Stranded RNA Genome Research Articles

Role of Culex and Anopheles mosquito species as potential vectors of rift valley fever virus in Sudan outbreak, 2007

BackgroundRift Valley fever (RVF) is an acute febrile arthropod-borne viral disease of man and animals caused by a member of the Phlebovirus genus, one of the five genera in the family Bunyaviridae. RVF virus (RVFV) is transmitted between animals and human by mosquitoes, particularly those belonging to the Culex, Anopheles and Aedes genera.MethodsExperiments were designed during RVF outbreak, 2007 in Sudan to provide an answer about many raised questions about the estimated role of vector in RVFV epidemiology. During this study, adult and immature mosquito species were collected from Khartoum and White Nile states, identified and species abundance was calculated. All samples were frozen individually for further virus detection. Total RNA was extracted from individual insects and RVF virus was detected from Culex, Anopheles and Aedes species using RT-PCR. In addition, data were collected about human cases up to November 24th, 2007 to asses the situation of the disease in affected states. Furthermore, a historical background of the RVF outbreaks was discussed in relation to global climatic anomalies and incriminated vector species.ResultsA total of 978 mosquitoes, belonging to 3 genera and 7 species, were collected during Sudan outbreak, 2007. Anopheles gambiae arabiensis was the most frequent species (80.7%) in White Nile state. Meanwhile, Cx. pipiens complex was the most abundant species (91.2%) in Khartoum state. RT-PCR was used and successfully amplified 551 bp within the M segment of the tripartite negative-sense single stranded RNA genome of RVFV. The virus was detected in female, male and larval stages of Culex and Anopheles species. The most affected human age interval was 15-29 years old followed by ≥ 45 years old, 30-44 years old, and then 5-14 years old. Regarding to the profession, housewives followed by farmers, students, shepherd, workers and the free were more vulnerable to the infection. Furthermore, connection between human and entomological studies results in important human case-vulnerability relatedness findings.ConclusionModel performance, integrated with epidemiologic and environmental surveillance systems should be assessed systematically for RVF and other mosquito-borne diseases using historical epidemiologic and satellite monitoring data. Case management related interventions; health education and vector control efforts are extremely effective in preparedness for viral hemorrhagic fever and other seasonal outbreaks.

Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

Activation of the latent kinase PKR is a potent innate defense reaction of vertebrate cells towards viral infections, which is triggered by recognition of viral double-stranded (ds) RNA and results in a translational shutdown. A major gap in our understanding of PKR's antiviral properties concerns the nature of the kinase activating molecules expressed by influenza and other viruses with a negative strand RNA genome, as these pathogens produce little or no detectable amounts of dsRNA. Here we systematically investigated PKR activation by influenza B virus and its impact on viral pathogenicity. Biochemical analysis revealed that PKR is activated by viral ribonucleoprotein (vRNP) complexes known to contain single-stranded RNA with a 5′-triphosphate group. Cell biological examination of recombinant viruses showed that the nucleo-cytoplasmic transport of vRNP late in infection is a strong trigger for PKR activation. In addition, our analysis provides a mechanistic explanation for the previously observed suppression of PKR activation by the influenza B virus NS1 protein, which we show here to rely on complex formation between PKR and NS1's dsRNA binding domain. The high significance of this interaction for pathogenicity was revealed by the finding that attenuated influenza viruses expressing dsRNA binding-deficient NS1 proteins were rescued for high replication and virulence in PKR-deficient cells and mice, respectively. Collectively, our study provides new insights into an important antiviral defense mechanism of vertebrates and leads us to suggest a new model of PKR activation by cytosolic vRNP complexes, a model that may also be applicable to other negative strand RNA viruses.

Antiviral effect of ribonuclease conjugated oligodeoxynucleotides targeting the IRES RNA of the hepatitis C virus

Hepatitis C virus (HCV) translation initiation is mediated by a highly structured and conserved RNA, termed the Internal Ribosome Entry Site (IRES), located at the 5′-end of its single stranded RNA genome. It is a key target for the development of new antiviral compounds. Here we made use of the recently developed HCV cell culture system to test the antiviral activity of artificial ribonucleases consisting of imidazole(s) linked to antisense oligodeoxynucleotides targeting the HCV IRES. Results from the cell culture model indicate that the naked antisense oligodeoxynucleotide displayed an efficient antiviral activity. Despite the increased activity observed with the addition of imidazole moieties when tested with the cell-free system, it appears that these improvements were not reproduced in the cellular model.

Properties and use of novel replication-competent vectors based on Semliki Forest virus

BackgroundSemliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important.ResultsWe analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture.ConclusionThe properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.

Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.

Molecular characterization of Lolium latent virus, proposed type member of a new genus in the family Flexiviridae

Lolium latent virus (LoLV) was recently detected in the USA for the first time in ryegrass hybrids (Lolium perenne x Lolium multiflorum). The genome of one USA isolate, LoLV-US1, has now been fully sequenced. The positive strand genomic RNA is 7674 nucleotides (nt) long and is organized in five open reading frames (ORFs) encoding the replication-associated protein, the movement-associated triple gene block proteins and the coat protein (CP). The genome organization is similar to that of viruses in the genera Potexvirus and Foveavirus; however, analysis of the complete LoLV genomic sequence, phylogenetic analyses of the deduced amino acid (aa) sequences of the polymerase and the CP, presence of a putative ORF 6, and the in vivo detection of two CPs in equimolar amounts, highlight features peculiar to LoLV. These characteristics indicate that LoLV forms a monotypic group separate from existing genera and unassigned species within the family Flexiviridae, for which we propose the genus name Lolavirus. One-step RT-PCR was developed for quick and reliable LoLV detection.

Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity

The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.

Nucleotidylylation of the VPg protein of a human norovirus by its proteinase-polymerase precursor protein

Caliciviruses have a positive strand RNA genome covalently-linked at the 5'-end to a small protein, VPg. This study examined the biochemical modification of VPg by the ProPol form of the polymerase of human norovirus strain MD145 (GII.4). Recombinant norovirus VPg was shown to be nucleotidylylated in the presence of Mn 2+ by MD145 ProPol. Phosphodiesterase I treatment of the nucleotidylylated VPg released the incorporated UMP, which was consistent with linkage of RNA to VPg via a phosphodiester bond. Mutagenesis analysis of VPg identified Tyrosine 27 as the target amino acid for this linkage, and suggested that VPg conformation was important for the reaction. Nucleotidylylation was inefficient in the presence of Mg 2+; however the addition of full- and subgenomic-length MD145 RNA transcripts led to a marked enhancement of the nucleotidylylation efficiency in the presence of this divalent cation. Furthermore, evidence was found for the presence of an RNA element near the 3'-end of the polyadenylated genome that enhanced the efficiency of nucleotidylylation in the presence of Mg 2+.

In vitro antitumor and antiviral activities of new benzothiazole and 1,3,4-oxadiazole-2-thione derivatives

A series of new benzothiazole derivatives 6a-h have been synthesized, in five steps, from substituted phenols via the 1,3,4-oxadiazole-2-thiones 5a-h. The in vitro antitumor activity of the compounds obtained was investigated and the benzothiazol derivatives 6d and 6e showed strong effects on leukaemia cell lines CCRF-CEM (CC50=12+/-2 micromol L(-1), 8+/-1 micromol L(-1), respectively). These compounds are leading candidates for further development. The title compounds were tested against representatives of several virus families containing single stranded RNA genomes, either positive-sense (ssRNA+), or negativesense (RNA-), and against double-stranded RNA genomes (dsRNA), as well as some Flaviviridae viruses.

Anti-hepatitis C Virus Activity of Tamoxifen Reveals the Functional Association of Estrogen Receptor with Viral RNA Polymerase NS5B

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. HCV genome replication occurs in the replication complex (RC) around the endoplasmic reticulum membrane. However, the mechanisms regulating the HCV RC remain widely unknown. Here, we used a chemical biology approach to show that estrogen receptor (ESR) is functionally associated with HCV replication. We found that tamoxifen suppressed HCV genome replication. Part of ESRalpha resided on the endoplasmic reticulum membranes and interacted with HCV RNA polymerase NS5B. RNA interference-mediated knockdown of endogenous ESRalpha reduced HCV replication. Mechanistic analysis suggested that ESRalpha promoted NS5B association with the RC and that tamoxifen abrogated NS5B-RC association. Thus, ESRalpha regulated the presence of NS5B in the RC and stimulated HCV replication. Moreover, the ability of ESRalpha to regulate NS5B was suggested to serve as a potential novel target for anti-HCV therapeutics.

Molecular cloning, sequence analysis and expression of genome segment 7 (S7) of Antheraea mylitta cypovirus (AmCPV) that encodes a viral structural protein

The Genome segment 7 (S7) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus (AmCPV) was converted to cDNA, cloned and sequenced. The nucleotide sequence showed that segment 7 consisted of 1789 nucleotides with an ORF of 530 amino acids and could encode a protein of approximately 61 kDa, termed P61. The 5' terminal sequence, AGTAAT and the 3' terminal sequence, AGAGC of the plus strand was found to be the same as genome segment 10 of AmCPV encoding polyhedrin. No sequence similarity was found by searching nucleic acid and protein sequence databases using BLAST. The secondary structure prediction showed the presence of 17 alpha-helices, 18 extended beta-sheets along the entire length of P61. The ORF of segment 7 was expressed in E. coli as His-tagged fusion protein, purified through Ni-NTA chromatography, and polyclonal antibody was raised in rabbit indicating that P61 is immunogenic. Immunoblot analysis using this antibody on viral infected cells as well as purified polyhedra showed that P61 is a viral structural protein. Motif scan search showed some similarity of P61 with Inosine monophosphate dehydrogenase (IMPDH) cystathionine-beta-synthase (CBS) domain at the C-terminus and it was hypothesized that by binding to single stranded viral RNA through its CBS domain P61 may help in virus replication or transcription.

Dynamic force microscopy imaging of plasmid DNA and viral RNA

Plasmid DNA and viral RNA were imaged in a liquid environment by dynamic force microscopy (DFM) and fine structures of DNA with heights of 1.82±0.66 nm were obtained in topographical images. In simultaneously acquired phase images, DNA could be imaged with better contrast at lower imaging forces. By splitting the cantilever oscillation signal into lower and upper parts, the contribution of the adhesion between tip and sample to the topographical images was eliminated, resulting in better signal-to-noise ratio. DFM of the single stranded RNA genome of a human rhinovirus showed loops protruding from a condensed RNA core, 20–50 nm in height. The mechanical rigidity of the RNA was determined by single molecule pulling experiments. From fitting RNA stretching curves to the Worm-Like-Chain (WLC) model a persistence length of 1.0±0.17 nm was obtained.

Molecular Insights into the Mechanisms of HIV-1 Reverse Transcriptase Resistance to Nucleoside Analogs

The causative agent of acquired immunodeficiency syndrome, HIV-1, depends on one of its enzymes, reverse transcriptase, to copy its single stranded RNA genome into a double stranded DNA nucleic acid suitable for integration in the host cell genome. In the last two decades, the advances in the knowledge of the kinetic mechanism of reverse transcription and in the determination of the crystallographic structures for the complexes of the enzyme with substrates and products were huge. However, all of this knowledge resulted in the design of RT inhibitors for which the virus, after a short period of exposure, becomes less susceptible, due to the development of resistance. The development of resistance is caused by the high frequency of viral mutation and the toxicity of those same drugs. Therefore, a closer look at all the available information might shed some light into this subject and help to develop new strategies to overcome the lack of long term clinical efficiency of these drugs. Here, we present a critical atomic level study of all the mutations that have been detected and reported so far, as a reaction of the enzyme to counteract the action of the inhibitors.

Molecular epidemiology of human picobirnaviruses among children of a slum community in Kolkata, India

Picobirnaviruses are a group of unclassified, non-enveloped, small spherical viruses, 35–41 nm in diameter without any apparent surface morphology. They have characteristic bisegmented double stranded RNA genome of two types namely large profile (2.3–2.6 kbp for the larger and 1.5–1.9 kbp for the smaller segment, respectively) or small profile (1.75 and 1.55 kbp for segments 1 and 2, respectively). Human picobirnaviruses ( n = 12 positives; 2/56 diarrhoeic children and 10/607 non-diarrhoeic children) with large ( n = 11) or small ( n = 1) genome pattern were observed in faecal specimens of children from a slum community by silver stained PAGE gels. Faecal specimen from four asymptomatic cases (P597_02_IND, K135_02_IND, A373_03_IND, A356_03_IND) and one diarrhoeic case (K135_03_IND) had genogroup I picobirnaviruses (1-CHN-97 like) showing amplicons within the 201 bp region, with primers PicoB25–PicoB43, targeting the conserved domain of RNA-dependent RNA polymerase (RdRp) gene. It was interesting to note that only the PBV strain P597_02_IND from Kolkata with large genome was closely related to a reported strain (similarity with 2-GA-91 from USA was 87% at the nucleotide level and 90% at the amino acid level). Sequence analysis showed three conserved amino acid domains as well as a highly conserved D-S—D motif, characteristic of RNA-dependent RNA polymerase gene of bisegmented, double stranded RNA viruses. Sequence data of the picobirnavirus A356_03_IND indicated strong heterogeneity with all other picobirnavirus strains sequenced till date. After nearly a decade a genogroup II picobirnavirus strain (R227_03_IND) was isolated from a diarrhoea case in the community, with small genome profile and amplified with specific primers PicoB23–PicoB24; but the sequence data showed that it was divergent from the hitherto reported prototype strain 4-GA-91 of genogroup II human picobirnaviruses.

A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame

BackgroundHepatitis C virus (HCV) is a pathogenic hepatic flavivirus with a single stranded RNA genome. It has a high genetic variability and is classified into six major genotypes. Genotype 1a and 1b cause the majority of infections in the USA. Viral genomic sequence information is needed to correlate viral variation with pathology or response to therapy. However, reverse transcription-polymerase chain reaction (RT-PCR) of the HCV genome must overcome low template concentration and high target sequence diversity. Amplification conditions must hence have both high sensitivity and specificity yet recognize a heterogeneous target population to permit general amplification with minimal bias. This places divergent demands of the amplification conditions that can be very difficult to reconcile.ResultsRT and nested PCR conditions were optimized independently and systematically for amplifying the complete open reading frame (ORF) from HCV genotype 1a and 1b using several overlapping amplicons. For each amplicon, multiple pairs of nested PCR primers were optimized. Using these primers, the success rate (defined as the rate of production of sufficient DNA for sequencing with any one of the primer pairs for a given amplicon) for amplification of 72 genotype 1a and 1b patient plasma samples averaged over 95% for all amplicons. In addition, two sets of sequencing primers were optimized for each genotype 1a and 1b. Viral consensus sequences were determined by directly sequencing the amplicons. HCV ORFs from 72 patients have been sequenced using these primers. Sequencing errors were negligible because sequencing depth was over 4-fold and both strands were sequenced. Primer bias was controlled and monitored through careful primer design and control experiments.ConclusionOptimized RT-PCR and sequencing conditions are useful for rapid and reliable amplification and sequencing of HCV genotype 1a and 1b ORFs.

Ultrastructure of maize necrotic streak virus infections.

Maize necrotic streak virus (MNeSV) has 32 nm isometric particles that encapsidate a single stranded RNA genome ca. 4.3 kb in size, and is a tentative member of the genus Tombusvirus. Since tombusviruses elicit the formation of cytopathological structures known as “multivesicular bodies” that assist in their classification at the genus level, the cytology of thin-sectioned maize leaf tissues 7, 15, and 45 days after inoculation was investigated. The prominent cytopathological features were: (i) an extensive accumulation of virus particles scattered throughout the cytoplasm or in crystalline arrays in the cytoplasm or, occasionally, in intercellular spaces; (ii) extensive plasmolysis and the appearance of small patches of amorphous, dark-staining material in the cytoplasm; (iii) the proliferation of membranes and the presence of empty double membrane-bound vesicular structures of various sizes. No apparent connection between these membranous elements and the nuclear envelope was detected, even when large clusters of vesicles were adpressed to the nuclei. Some of the largest double-membrane bound structures had rows of small vesicles located inside peripheral dilations of the bounding membrane. Although most of these structures appeared to originate from disintegrating mitochondria, they bore little resemblance to genuine multivesicular bodies like those arising from the modified mitochondria that follow infection by certain members of the family Tombusviridae, i.e. Carnation Italian ringspot virus, Pelargonium necrotic spot virus (PNSV), Galinsoga mosaic virus (GaMV), and Turnip crinkle virus (TCV). Immunogold labelling using antibodies to virus particles was detected over cytoplasm associated with virus particles and patches of electrondense amorphous material, suggesting that this material consists of virus coat protein.

A Single Amino Acid Mutation in thePlum pox virusHelper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities

ABSTRACT The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL(134)H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL(134)H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.

Molecular characterization of banana virus X (BVX), a novel member of the Flexiviridae family

A novel virus was identified in banana (Musa spp). Analysis of the last 2917 nucleotides of its positive strand genomic RNA showed five open reading frames corresponding, from 5' to 3', to a truncated ORF coding for a replication-associated protein, three ORFs coding for a movement-associated triple gene block (TGB) and a capsid protein (CP) gene. This genome organization is similar to that of some members of the Flexiviridae family such as potexviruses and foveaviruses. This virus was named Banana virus X (BVX). Comparative sequence analysis showed that BVX is only distantly related to other members of the Flexiviridae family, in which it appears to define a new genus. BVX produces defective RNAs derived from its genomic RNA by non-homologous recombination. Three distinct pairs of donor/acceptor recombination sites involving short direct nucleotide repeats were characterized, accounting for deletions of 1268, 1358 and 1503 nucleotides. Contrary to the situation encountered for Potexviruses, these recombination sites are located within the TGB1 and CP genes and result in a truncated TGB1 protein.

Conservation of functional domains and limited heterogeneity of HIV-1 reverse transcriptase gene following vertical transmission.

BackgroundThe reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the life cycle of the virus by converting the single stranded RNA genome into double stranded DNA that integrates into the host chromosome. In addition, RT is also responsible for the generation of mutations throughout the viral genome, including in its own sequences and is thus responsible for the generation of quasi-species in HIV-1-infected individuals. We therefore characterized the molecular properties of RT, including the conservation of functional motifs, degree of genetic diversity, and evolutionary dynamics from five mother-infant pairs following vertical transmission.ResultsThe RT open reading frame was maintained with a frequency of 87.2% in five mother-infant pairs' sequences following vertical transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' sequences. Both mothers and infants RT sequences were under positive selection pressure, as determined by the ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 132 mother-infant RT sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked mother-infant pairs. The functional domains of RT which are responsible for reverse transcription, DNA polymerization and RNase H activity were mostly conserved in the RT sequences analyzed in this study. Specifically, the active sites and domains required for primer binding, template binding, primer and template positioning and nucleotide recruitment were conserved in all mother-infant pairs' sequences.ConclusionThe maintenance of an intact RT open reading frame, conservation of functional domains for RT activity, preservation of several amino acid motifs in epidemiologically linked mother-infant pairs, and a low degree of genetic variability following vertical transmission is consistent with an indispensable role of RT in HIV-1 replication in infected mother-infant pairs.

Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70–94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles.