Novel variants of human herpesvirus 2 from Brazilian HIV-1 coinfected subjects

BACKGROUND Human herpesvirus 2 (HHV-2) have DNA genome with a limited genetic variability and have been classified into two clades. OBJECTIVES To identify and characterise six HHV-2 isolates derived from Brazilian women. METHODS HHV-2 isolates were performed polymerase chain reaction (PCR) and sequencing of 2250 pb of the glycoprotein B (gB) coding regions. FINDINGS Four HHV-2 isolates were classified into clade B, while the remaining two, derived from HIV-1 co-infected women, showed a notable genetic divergence (> 1%). MAIN CONCLUSION The results reveal novel HHV-2 variants. The impact of these novel variants on HHV-2 pathogenesis and HIV/HHV-2 coinfection need to be investigated.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Molecular characterization of duck enteritis virus CHv strain UL49.5 protein and its colocalization with glycoprotein M

The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.

Cloning and expression analysis of multiple proteins encoding P gene of Newcastle disease virus.

Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.

Influenza A H1N1 virus in Indian pigs & its genetic relatedness with pandemic human influenza A 2009 H1N1.

Background & objectives: With the emergence of a new reassortant influenza A H1N1 virus that caused the 2009 pandemic it was felt necessary that pigs should be closely monitored for early detection of any influenza virus infection. Therefore, we investigated disease outbreaks with clinical history suggestive for swine influenza reported to our laboratory by owners of affected pig farms in Uttar Pradesh. Methods: Detection of swine influenza A virus (SIV) was attempted by isolation in embryonated chicken eggs. Presence of virus was detected by haemagglutination (HA) test and RT-PCR for amplification of different gene segments, cloning and sequencing. BLAST analysis of sequence data, phylogenetic analysis and mutation analysis based on HA, NA and matrix genes was done. Results: SIV could be isolated from one farm and all eight gene segments amplified by RT-PCR. BLAST analysis of partial nucleotide sequences and phylogenetic analysis using nucleotide sequence of HA (601 nt), NA (671 nt) and M (1031 nt) genes indicated close genetic relationship of the Indian swine isolate (A/Sw/UP-India-IVRI01/2009) with human pandemic 2009 (H1N1). The HA gene showed close relationship with the viruses of “North American Swine” lineage, whereas the NA and M genes clustered with the viruses of “Eurasian Swine” lineage, indicating a novel HA-NA reassortant. The remaining of 5 genes (NP, PA, PB1, PB2 and NS) belonged to “North American Swine” lineage. Interpretation & conclusions: This is perhaps the first report describing swine influenza among Indian pigs caused by an influenza A H1N1 virus sharing close homology with the human pandemic (H1N1) 2009 virus. Further reassortment with circulating influenza viruses must be closely monitored.

UL146 variability among clinical isolates of Human Cytomegalovirus from Japan

Human Cytomegalovirus (HCMV) is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR) in only 17/56 strains (30 percent), while the success rate for UL145/UL147 gene was 18/56 strains (32 percent). After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1 percent to 52.9 percent at the DNA level and from 34.5 percent to 67 percent at the amino acid level. Seven groups had the interleukin-8 (IL-8) motif ERL (Glu-Leu-Arg) CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.

Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil

Group B streptococci (GBS) infections occur worldwide. Although serotyping has been used for epidemiologic purposes, this does not accurately characterize enough members of a genetically heterogeneous bacterial population. The aims of this work were to evaluate the genetic diversity of 45 type Ia GBS strains isolated in Brazil by pulsed-field gel electrophoresis as well as to evaluate antimicrobial susceptibility profiles and identify virulence genes. Twenty-four strains were assigned to cluster A. All strains under study contained the hylB and scpB genes. The bca gene was detected in only 10 strains and none of the streptococci carried the bac gene. Thirty-nine strains were resistant to tetracycline.

Characterization and phylogenetic relationships of strains of Dengue type 1 virus from Costa Rica

Phylogenetic analysis of the envelope gene sequence (E) of three isolated Dengue virus type 1 (DEN1) sequences from the Costa Rican 1993 epidemic was performed. All the strains fell into genotype V. Genotype V has grouped all the strains isolated from America, three strains from Africa and two strains from Asia. Costa Rican strains are closely related to the strain from Angola and to the Brazil 1990 strain. Costa Rican strains are able to cause DHF but with low clinical disease severity and low incidence maybe because of low virulence. Rev. Biol. Trop. 55 (2): 365-372. Epub 2007 Jun 29.

Tres aislamientos del gen de la envoltura (E) de Dengue tipo 1 del período epidémico de 1993 en Costa Rica se analizaron filogenéticamente. Todas las cepas pertenecen al genotipo V. El genotipo V ha agrupado todos los aislamientos de América, tres cepas de África y dos cepas de Asia. Los aislamientos costarricenses están cercanamente relacionados a la cepa de Angola y al aislamiento brasileño de 1990. Las cepas costarricenses son capaces de causar Fiebre de Dengue Hemorrágico pero con baja severidad clínica y baja incidencia quizá debido a una baja virulencia.

L1 sequence of a new human papillomavirus type-58 variant associated with cervical intraepithelial neoplasia

The present study on molecular characterization of a human papillomavirus (HPV) isolated in Central Brazil describes the L1 gene sequence from a new variant of HPV-58, the isolate Bsb-02. The sample was from a smear obtained from a woman with cervical intraepithelial neoplasia grade II. The whole L1 gene from isolate Bsb-02 was sequenced automatically, showing 99.1 percent nucleotide identity with the gene from the HPV-58 reference. The clustering between Bsb-02 and HPV-58 reference sequence was also supported by phylogenetic analysis. Fourteen nucleotide substitutions were observed: eight were synonymous and six were associated with amino acid substitutions. A10V and V144I have not been previously described. At GenBank, the only complete L1 sequence from HPV-58 in addition to the HPV-58 reference one is that of Bsb-02. These data provide information that may be relevant to HPV diagnosis and to rational vaccine strategies. HPV variants may also be associated with host immune responses and with the risk of cervical neoplasia.

Regulación genética de los papilomavirus humanos genitales / Genetic regulation of human genital papillomavirus

Los papilomavirus humano (PVH) infectan epitelios estratificados queratinizados con una alta especificidad y están asociados con la aparición y persistencia de neoplasias benignas y malignas. Los elementos que dirigen la expresión genética de estos virus se localizan en una región no codificadora conocida como región larga de control (RLC). Al inicio del ciclo viral, una combinación particular de factores celulares que interactúan con la RLC promueven la transcripción temprana de los oncogenes virales E6 y E7. Estos favorecen la división celular interrumpiendo los mecanismos regulatorios celulares: E6 se une a la proteína supresora de tumor p35 y E7 se une a p105RB. La continuidad en la transcripción temprana conlleva al aumento gradual de las proteínas virales E1 y E2. La proteína E2 impide la transcripción temprana y confiere especificidad de unión a E1, la cual promueve la replicación viral. El cese paulatino de la transcripción de los oncogenes virales a través de la represión por E2, libre la regulación del crecimiento celular mediada por p53 y p105RB, permitiendo que la diferenciación celular progrese. Es entonces cuando el promotor tardío funciona para la producción de las proteínas de la cápside viral L1 y L2, permitiendo la maduración de viriones en los estratos superiores del epitelio. La disrupción del gen E2 durante un evento de integración del genoma viral, impide la progreción del ciclo vira y la entrada del programa de diferenciación de la célula epitelial, sosteniendo el estado transformado producido por E6 y E7

Human papillomavirus (HPV) specifically infect stratified epithelial cells, causing benign and malignant neoplasia. Several elements directing this virus' genetic expression are present in a non-coding region called LCR. HPV infection starts in the basal cells of stratified epithelia, where a particular combination of cellular factors interacting with the LCR starts the transcription of the viral E6 and E7 oncogenes. The E6 and E7 genes alter the cell cycle because they interact and inactivate tumor suppressor proteins: E6 binds and degrades protein p53 and E7 associates with p105RB. E1 and E2 are the next synthesized proteins. E2 blocks the early transcription and permits E1 specific binding to the viral origin of replication located within the lcr, initiating the viral genome replication. Following the course of viral infection, the E2-induced E6 and E7 down-regulation releases p53 and p105RB proteins, and the differentiation process can continue. Then, a putative late promoter can activate the capsid genes L1 and L2. At this step, mature virions can be detected in the upper layers of the epithelium. Disruption in E2 gene transcription is usually associated to genital malignant neoplasia. In the absence of E2, E6 and E7 remain constitutively expressed, sustaining the immortality of the infected cell and blocking the epithelial differentiation program.

Aspectos moleculares de los papilomavirus humanos y su relación con el cáncer cérvico-uterino / Molecular aspects of human papillomaviruses and their relationship to uterine-cervix carcinoma

Los papilomavirus son responsables del desarrollo de lesiones epiteliales benignas y malignas en mamíferos. Más de 60 tipos diferentes de papilomavirus humanos (HPVs) han sido aislados hasta ahora: algunos de ellos participan de manera importante en el desarrollo del cáncer cervical. El DNA de los tipos de 90 por ciento de los carcinomas genitales. La integración del DNA viral en el genoma celular puede ser un paso importante en el desarrollo de la enfermedad. Dos genes tempranos de los HPVs (E6 y E7) están involucrados en la inmortalización celular. Otro gen temprano (E2) participa en el control de la expresión de los oncogenes, al unirse directamente a regiones conservadas del genoma viral como celular, interactúan con la región regulatoria de los HPVs, y participan en la regulación de la transcripción de los oncogenes E6 y E7. Factores celulares, tales como el sistema inmune, oncogenes o antioncogenes, parecen jugar un papel importante en la carcinogénesis asociada con los HPVs.

Analysis of Japanese encephalitis (JE) virus genome and implications for recombinant JE vaccine.

From the information of nucleotide sequences and deduced amino acid sequences of flaviviruses including JEV, we can postulate processing mechanisms of a polyprotein translated from single long open reading frame of the genome and mechanisms of construction of antigenic structures of structural proteins with biologically active forms after these proteins are translated. The results of comparative analysis of amino acid sequences among flaviviruses and epitope analysis on the E proteins which are the most important antigens for protective immunity suggest that the E protein of flaviviruses may have a similar structure closely related to each other. PrM and E proteins which had predictable signal sequences upstream on the N terminals were expressed with antigenically active form and molecular size the same as the authentic ones by the recombinant viruses. However, the recombinant viruses which had no such signal sequence expressed unprocessed proteins with antigenically denatured forms. These results suggest that normal proteolytic processing is needed to construct biologically active structures of JEV structural proteins. The E proteins which were expressed by the recombinant viruses as antigenically active form could elicit nutralizing and HI antibodies in animals and protective immunity in mice. The recombinant vaccinia viruses which express the E protein could induce strong immunologic memory against the E protein in mice. These results indicate that the development of a new type of vaccine against JEV will become possible in future.

Overview of flavivirus molecular biology and future vaccine development via recombinant DNA.

Studies in many laboratories over the last several years have elucidated the structures of several different flavivirus genomes. Conserved features include the production of at least 10 different virus encoded proteins from a single long open reading frame by a combination of host and virus-encoded proteases. The established gene order is 5'-C- prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS 5-3' and these proteins exhibit varying degrees of homology in comparisons among flaviviruses. Conserved RNA sequences and structures have also been identified for the mosquito-borne flaviviruses but are absent in sequenced tick-bone viruses. Relevant to the development of efficacious flavivirus vaccines, studies aimed at defining the antigenic determinants necessary for eliciting protective immunity have focused primarily on the structural proteins, in particular the E protein, as well as the nonstructural secreted glycoprotein, NS1. Other work, which has led to the derivation of live-attenuated flavivirus strains, should eventually allow the genetic determinants of flavivirus attenuation and pathogenesis to be understood at the molecular level. The successful recovery infectious flaviviruses from cloned cDNA raises the possibility of manipulating these viral genomes as cDNA to construct or propagate candidate live-attenuated vaccine strains. Several applications of this technology are discussed.