Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71.

BACKGROUND: Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China. It is difficult to distinguish between EV71 and coxsackievirus A16 (CVA16) infections in clinical HFMD patients. Routine laboratory diagnosis of EV71 infection is time-consuming and requires expensive instruments. In this study, we have developed a one-step, single tube, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of EV71. METHODS: Six primers that can recognize 6 distinct regions on the VP2 gene of EV71 were designed for RT-LAMP assay. The amplification was completed by incubating all reagents in a single tube with reverse transcriptase and Bst DNA polymerase under the isothermal condition (60°C) for 60 min, and could be evaluated by using GoldView staining under a handheld ultraviolet torch lamp or electrophoresis analysis. RESULTS: A total of 123 specimens collected from suspicious patients with HFMD were simultaneously detected by RT-LAMP and PCR fluorescence probing assay. The RT-LAMP amplified products containing EV71 were digested by HinfI and TaqI restriction endonucleases; in contrast, non-specific products with CVA16, coxsackievirus A4 and coxsackievirus B3 could not be detected in RT-LAMP assay. Meanwhile, RT-LAMP assay could amplify EV71 virus with a detection limit of 1 PFU/ml within 60 min. Compared with PCR fluorescence probing assay, RT-LAMP assay exhibited 98.4% identity during the detection of EV71 viral RNA without the missing of positive samples. CONCLUSION: Our results indicated that RT-LAMP is a rapid, sensitive, specific and accurate method for the detection of EV71 in clinical specimens. Therefore, this developed method has potential application for rapid and comprehensive surveillance for EV71 infection, especially in developing country.

An enhanced isothermal amplification assay for viral detection.

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

Detection of a human intracisternal A-type retroviral particle antigenically related to HIV.

Sjögren's syndrome is an autoimmune disease that is characterized by dryness of the mouth and eyes. The loss of salivary and lacrimal gland function is accompanied by lymphocytic infiltration. Because similar symptoms and glandular pathology are observed in certain persons infected with human immunodeficiency virus (HIV), a search was initiated for a possible retroviral etiology in this syndrome. A human intracisternal A-type retroviral particle that is antigenically related to HIV was detected in lymphoblastoid cells exposed to homogenates of salivary tissue from patients with Sjögren's syndrome. Comparison of this retroviral particle to HIV indicates that they are distinguishable by several ultrastructural, physical, and enzymatic criteria.

Effects of Poly(1-vinyluracil) and Poly(9-vinyladenine) on viral RNA-directed DNA polymerase.

The effects of poly(1-vinyluracil) [poly(vU)] and poly(9-vinyladenine) [poly(vA)] on the RNA-dependent DNA polymerase activity of murine leukemia virus (Moloney strain) were studied. Vinyl polymers themselves cannot act as templates for the polymerase. However, if a vinyl polymer is added to a polymerase reaction mixture in which a complementary polynucleotide serves as the template, the reaction is inhibited: thus with polyribocytidylic acid as template and oligodeoxyguanylic acid as primer, neither poly(vU) nor poly(vA) had a significant effect; when polyribouridylic acid was used as template and oligodeoxyadenylic acid as primer, poly(vA) inhibited polymerase activity while poly(vU) had little effect; when polyriboadenylic acid was a template and oligodeoxy thymidylic acid was a primer, poly(vU) was an inhibitor. Complex effects were noted with the latter system and poly(vA); either stimulation or inhibition of the reaction was observed, depending on the concentration of poly(vA). The stimulation brings about a decrease in the amount of lower-molecular-weight materials in the product and is caused by the interaction of poly(vA) with the template-primer. Thus vinyl polymers differ from polynucleotides in their mechanism of inhibition of viral polymerase, since the latter inhibit the enzyme by binding to it.

Presence of DNA polymerase in lymphosarcoma in northern pike (Esox lucius).

Northern poke lymphosarcoma DNA polymerase was partially purified from particulate fractions banding at 1.15 to 1.16 g/ml from homogenates prepared from frozen necropsies of tumor-bearing pike. The enzyme behaves as a typical reverse transcriptase, in that it prefers ribotemplates to deoxytemplates. The isoelectric point (pl 5.5) is similar to that of avian myeloblastosis virus polymerase. The pike enzyme elutes from a phosphocellulose column at 0.22 M potassium phosphate, the same as avian myeloblastosis virus DNA polymerase. The enzyme activity is inhibited by pyran, a specific inhibitor of viral DNA polymerases. The most striking difference between the pike lymphoma polymerase and the other viral DNA polymerases tested is the low maximum temperature of 20 degrees, compared to 30 degrees for Rauscher leukemia virus polymerase and 38 degrees for avian myeloblastosis virus and Rous sarcoma virus.

Ionophorous polymers. Interaction with polynucleotides and effects on RNA-directed DNA polymerase activity.

Poly(vinylbenzo-18-crown-6), a water-soluble polymer endowed with ion-binding crown moieties as pendent groups, forms insoluble complexes with polyadenylate in the presence of K+; the corresponding monomeric benzo-18-crown-6, does not form a precipitate under the same conditions. In the presence of Na+ and Mn2+ which in aqueous solution complex weakly to crown compounds, no coprecipitation of the crown polymer and polyadenylate occurs; nevertheless, the crown polymer strongly binds to immobilized polyadenylate even under these conditions. The interactions of crown polymer with the poly-nucleotide result in a loss of templating ability of the latter. Using RNA-dependent DNA polymerase of murine leukemia virus it was found that (1) enzymatic action is efficiently inhibited even in the absence of ions which coprecipitate crown polymer and template, (2) inhibition is reversed by addition of excess polynucleotide and (3) monomeric crown does not inhibit the reaction.

Effects of polyethylene glycol on reverse transcriptase and other polymerase activities.

Polyethylene glycol enhances reverse transcription, augmenting both the rate and duration of polymerization. The effective mean molecular weight of polyethylene glycol is 6000 and the optimal concentration is 12% (w/w). Polyethylene glycol is effective on the reverse transcriptase reaction of all ten type B, C, and D viruses tested under a variety of exogenous, endogenous, and reconstitution assay systems, including the highly efficient conditions involving calf thymus DNA oligonucleotide primers. By three methods of synthesis, polyethylene glycol increased the yields of complementary [3H]DNA by a factor of 1.8--6.5. Polyethylene glycol does not alter the divalent cation requirements of the specificities of the enzyme. Complementary [3H]DNAs made in the presence of polyethylene glycol are indistinguishable in terms of size and sequence complementarity from those made in the absence of the polymer. The stimulatory effect was partly due to the ability of polyethylene glycol to stabilize reverse transcriptase. Preliminary tests indicate that polyethylene glycol also stimulates other nucleotide polymerases, such as the DNA-dependent DNA and RNA polymerases of Escherichia coli and the terminal transferase of calf thymus.

Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process.

The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.

Kinetic analysis of the catalysis of strand transfer from internal regions of heteropolymeric RNA templates by human immunodeficiency virus reverse transcriptase.

The kinetic mechanism of HIV reverse transcriptase catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142 nucleotide RNA template (donor), primed with a specific 20 nucleotide DNA oligonucleotide that was used to initiate synthesis. An RNA with homology to an internal region of the donor was used as acceptor template. Using 32P-labeled DNA oligonucleotide, the primer-extension products made from full-length synthesis on the donor (108 bases in length) or homologous transfer to and extension on the acceptor (155 bases) were monitored. Results indicated that the maximum efficiency of transfer (the ratio of transfer products to donor-directed+transfer products x 100) in this particular system was about 25% while the theoretical Vmax for the rate of appearance of transfer products at infinite acceptor concentration was about 20-fold lower than the measured rate for full-length donor-directed products. The Km for acceptor template in the transfer reaction was about 8 nM. Experiments using the above donor template hybridized to a specific DNA that has been shown to transfer to the acceptor indicated that RNase H-mediated rapid release of this DNA from the donor while subsequent association with the acceptor was relatively slow.

Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain.

Reverse transcriptase (RT) serves as the replicative polymerase for retroviruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.

Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA.

Bovine tRNA(Trp) can be partially hybridized to the avian myeloblastosis virus (AMV) 35 S RNA at 37 degrees C, in the presence of AMV RNA-dependent DNA polymerase (reverse transcriptase). This template-primer complex is active in the synthesis of viral cDNA. The size of the cDNA products synthesized in the in vitro reconstituted AMV system was determined by urea-polyacrylamide gel electrophoresis using a tRNA labelled at the 3'-end by yeast tRNA nucleotidyl transferase. The synthesized cDNA has a size of about 100 nucleotides and was shown by Southern blotting to be complementary to a specific sequence of the 5'-end of the retroviral genome. These results indicate that reverse transcriptase is able to anneal the exogenous primer tRNA at the 'primer-binding site' near the 5'-end of the long terminal repeat (LTR) of AMV RNA.

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV-1 reverse transcriptase: tRNA(Lys,3) activation.

HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.

Inactivation of human immunodeficiency virus by Betadine products and chlorhexidine.

Eleven povidone-iodine-containing products (Betadine) and chlorhexidine gluconate solution were tested for their ability to inactivate human immunodeficiency virus (HIV) in a cell culture system. All Betadine products completely inactivated the virus at povidone-iodine concentrations of greater than or equal to 0.5% (10- to 20-fold dilutions of stock) except for Betadine Lubricating Antiseptic Gel, which required 2.5% for efficacy (1:2 dilution). Chlorhexidine gluconate completely inactivated HIV at concentrations of greater than or equal to 0.2% (1:100 dilution of laboratory stock; 1:20 dilution of commercial stock). Betadine douche and medicated douche did not inactivate HIV at the concentrations recommended for clinical use (0.33% and 0.25%, respectively) but were effective at povidone-iodine concentrations of 0.5%. Inactivation appeared to be immediate since no difference in efficacy based on length of exposure to the microbicide was detected. Thus, both microbicides are highly effective at killing HIV in vitro.

Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus.

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A,1 rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A280 units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.

The ribonuclease H activity of HIV-1 reverse transcriptase: further biochemical characterization and search of inhibitors.

A recombinant homodimer p66/p66 of the HIV-1 reverse transcriptase (RT) was expressed in and purified from a protease-deficient strain of the yeast Saccharomyces cerevisiae. The RNase H activity associated with the homodimer was biochemically characterized. The effect of cations and the hybrid substrate specificity were studied. Some compounds which have been found to inhibit retroviral replication were tested as potential inhibitors of the retroviral DNA polymerase and RNase H activities. Most of these compounds inhibited preferentially the DNA polymerase activity. On the other hand, only suramin was found to inhibit RNase H more efficiently than DNA polymerase. As in the case of the DNA polymerase activity, the thiol-reacting agent N-ethylmaleimide (NEM) did not affect the RNAse H activity of HIV RT. When the effect of NEM was tested against E coli RNase H, a weak inhibitory effect was detected. Surprisingly, NEM strongly inhibits the same bacterial RNase H in the presence of a recombinant form of HIV RT devoid of nuclease activity. These results strongly suggest an interaction between E coli RNase H and HIV-1 RT.

Inhibition of HIV replication by liposomal encapsulated amphotericin B.

This report shows the potential of using a liposomal encapsulated preparation of amphotericin B (a polyene macrolide antibiotic) for the in vitro inhibition of HIV. There was no significant difference between the effective doses of the free form of drug when compared to the liposomal encapsulated preparation in inhibiting the growth of HIV. Virus expression was suppressed at a concentration of 5-10 micrograms/ml of the drugs. The liposomal preparation showed greatly reduced cytotoxicity in experiments using cultures of murine leukocytes. These results show the potential usefulness of liposomal encapsulated drugs in the treatment of patients with AIDS or AIDS related complex.

Sulfonic acid polymers as a new class of human immunodeficiency virus inhibitors.

Four sulfonic acid polymers [poly(4-styrenesulfonic acid)(PSS), poly(anetholesulfonic acid)(PAS), poly(vinylsulfonic acid)(PVS), poly(2-acrylamido-2-methyl-1-propanesulfonic acid)(PAMPS)] have been found to inhibit the cytopathicity of HIV-1 and HIV-2 in MT-4 cells at concentrations that are not toxic to the host cells. The sulfonic acid polymers also inhibited syncytium formation in co-cultures of MOLT-4 cells with HIV-1- or HIV-2-infected HUT-78 cells. They also inhibited binding of anti-gp120 mAb to HIV-1 gp120 and blocked adsorption of HIV-1 virions to MT-4 cells. PSS and PAS, but not PVS and PAMPS, interfered with the binding of OKT4A/Leu3a to the CD4 receptor. The anti-HIV activity of these polyanionic compounds can be ascribed to inhibition of the gp120-CD4 interaction. Sulfonic acid polymers represent a lead of anti-HIV compounds that warrant further evaluation of their therapeutic potential.

A solid phase reverse transcriptase micro-assay for the detection of human immunodeficiency virus and other retroviruses in cell culture supernatants.

A simple and rapid solid-phase reverse transcriptase assay was developed based on the use of poly(rA):oligo(dT)12-18 as template primer immobilized on DEAE cellulose paper squares to detect human immunodeficiency virus (HIV) and/or other retroviruses in cell culture supernatants. It was found that PEG (per se) -up to 4% concentrations (w/v)--did not inhibit reverse transcriptase activity. Optimal conditions of the assay were determined. This solid-phase technique is much faster and more convenient than the methods described previously.

A versatile method for the removal of melanin from ribonucleic acids in melanocytic cells.

Melanin pigments often co-purify during preparation of nucleic acids from cells or tissues of melanocytic origin. Contaminating melanin can severely impede subsequent analyses of RNA. We attempted to eliminate melanin in RNA preparations using selected gel matrices. We show here that co-purified melanin pigments can be largely eliminated from RNA samples after passing through polyacrylamide-based beads (Bio-Gel P-60). After isolation from the pigment-containing cells or tissues, RNA was subsequently processed through batch or column purification under acidic pH conditions. The resulting RNA was devoid of contaminating melanin pigments and amenable to molecular reactions such as polymerase chain reaction and cDNA synthesis by reverse transcriptase. Although the process results in some loss of input RNA, this purification procedure is simple, robust and can easily be adopted in any laboratory for the molecular analysis of RNA that requires removal of melanin contamination.

A rapid method for the large scale preparation of bovine leukemia virus antigen.

Bovine leukemia virus (BLV) is the etiologic agent responsible for enzootic bovine leukosis. Detection of cattle seropositive for BLV and laboratory studies of BLV require the preparation of large quantities of BLV antigen. A convenient and economical method for concentrating virus from large volumes of cell culture medium involves the precipitation of the virus in the cold by polyethylene glycol (PEG). The present work demonstrates the feasibility of rapidly precipitating BLV with 10% PEG and subsequently separating the resuspended virus from the residual PEG on a disposable desalting column.