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At least 19 recordsLinked to original sources
PubMed2025

Viral viability markers of SARS-CoV-2: a comparison of cell culture, genomic RNA RT-PCR, and subgenomic RNA RT-PCR.

UNLABELLED: Accurate methods to assess viral viability are crucial for determining isolation duration and antiviral therapy in immunocompromised patients. Although cell culture (CC) is the gold standard, it has limitations. Cycle threshold (Ct) values from genomic RNA (gRNA) RT-PCR and subgenomic RNA (sgRNA) RT-PCR have been proposed as markers of active viral replication. This study evaluated the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. This study aimed to evaluate the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. We conducted a prospective study on immunocompromised patients with suspected SARS-CoV-2 infection at a tertiary hospital (May 2021 to May 2023). Nasopharyngeal swabs were inoculated into Vero E6 cells and tested for gRNA using RT-PCR (Cobas 6800, Roche) and for sgRNA (non-commercial RT-PCR). The sensitivity (S), specificity (SP), positive (PPV) and negative predictive value (NPV), and accuracy were calculated using CC as the gold standard. Among 285 samples from 108 patients, gRNA RT-PCR had high S and NPV (1.0) but low SP (0.24) and moderate PPV (0.63). Ct analysis improved performance in extreme but not intermediate values. A Ct ≤ 30 maximized S but had low SP; Ct ≤ 25 yielded S (0.88), SP (0.89), PPV (0.92), NPV (0.84), and accuracy (0.88); sgRNA showed the highest S (0.99), SP (0.96), PPV (0.97), NPV (0.99), and accuracy (0.98). sgRNA detection is the best marker for identifying viable SARS-CoV-2, aiding decisions on isolation, antiviral treatment, or delaying chemotherapy in immunocompromised patients. IMPORTANCE: Identifying whether a patient still has contagious SARS-CoV-2 is essential for managing isolation, antiviral treatment, and other clinical decisions-especially in immunocompromised individuals. While viral culture is the gold standard for confirming viral viability, it is slow, expensive, and not widely available. Many hospitals rely on RT-PCR tests, but these detect viral genetic material whether or not the virus is still active. This study shows that detecting subgenomic RNA (sgRNA), a molecule only present when the virus is actively replicating, is a highly accurate way, a molecule only present when the virus is actively replicating, is a highly accurate way to determine whether the virus is still viable. Compared to standard PCR or viral culture, sgRNA testing better predicts who is truly infectious. These findings support sgRNA as a useful tool to guide clinical management and infection control in vulnerable patients.

Humans↗
PubMed1979

Evidence for the identity of shared 5'-terminal sequences between genome RNA and subgenomic mRNA's of B77 avian sarcoma virus.

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.

Avian Sarcoma Viruses↗
PubMed1977

Togavirus RNA: reversible effect of urea on genomes and absence of subgenomic viral RNA in Kunjin virus-infected cells.

Electrophoretic analyses showed that no RNase-sensitive RNA smaller than the genome was specified by the flavivirus Kunjin in infected Vero cells during the period of maximum RNA and protein synthesis. In contrast, RNA extracted from Sindbis virus-infected cells under similar conditions included the expected 42S RNA (equivalent to the genome) and the smaller 26S (interjacent) RNA. Treatment of the genome of both togaviruses with 12 M urea produced a reversible (possibly conformational) change; measurement of the molecular weights of the treated RNAs by co-electrophoresis with fully denatured ribosomal RNA markers in SDS-polyacrylamide gels yielded a value of 2.1 X 10(6) if 8 M urea was incorporated in the gels and 4.2 X 10(6) if urea was omitted from the gels. These results indicate that flavivirus messenger RNA is represented solely by the intact genome of m.wt. 4.2 X 10(6).

Arboviruses↗
PubMed1979

An MSV-specific subgenomic mRNA in MSV-transformed G8-124 cells.

An intracellular subgenomic RNA species from MSV-transformed G8-124 cells was characterized by electron microscopy of RNA:cDNA heteroduplexes using long cDNAs both MSV and MuLV. This subgenomic RNA, 3.1 kb long, consisted of 5'-derived sequences of about 0.4 kb joined to 2.7 kb of RNA derived from the 3' end of the RNA genome. The 3'-derived sequences included the residual sequences from the MuLV pol region and the acquired cellular sequences of MSV. The genome of MSV was shown to retain approximately 0.13 kb from the 5' end of the MuLV env region, including sequences which span the point in the MuLV env mRNA. No subgenomic MSV RNA could be detected, however, which consisted of a 5'-derived leader sequence spliced to the retained env region sequences. Nor could a subgenomic MSV RNA be detected in which a 5'-derived leader sequence was joined directly to the acquired cellular sequences. Although its translation products are unknown, the subgenomic MSV RNA was present in preparations of poly(A)+ polysomal RNA, consistent with this RNA functioning as a messenger. The structure of this 3.1 kb MSV subgenomic RNA suggests a possible role in the expression of 3'-encoded MSV information, possibly including transformation-specific sequences.

Cell Transformation, Viral↗
PubMed2025

Performance of Subgenomic RT-PCR for Predicting SARS-CoV-2 Infectivity Compared to Genomic RT-PCR and Culture Isolation.

SARS-CoV-2 clinical samples can be detected as positive for a long period of time using real-time RT-PCR, even when patients are no longer infectious. Viral culture is the gold standard for assessing a patient's infectivity, but it is a time-consuming technique and lacks sensitivity. SARS-CoV-2 subgenomic RNA (sgRNA) detection has been used as a proxy for assessing the infectivity but only a limited number of studies have described its use in vitro and in clinical samples. This study aimed to evaluate the correlation between results from viral culture, genomic RT-PCR (gRT-PCR), and subgenomic RT-PCR (sgRT-PCR) during in vitro infection and in clinical samples. In vitro viral replication kinetics showed that both genomic RNA (gRNA) and subgenomic RNA (sgRNA) levels remained stable up to 21 days in the absence of replication-competent virus. Using clinical samples, sgRNA was detected in 87.5% of culture-positive samples, demonstrating better performances than gRT-PCR (Positive predictive value (PPV) 93.3% and Negative predictive value (NPV) of 87.5%) and an almost perfect agreement with culture results (Cohen κ = 0.81 [95% CI: 0.66-0.95]). These findings suggest that testing for sgRNA and/or using a gRNA Ct cut-off of 21.2 could be used as a proxy to determine the presence of SARS-CoV-2 replication-competent virus.

Humans↗
PubMed1979

Virus-like 30S RNA in mouse cells.

Uninfected JLS-V9 mouse cells are known to express high levels of viral sequences that hybridize to complementary DNA made by the BrdU-induced virus of JLS-V9 cells. The genome in the BrdU-induced virus has been found to consist mainly of an RNA species that migrates as 30S RNA material during electrophoresis through agarose gels. This virus-like 30S RNA, designated VL30 RNA, apparently represents a new class of endogenous defective retroviruses that are not generally evident because of their defectiveness and lack of biological function. Fingerprint analysis and hybridization studies show that VL30 RNA does not have homology with the standard nondefective murine leukemia viruses. Upon superinfection with a nondefective murine leukemia virus, or upon induction of endogenous virus with BrdU, VL30 RNA is rescued into virions by phenotypic mixing. When VL30 RNA is rescued by BrdU induction, the VL30 RNA is mainly organized as a 50S complex, but when VL30 is rescued by superinfection, VL30 is also found in 70S RNA. Rescued VL30 RNA sequences can be reverse transcribed by the virion-associated DNA polymerase in an endogenous reaction. Many mouse cells express the sequences, whereas heterologous cells such as rat or rabbit cells do not contain them. By using hybridization of a complementary DNA probe to cellular RNA immobilized on paper, no subgenomic RNA related to the VL30 RNA could be found in cells expressing the VL30 sequences. From 20 to 50 copies of these sequences were found to be contained in the mouse genome. VL30 RNA is probably present in most stocks of leukemia and sarcoma viruses made in mouse cells.

Animals↗
PubMed1979

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.

DNA-Directed RNA Polymerases↗
PubMed1979

The smallest genome RNA segment of influenza virus contains two genes that may overlap.

The genome of influenza virus consists of eight segments of single-stranded RNA, each of which encodes a different polypeptide. In addition to the eight recognized gene products, the virus specifies a distinct smaller nonstructural polypeptide (NS2), which is translated from a separate species of virus-specific mRNA. The location on the virus genome of the gene encoding this polypeptide was investigated by hybridization of the NS2 mRNA with isolated subgenomic RNA species, and by correlation of the inheritance of a strain-specific NS2 with inheritance of particular genome RNA segments during recombination between two different virus strains. The genetic information for NS2 was found to reside in the smallest genome RNA segment of the virion, which also encodes the NS1 polypeptide. Considering the sizes of the molecules involved, it is likely that the coding sequences for the two polypeptides overlap.

Genes, Viral↗
PubMed2026

Naturally occurring mutations in replication proteins of a small RNA virus that alter the number, sizes, and relative abundances of subgenomic RNAs.

Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in infected cells. sgRNAs are synthesized by virus-encoded replication proteins (RPs), but whether RPs regulate the number and sizes of sgRNAs remains largely unknown. We report multiple naturally occurring mutations within the RPs of turnip crinkle virus (TCV) that alter the number, sizes, and relative abundances of TCV sgRNAs. TCV is a (+) RNA virus that normally produces two sgRNAs: the 1,724-nucleotide (nt) sgRNA1 expressing movement proteins, and the 1,449-nt sgRNA2 expressing capsid protein. A single amino acid change, A113V, within a region shared by TCV RPs p28 and p88, diminished sgRNA1 levels and delayed viral systemic spread. Interestingly, three second-site RP mutations emerged in infected plants that, alone or in combination with A113V, resulted in over-production of sgRNA1 or accumulation of two alternative sgRNAs of 1,876 and 1,601 nt, and rescued A113V defects. The alternative sgRNAs originated from nearly identical recombination events, their size difference reflecting varying 5' extensions. They may have accumulated to high levels through selective stabilization of their (-)-strand intermediates that were in turn derived from transcriptional pausing and recombination. Our findings reveal previously unrecognized constraints on viral RPs that ensure production of sgRNAs with precise sizes and abundances.

Subgenomic RNA↗
PubMed1977

Translation of 35S and of subgenomic regions of avian sarcoma virus RNA.

Rabbit antiserum monospecific for an internal structural protein, p27, of avian sarcoma viruses (ASV) was found to immunoprecipitate polypeptides with molecular weights (Mr) of 180,000 and 76,000 from cell-free reticulocyte lysates programmed by ASV 35S RNA and also from lysates of ASV-infected cells. In addition, the Mr 180,000 protein was also precipitated by antiserum raised against virion DNA polymerase, suggesting that is a product of the two genes nearest the 5' end of virion 35S RNA. We have also investigated the ability of subgenomic portions of virion RNA to program cell-free protein synthesis. A 10-12S poly(A)-containing fragment of RNA from both nondefective and transformation-defective ASV directed the synthesis of a polypeptide of Mr 29,000 immunologically unrelated to the gs antigens; 20-24S poly(A)-containing RNA from nondefective ASV directed the synthesis of a polypeptide of Mr 60,000 not found when a similar RNA preparation from transformation-defective ASV was translated, suggesting that it is the product of the ASV src gene. These results indicate that internal initiation sites for protein synthesis exist on the 35S RNA genome.

Avian Sarcoma Viruses↗
PubMed2026

Transgene sequence codon optimization and composition determines replication competence of self-amplifying RNA.

Self-amplifying RNA (saRNA) is an emerging RNA therapeutic modality that can facilitate higher magnitude and more durable protein expression at substantially lower doses than nonreplicating mRNA. Unlike conventional messenger RNA (mRNA), alphavirus-derived saRNA must support a replicase-driven RNA amplification step in addition to translation, raising the possibility that transgene coding sequences impose sequence-level constraints on replication. Here, saRNA replication was found to be dependent on the codon composition of the transgene; multiple therapeutic transgenes were replication defective despite an intact Venezuelan Equine Encephalitis Virus (VEEV)-derived saRNA backbone. Replication defects were rescued by synonymous codon re-optimization of the same transgenes, indicating that nucleotide-level features of the coding sequence, rather than the encoded protein, govern replication competence. Comparative compositional analyses identified a distinct signature associated with productive replication, characterized by elevated GC (>53%) and GC3 (>63%) content, higher codon adaptation to human (>0.75), and reduced UpA (<43/kb) and UpU (<41/kb) dinucleotide density. Moreover, deliberate compositional perturbation of an otherwise replication-competent transgene shifted these features and abolished replication, supporting a causal and combinatorial role for sequence composition in defining saRNA replication outcome. These findings define an underappreciated constraint in saRNA therapeutics and motivate saRNA-specific payload design frameworks that incorporate alphavirus-associated compositional biases during transgene sequence optimization.

Codon↗
PubMed1977

Size and genetic content of viral RNAs in avian oncovirus-infected cells.

Viral complementary DNA (cDNA) sequences corresponding to the gag, pol, env, src, and c regions of the Rous sarcoma virus genome were selected by hybridizing viral cDNA to RNA from viruses that lack the env or src gene or to polyadenylic acid [poly(A)]-containing RNA fragments of different lengths and isolating either hybridized or unhybridized DNA. The specificities, genetic complexities, and map locations of the selected cDNA's were shown to be in good agreement with the size and map locations of the corresponding viral genes. Analyses of virus-specific RNA, using the specific cDNA's as molecular probes, demonstrated that oncovirus-infected cells contained genome-length (30-40S) RNA plus either one or two species of subgenome-length viral RNA. The size and genetic content of these RNAs varied, depending on the genetic makeup of the infecting virus, but in each case the smaller RNAs contained only sequences located near the 3' end of the viral genome. Three RNA species were detected in Schmidt-Ruppin Rous sarcoma virus-infected cells: 39S (genome-length) RNA; 28S RNA, with an apparent sequence of env-src-c-poly(A); and 21S RNA, with an apparent sequence of src-c-poly(A). Cells infected with the Bryan high-titer strain of Rous sarcoma virus, which lacks the env gene, contained genome-length (35S) RNA and 21S src-specific RNA, but not the 28S RNA species. Leukosis virus-infected cells contained two detectable RNA species: 35S (genome-length) RNA and 21S RNA, with apparent sequence env-c-poly(A). Since gag and pol sequences were detected only in genome-length RNAs, it seems likely that the full-length transcripts function as mRNA for these two genes. The 28S and 21S RNAs could be the active messengers for the env and src genes. Analyses of sequence homologies among nucleic acids of different avian oncoviruses demonstrated substantial similarities within most of the genetic regions of these viruses. However, the "common" region of Rous-associated virus-0, an endogenous virus, was found to differ significantly from that of the other viruses tested.

Animals↗
PubMed1979

Nucleotide sequence of the 3'-noncoding region of alfalfa mosaic virus RNA 4 and its homology with the genomic RNAs.

A 226-nucleotide fragment was derived from alfalfa mosaic virus RNA 4 (ALMV RNA 4), the subgenomic messenger for viral coat protein, and its sequence was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. The fragment contains the 3'-terminal 45 nucleotides of the coat protein cistron and the complete 3'-noncoding region of 182 nucleotides. The total length of RNA 4 was calculated to be 881 nucleotides. AlMV RNAs 1, 2 and 3 were elongated with a 3'-terminal poly(A) stretch and subjected to sequence analysis by using a specific primer, reverse transcriptase and chain terminators. This revealed and extensive homology between the 3'-terminal 140 to 150 nucleotides of all four ALMV RNAs. Despite a number of base substitutions, the secondary structure of the homologous region is highly conserved. The observed homology indicates that, as with RNA 4, the sites with a high affinity for the viral coat protein are located at the 3'-termini of the genomic RNAs.

Base Sequence↗
PubMed1978

Coat protein binds to the 3'-terminal part of RNA 4 of alfalfa mosaic virus.

All four RNAs of alfalfa mosaic virus contain a limited number of sites with a high affinity for coat protein [Van Boxsel, J. A. M. (1976), Ph.D. Thesis, University of Leiden]. In order to localize these sites in the viral RNAs, RNA 4 Tthe subgenomic messenger for coat protein) was subjected to a very mild digestion with ribonucleast T1. The ten major fragments, apparently resulting from five preferential hits, were separated and tested for messenger activity in a wheat germ cell-free system, as well as for the capacity to withdraw coat protein from intact particles. Fragments which stimulated amino acid incorporation were assumed to contain the 5 terminus. Strong evidence was obtained for the location of sites with a high affinity for coat protein near the 3' terminus. The smallest fragment which has the 3'-terminal cytosine comprises only 10% of the length of intact RNA 4 but still possesses these sites. Evidence is presented that the complete coat protein cistron is in the complementing 90% fragment. Possibly, the high-affinity sites are entirely located in the 3'-terminal extracistronic part of RNA 4. They will have the same position in RNA 3 and, possibly, also in the other parts of the genome of alfalfa mosaic virus. The need of this genome for coat protein in order to become infectious may therefore find its explanation in the fact that a conformational change at the 3' ends of the genome parts brought about by the coat protein is required for recognition by the viral replicase.

Medicago sativa↗
PubMed1978

Purification of virus-specific RNA from chicken cells infected with avian sarcoma virus: identification of genome-length and subgenome-leghth viral RNAs.

Avian sarcoma virus (ASV)-specific RNA was purified from ASV-infected cells by using hybridization techniques which employ polydeoxycytidylic acid-elongated DNA complementary to ASV RNA as well as chromatography on polyinosinic acid-Sephadex columns. The purity and nucleotide sequence composition of purified, virus-specific RNA were established by rehybridization experiments and analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. Polyadenylic acid-containing RNA purified from ASV-infected cells contained approximately 1 to 4% virus-specific RNA, compared with 0.06 to 0.15% observed in uninfected cells. Sucrose gradient analysis of virus-specific RNA isolated from ASV-infected cells revealed two major classes of polyadenylated viral RNA with sedimentation values of 36S and 26-28S. Cells infected with transformation-defective ASV (virus containing a deletion of the sarcoma gene) contained 34S and 20-22S viral RNA species. Double-label experiments employing infected cells labeled initially for 48 h with [3H]uridine and then for either 30, 60, or 240 min with [32P]phosphate showed that the intracellular accumulation of genome-length RNA (36S) was significantly faster than that of the 26-28S viral RNA species.

Alpharetrovirus↗
PubMed1978

Influence of a few coat protein subunits on the base-paired structure of the RNA species of alfalfa mosaic virus.

Differentiated thermal melting profiles were made of all the three RNA species (RNAs 1, 2 and 3), constituting the genome of alfalfa mosaic virus and of the subgenomic coat protein messenger RNA (RNA 4) of this virus. Whereas all profiles showed multiphasicity the profile of RNA 4 was most clearly subdivided showing three clear transitions with tm values of 25.5, 35.5 and 53 degrees C. The first transition disappeared upon addition of 6 coat protein molecules per molecule of RNA 4. The effect of coat protein on the melting profiles of the genome RNAs was much less clear.

Capsid↗
PubMed2026

A structural bridge between dengue virus tandem xrRNAs facilitates coordination of exonuclease resistance.

Orthoflavivirus RNA genomes resist host 5'-3' exoribonucleases to produce subgenomic flaviviral RNAs (sfRNAs). This resistance is conferred by exoribonuclease-resistant RNA (xrRNA) structures within the viral 3' untranslated region that often occur in tandem, and whose function can be coupled. In dengue virus serotype 2 (DENV2), this coupling results in changing patterns of sfRNA identity and abundance associated with the ability of the virus to adapt to host vs. vector infections. The physical basis of this coupling was unknown. Using a combination of virology, biochemistry, bioinformatics, structural biology, and biophysics, we explored the structural and sequence determinants of tandem xrRNA coupling in DENV2. We discovered that the spatial proximity, order, and structural integrity of the tandem xrRNAs are all important for coupling. Furthermore, an unpaired A-rich linker that lies between the two xrRNAs is essential in stabilizing a specific structure that correlates to coupling. This A-rich sequence likely forms tertiary contacts with an adjacent stem-loop structure to form a physical bridge between the two xrRNAs, a finding that is supported by a mid-resolution cryo-electron microscopy (cryo-EM) map of the DENV2 tandem xrRNAs. Disruption of the structure of this bridge by mutation changes the relative orientation or spacing between the tandem xrRNAs, which is correlated to their functional coupling. These findings help provide an explanation for the coupling between tandem xrRNAs, suggesting a new mechanistic hypothesis in which the two tandem xrRNAs can simultaneously encounter Xrn1.IMPORTANCEDengue virus (DENV) generates non-coding subgenomic flaviviral RNAs (sfRNAs) that affect several cellular pathways and are important for successful infection. These sfRNAs are formed by structured RNA elements in the viral genome called exoribonuclease-resistant RNAs (xrRNAs), which fold into a distinct three-dimensional topology to block degradation by host cell exoribonucleases and often occur in tandem. Specific patterns of sfRNAs made during infection are important for host vs. vector fitness, and in DENV2, this pattern depends on functional coupling between tandem xrRNAs. However, the source of this functional coupling was unknown. We determined that an unpaired A-rich linker between the tandem xrRNAs is necessary for creating a structural bridge between the tandem xrRNAs. This bridge appears to favor a specific orientation between the tandem xrRNAs that is correlated to coupling and therefore to the patterns and relative abundance of sfRNAs produced during infection.

Dengue Virus↗
PubMed1977

Biological and biochemical characterization of a latent subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture.

The present investigation describes the biological and biochemical properties of a persistent SSPE virus infection. Persistently infected cells were derived by cocultivation of infected brain cells and uninfected Vero cells, and cultures were maintained by normal subculturing methods. No infectious virus was ever released from these cultures, and all attempts to induce infectious virus release were unsuccessful. Biological assays showed that infected cells contained nucleocapsid and salt-dependent hemagglutinin antigens, whereas the normal hemagglutinin appeared not to be present. Electron microscopic examination demonstrated the presence of both intranuclear and cytoplasmic nucleocapsids together with the release of virus particles (defective?) from the cell membrane. Biochemical analysis demonstrated that approximately 90% of the intracellular genomic RNA was defective or subgenomic although a small quantity of infectious genomes was present. It is proposed that the large quantities of defective genomes in the infected cells are the major factor in the maintenance of this persistent infection.

Animals↗