

Human Polyomavirus JC (JCV) Infection of Human B Lymphocytes: A Possible Mechanism for JCV Transmigration across the Blood‐Brain Barrier

It has been suggested that JC virus (JCV) might travel to the central nervous system in infected B cells. Moreover, recent data suggest the presence of JCV in bone marrow plasma cells. However, the evidence for infection and replication of JCV in B cells is unclear. To address this question, we infected Epstein-Barr virus-transformed B cells with JCV and found that the viral genome decreased >1000-fold from days 0 to 20 after infection, which concurred with the absence of viral early and late messenger RNA transcripts and proteins. However, immunofluorescent images of B cells infected with fluorescein isothiocyanate-conjugated JCV demonstrated that JCV enters the B cells, and DNase protection assay confirmed the presence of intact JCV virions inside the B cells. Moreover, JCV-infected B cells were able to transmit infection to naive glial cells. These data confirm that JCV nonproductively infects B cells and possibly uses them as a vehicle for transmigration across the blood-brain barrier.

Enzymology of RNA cap synthesis

The 5' guanine-N7 methyl cap is unique to cellular and viral messenger RNA (mRNA) and is the first co-transcriptional modification of mRNA. The mRNA cap plays a pivotal role in mRNA biogenesis and stability, and is essential for efficient splicing, mRNA export, and translation. Capping occurs by a series of three enzymatic reactions that results in formation of N7-methyl guanosine linked through a 5'-5' inverted triphosphate bridge to the first nucleotide of a nascent transcript. Capping of cellular mRNA occurs co-transcriptionally and in vivo requires that the capping apparatus be physically associated with the RNA polymerase II elongation complex. Certain capped mRNAs undergo further methylation to generate distinct cap structures. Although mRNA capping is conserved among viruses and eukaryotes, some viruses have adopted strategies for capping mRNA that are distinct from the cellular mRNA capping pathway.

Real-time RT-qPCR assay for the analysis of human influenza A virus transcription and replication dynamics

A quantitative real-time reverse transcriptase PCR (RT-qPCR) assay was developed for the analysis of influenza A virus transcription and replication dynamics in mammalian cell culture. The assay is based on a polarity- and sequence-specific reverse transcription used to distinguish specifically between viral genomes (vRNA(−)), replicative intermediates (cRNA(+)) and viral messenger RNAs (vmRNA(+)) of segments 4 (HA), 6 (NA), 7 (M) and 8 (NS) during the life cycle of influenza virus. Synthetic viral RNAs used as reference standards for validation and quantitation were prepared for each viral RNA type and segment. Assay validation demonstrated linearity over five orders of magnitude, sensitivity of 1.0 × 10 3 to 8.9 × 10 3 of viral RNA molecules, repeatability and reproducibility of less than 0.8–3.1% CV (coefficient of variation). Dynamics of influenza A virus infection in adherent MDCK cells, a substrate considered for human influenza vaccine manufacturing, were analyzed. In general, mainly vmRNA(+) were synthesized during early phases of infection at about 0.6 hpi, followed immediately by cRNA(+) synthesis and after a short delay of about 1.9 hpi viral genome replication could be detected. The vRNA(−)s were synthesized in equimolar amounts and similar dynamics whereas preferential synthesis of NS1 vmRNA(+) in early transcription phases and a delay for M1 vmRNA(+) was found.

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health. High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective. In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON) was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1) reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected p27(-/-) (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus-host interactions and the identification of drug targets for a broad range of influenza viruses.

Subcellular localization of the barley stripe mosaic virus triple gene block proteins.

Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.

Mesoionic Heterocyclic Compounds as Candidate Messenger RNA Cap Analogue Inhibitors of the Influenza Virus RNA Polymerase Cap-Binding Activity

An unusual feature of influenza viral -messenger RNA (mRNA) synthesis is its dependence upon host cell mRNAs as a source of capped RNA primers. A crucial activity of the influenza polymerase is to steal these primers by binding and cleaving the caps from host mRNAs. The recent structural analysis of the cap-binding fragment of the influenza virus PB2 protein has highlighted the importance of the mesoionic properties of the N7-methylguanine (N(7m)G) component of the mRNA cap in this interaction. A series of mesoionic heterocycles with 5,6-fused ring systems analogous to the N(7m)G component of mRNA cap structures were synthesized and examined for the ability to inhibit the cap-binding activity of the influenza virus RNA polymerase complex using a bead-based in vitro cap-binding assay. None of the compounds tested were able to significantly inhibit binding and subsequent endonucleolytic cleavage of a synthetic radiolabelled capped mRNA substrate by recombinant influenza virus polymerase in vitro. Compounds analogous to the mesoionic N(7m)G component of mRNA cap structures comprise a large class of potential inhibitors of the influenza virus polymerase. Although this preliminary assessment of a small group of related analogues was unsuccessful, further screening of this class of compounds is warranted.

The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit

The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

Viral and cellular messenger RNA targets of viral microRNAs

Given the propensity of viruses to co-opt cellular pathways and activities for their benefit, it is perhaps not surprising that several viruses have now been shown to reshape the cellular environment by reprogramming the host's RNA-interference machinery. In particular, microRNAs are produced by the various members of the herpesvirus family during both the latent stage of the viral life cycle and the lytic (or productive) stage. Emerging data suggest that viral microRNAs are particularly important for regulating the transition from latent to lytic replication and for attenuating antiviral immune responses.

A Case of a Synchronous Peripheral T-cell Lymphoma in a Patient with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) patients tends to have a higher risk of developing lymphoid malignancies. The majority of such tumors are of a B cell origin. However, it is known that the T cell lymphoma subtypes in SLE patients are quite rare. Here, we describe a case of peripheral T cell lymphoma, unspecified (PTCL-U) that occurred in a 50-year-old female SLE patient. The lymphoma was located at the bilateral cervical and mediastinal lymph nodes. The staging workup revealed no evidence of any other organ involvement. Epstein-Barr virus messenger RNA was detected in the serum, but not in the lymph nodes. She received front-line chemotherapy with the CHOP regimen and she achieved complete remission. She then subsequently received high-dose chemotherapy with autologous peripheral stem cell transplantation. The patient currently remains in a clinical and serological state of remission for the SLE and PTCL until the time of this report 18 months after chemotherapy, and this was followed by autologous peripheral blood stem cell transplantation.

RNA‐based viral immunity initiated by the Dicer family of host immune receptors

Suppression of viral infection by RNA in a nucleotide sequence homology-dependent manner was first reported in plants in early 1990 s. Studies in the past 15 years have established a completely new RNA-based immune system against viruses that is mechanistically related to RNA silencing or RNA interference (RNAi). This viral immunity begins with recognition of viral double-stranded or structured RNA by the Dicer nuclease family of host immune receptors. In fungi, plants and invertebrates, the viral RNA trigger is processed into small interfering RNAs (siRNAs) to direct specific silencing of the homologous viral genomic and/or messenger RNAs by an RNaseH-like Argonaute protein. Deep sequencing of virus-derived siRNAs indicates that the immunity against viruses with a positive-strand RNA genome is induced by Dicer recognition of dsRNA formed during the initiation of viral progeny (+)RNA synthesis. The RNA-based immune pathway in these organisms overlaps the canonical dsRNA-siRNA pathway of RNAi and may require amplification of viral siRNAs by host RNA-dependent RNA polymerase in plants and nematodes. Production of virus-derived small RNAs is undetectable in mammalian cells infected with RNA viruses. However, infection of mammals with several nucleus-replicating DNA viruses induces production of virus-derived microRNAs capable of silencing host and viral mRNAs as found for viral siRNAs. Remarkably, recent studies indicate that prokaryotes also produce virus-derived small RNAs known as CRISPR RNAs to guide antiviral defense in a manner that has yet to be defined. In this article, we review the recent progress on the identification and mechanism of the key components including viral sensors, viral triggers, effectors, and amplifiers, of the small RNA-directed viral immunity. We also highlight some of the many unresolved questions.

Quantitative PCR analysis of the salivary gland hypertrophy virus (GpSGHV) in a laboratory colony of Glossina pallidipes

Many species of tsetse flies can be infected by a virus that causes salivary gland hypertrophy (SGH) and virus isolated from Glossina pallidipes (GpSGHV) has recently been sequenced. Flies having SGH have a reduced fecundity and fertility. To better understand the impact of this virus in a laboratory colony of G. pallidipes, where the majority of flies are infected but asymptomatic, and to follow the development of SGH in symptomatic flies in relation to virus copy number, a quantitative PCR (qPCR) method was developed. The qPCR analyses revealed that in asymptomatic flies virus copy number averaged 1.68E+5, 2.05E+5 and 1.07E+7 log 10 in DNA from an excised leg, salivary glands and a whole fly, respectively. In symptomatic flies the virus copy number in the same organs averaged 1.34E+7, 1.42E+10 and 1.5E+9, respectively. Despite these statistically significant differences ( p ≪ 0.0001) in virus copy number between asymptomatic and symptomatic flies, there was no correlation between age and virus copy number for either sets in adult flies. A clear correlation between virus copy number in pupae and their mothers was observed. Reverse transcription quantitative PCR (RT-qPCR) of the viral messenger RNA encoding ODV-E66, an envelope protein, revealed a clear correlation between virus copy number and the level of gene expression with values of 2.77 log 10 in asymptomatic males and 6.10 log 10 in symptomatic males. Taken together these results confirm the close relationship between virus copy number and SGH syndrome. They demonstrate the vertical transmission of GpSGHV from mother to progeny, and suggest that the development of SGH may be correlated to the virus copy number acquired by the larva during its intra-uterine development.