Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real- time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 ℃ isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36⌬50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta in trans in BAC36⌬50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36⌬50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36⌬50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36⌬50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication.

Genomewide analyses of the mammalian transcriptome have revealed that large tracts of sequence previously annotated as noncoding are frequently transcribed and give rise to stable RNA. Although the transcription of individual genes of the Kaposi's sarcoma-associated herpesvirus (KSHV) has been well studied, little is known of the architecture of the viral transcriptome on a genomewide scale. Here we have employed a genomewide tiling array to examine the lytic transcriptome of the Kaposi's sarcoma-associated herpesvirus, KSHV. Our results reveal that during lytic growth (but not during latency), there is extensive transcription from noncoding regions, including both intergenic regions and, especially, noncoding regions antisense to known open reading frames (ORFs). Several of these transcripts have been characterized in more detail, including (i) a 10-kb RNA antisense to the major latency locus, including many of its microRNAs as well as its ORFs; (ii) a 17-kb RNA antisense to numerous ORFs at the left-hand end of the genome; and (iii) a 0.7-kb RNA antisense to the viral homolog of interleukin-6 (vIL-6). These studies indicate that the lytic herpesviral transcriptome resembles a microcosm of the host transcriptome and provides a useful system for the study of noncoding RNAs.

Cardiovascular diseases, especially ones involving narrowed or blocked blood vessels with diameters smaller than 6 millimeters, are the leading cause of death globally.

Amplification of the human cytomegalovirus (HCMV) lytic origin (oriLyt) in human fibroblasts is dependent upon six core replication proteins and UL84, IE2, and UL36-38. Using a telomerase-immortalized human fibroblast cell line (T-HFs), oriLyt-dependent DNA replication no longer required the gene products of UL36-38. To determine the role of IE2 in DNA replication in human fibroblasts, we examined potential IE2-binding sites within HCMV oriLyt. We now show that a strong bidirectional promoter (oriLyt PM ) (nucleotides 91754 to 92030) is located in the previously identified core region of the origin and is required for efficient amplification of oriLyt. It was determined that a 14-bp novel DNA motif (oriLyt promoter activation element), which was initially identified as a binding element for the immediate-early protein IE2, was essential for oriLyt PM activity. In Vero cells the oriLyt PM was constitutively active and strongly repressed by IE2, but it was reactivated by UL84. In contrast, transfection of the oriLyt PM into human fibroblasts resulted in a very low basal level of promoter activity that was dramatically up-regulated upon infection with HCMV. Cotransfection assays demonstrated that the transfection of UL84 along with IE2 transactivated the oriLyt PM in human fibroblasts. Further activation was observed upon cotransfection of the set of plasmids expressing the entire replication complex. Efficient oriLyt amplification in the absence of IE2 in human fibroblasts was observed by replacing the oriLyt PM with the simian virus 40 early promoter. Under these conditions, however, UL84 was still required for amplification of oriLyt. These results suggest that the mechanism of initiation of HCMV lytic replication in part involves transcriptional activation.Human cytomegalovirus (HCMV) contains a single lytic origin for DNA replication, oriLyt, located near the center of the U L region and upstream of the open reading frame (ORF) encoding the single-stranded DNA-binding protein ppUL57 (1,19,40). The HCMV oriLyt is remarkable among viral replication origins for its apparent size and complexity. The entire oriLyt region of HCMV is located from nucleotides (nt) 90500 to 93930 and is composed of a core (nt 91751 to 93299) which contains two essential regions (1,40,66). These essential regions (I and II) contain a pyrimidine-rich sequence (Y-block), various reiterated sequences, several transcription factor-binding sites, direct and inverted repeat sequences, base composition biases and strand asymmetries, and RNA-DNA hybrid structures and is a site of active transcription (the small replication transcript [SRT]) (1,22,40,49,66). Despite the apparent exhaustive investigation of elements within oriLyt that contribute to DNA synthesis, few data exist with respect to the actual function of these elements in initiation of DNA replication.Some information concerning the initiation of DNA synthesis can be inferred from the required virus-encoded transacting factors necessary to replicate oriLyt. In human fibroblasts, oriLy...

We used a transient-transfection replication assay to identify two functional copies of the human herpesvirus 8 (HHV8) lytic origin of DNA replication (oriLyt). BCLB-1 cells were transfected with HHV8 subgenomic fragments containing the putative lytic origin along with a plasmid expressing viral transactivator open reading frame (ORF) 50. The HHV8 left-end oriLyt (oriLyt-L) lies between ORFs K4.2 and K5 and is composed of a region encoding various transcription factor binding sites and an A؉T-rich region and a G؉C repeat region. The right-end oriLyt (oriLyt-R) maps between ORF 69 and vFLIP, a region similar to the RRV oriLyt, and is an inverted duplication of oriLyt-L.Human herpesvirus 8 (HHV8) is a gamma-2 class herpesvirus related to Epstein-Barr virus (EBV). HHV8 is the probable cause of Kaposi's sarcoma and is involved in the pathogenesis of primary effusion lymphoma and multicentric Castleman's disease (1-4). HHV8 appears to be distinct from EBV in that it encodes a number of genes of cellular origin (20-22, 27, 28).HHV8 is typically latent in cultured B cells, and only a small number of cells undergo spontaneous lytic reactivation (18,19,26). In cell culture, HHV8 can be induced to enter the lytic cycle and produce infectious virus by treatment with tetradecanoyl phorbol acetate (TPA) and/or n-butyrate (26). In addition, transfection of a plasmid that expresses the viral transactivator open reading frame (ORF) 50 is sufficient for induction of the viral lytic cycle (9,17,33,34). Induction of the viral lytic cycle consists of the expression of viral enzymes that participate in replication of the viral genome, resulting in an increase in viral DNA and production of infectious virus (9, 33). HHV8 ORF 50 appears to have a function similar to that of the EBV viral transactivator BZLF1 or Zta with respect to induction of the viral lytic cycle. Zta is a key viral protein that participates in reactivation and initiation of lytic EBV DNA replication (5,6,8,12,15,29).Recently, it was shown that the six core replication proteins for HHV8 are sufficient for replication of the cloned EBV origin of replication (36). In addition, a replication complex containing the HHV8 K8 protein was shown to colocalize with promyelocytic leukemia protein oncogenic domains (PODs) in lytic-cycle-induced or HHV8-infected dermal microvascular endothelial cells (11). Despite this recent elucidation of the core transacting factors, the discovery of a lytic origin for HHV8 has not been discovered.The primarily latent nature of HHV8 in cell culture makes studies concerning the cis-acting lytic factors for this virus somewhat difficult. For this reason, we initially chose to examine the lytic replication machinery of the closely related virus rhesus rhadinovirus (RRV). In the RRV system, lytic replication occurs upon infection of rhesus fibroblasts (24, 32). The lytic origin for RRV is located at the right end of the genome and consists of a number of GϩC-and AϩT-rich repeat regions, along with various transcription factor binding sites, and m...

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.A common feature of herpesvirus lytic DNA replication is the requirement for six core replication proteins. These proteins-a DNA polymerase, helicase, primase, primase-associated factor, DNA processivity factor, and a single-stranded DNA-binding protein-carry out the necessary enzymatic processes to efficiently synthesize DNA (11,21,23). These enzymatic replication events follow the obligatory initiation of DNA synthesis that is mediated by proteins that are unique to each herpesvirus system and have been traditionally referred to as initiator proteins or origin-binding proteins (OBPs). In some cases, these initiator proteins themselves have an inherent catalytic property that may unwind a specific region of DNA and facilitate the assembly of the entire core replication machinery.Although the exact mechanism by which these OBPs initiate DNA synthesis has not been elucidated, several common features have been defined. (i) It appears that all characterized initiator proteins interact directly or indirectly with DNA within the origin of lytic replication. (ii) All identified initiator proteins have been shown to interact with themselves in transfected cells and contain leucine zipper motifs (5,7,8,14,15). Stable self-interaction of these proteins is seemingly necessary for their intrinsic enzymatic activity and may serve to regulate the initiation of DNA synthesis.For HCMV, the transient-cotransfection replication as...

The mammalian transcriptome is studded with putative noncoding RNAs, many of which are antisense to known open reading frames (ORFs). Roles in the regulation of their complementary mRNAs are often imputed to these antisense transcripts, but few have been experimentally examined, and such functions remain largely conjectural. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two transcripts that lack obvious ORFs and are complementary to the gene (RTA) encoding the master regulator of the latent/lytic switch. Here, we show that, contrary to expectation, these RNAs do not regulate RTA expression. Rather, they are found on polysomes, and genetic analysis indicates that translational initiation occurs at several AUG codons in the RNA, leading to the presumptive synthesis of peptides of 17 to 48 amino acids. These findings underscore the need for circumspection in the computational assessment of coding potential and raise the possibility that the mammalian proteome may contain many previously unsuspected peptides generated from seemingly noncoding RNAs, some of which could have important biological functions. Irrespective of their function, such peptides could also contribute substantially to the repertoire of T cell epitopes generated in both uninfected and infected cells.