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A VP3/VP1 gene polymerase chain reaction assay for detection of chicken anemia virus in broiler samples
Nogueira, E.O.;Brentano, L.;Ferreira, A.J.P.;
Arquivo Brasileiro de Medicina Veterinária e Zootecnia , 2005, DOI: 10.1590/S0102-09352005000800001
Abstract: a pcr assay was designed for amplification of the highly conserved vp3 gene and a 5' region of the vp1 gene, for the diagnosis of cav in organ samples of broiler flocks suspected of chicken infectious anemia. a comparison of the vp3/vp1 pcr with in vivo virus isolation revealed 100% agreement of the results, with 13 positive and 3 negative samples in both assays, indicating that the vp3/vp1 pcr is a specific diagnostic method. tissues from additional 24 broiler chicken flocks, with cav-like lesions and clinical history were then tested only by the vp3/vp1 pcr and a reference pcr with published primers for the vp1 gene. nineteen samples resulted positive and one negative in both pcr, while another 4 samples were positive only in the vp3/vp1 pcr. these results indicate that the vp3/vp1 pcr is a sensitive, specific diagnostic test, suitable as an alternative to the expensive and time consuming in vivo virus isolation method, specially considering the difficult diagnosis of cav strains not readily adaptable to msb-1 cell culture.
Correlation between pre-treatment quasispecies complexity and treatment outcome in chronic HCV genotype 3a
Isabelle Moreau, John Levis, Orla Crosbie, Elizabeth Kenny-Walsh, Liam J Fanning
Virology Journal , 2008, DOI: 10.1186/1743-422x-5-78
Abstract: The degree of sequence heterogeneity within the hypervariable region 1 was assessed by analyzing 20–30 individual clones in serial serum samples. Genetic parameters, including amino acid Shannon entropy, Hamming distance and genetic distance were calculated for each sample. Treatment outcome was divided into (1) sustained virological responders (SVR) and (2) treatment failure (TF).Our results indicate, (1) quasispecies complexity and diversity are lower in the SVR group, (2) quasispecies vary temporally and (3) genetic heterogeneity at baseline can be use to predict treatment outcome.We discuss the results from the perspective of replicative homeostasis.The Hepatitis C virus (HCV), is the causative agent of chronic hepatitis C and infects at least 170 million individuals worldwide [1-3]. The virus has been classified into six major genotypes and more than 70 subtypes based on sequence diversity [4-10]. The most important feature of HCV is that it causes chronic infection in about 50–80% of individuals [3,11-13].The HCV genome exhibits significant genetic heterogeneity due to accumulation of mutations during viral replication, attributed to a limited fidelity of the RNA dependent RNA polymerase [14,15]. This phenomenon generates a dynamic population of heterogeneous but closely related variants designated as quasispecies [14-17]. The massive genetic heterogeneity present in quasispecies has important biological consequences and enables HCV to escape immune clearance and to establish chronic infection [18-22]. Furthermore, the quasispecies distribution may influence the outcome of anti-viral therapy and be important in the development of resistance to anti-viral therapy [23-27]. It is well established that HCV genotype influences both response to therapy and disease severity as well as the viral-host interactions [19,28-30]. Patients infected with HCV genotypes 2 or 3 respond more favourably than genotype 1 to pegylated α2a-Interferon and ribavirin anti-viral therapy
In–Depth Characterization of Viral Isolates from Plasma and Cells Compared with Plasma Circulating Quasispecies in Early HIV-1 Infection  [PDF]
Judith Dalmau, Francisco M. Codo?er, Itziar Erkizia, Maria Pino, Christian Pou, Roger Paredes, Bonaventura Clotet, Javier Martinez-Picado, Julia G. Prado
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0032714
Abstract: Background The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies. Methodology/Principal Findings We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA. Conclusion This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.
Quasispecies Theory and the Behavior of RNA Viruses  [PDF]
Adam S. Lauring,Raul Andino
PLOS Pathogens , 2010, DOI: 10.1371/journal.ppat.1001005
Abstract: A large number of medically important viruses, including HIV, hepatitis C virus, and influenza, have RNA genomes. These viruses replicate with extremely high mutation rates and exhibit significant genetic diversity. This diversity allows a viral population to rapidly adapt to dynamic environments and evolve resistance to vaccines and antiviral drugs. For the last 30 years, quasispecies theory has provided a population-based framework for understanding RNA viral evolution. A quasispecies is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. Many predictions of quasispecies theory run counter to traditional views of microbial behavior and evolution and have profound implications for our understanding of viral disease. Here, we discuss basic principles of quasispecies theory and describe its relevance for our understanding of viral fitness, virulence, and antiviral therapeutic strategy.
The generalised quasispecies  [PDF]
Rapha?l Cerf,Joseba Dalmau
Quantitative Biology , 2015,
Abstract: We study Eigen's quasispecies model in the asymptotic regime where the length of the genotypes goes to infinity and the mutation probability goes to 0. We give several explicit formulas for the stationary solutions of the limiting system of differential equations.
Molecular Comparison and Evolutionary Analyses of VP1 Nucleotide Sequences of New African Human Enterovirus 71 Isolates Reveal a Wide Genetic Diversity  [PDF]
Ma?l Bessaud, Richter Razafindratsimandresy, Antoine Nougairède, Marie-Line Joffret, Jagadish M. Deshpande, Audrey Dubot-Pérès, Jean-Michel Héraud, Xavier de Lamballerie, Francis Delpeyroux, Jean-Luc Bailly
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0090624
Abstract: Most circulating strains of Human enterovirus 71 (EV-A71) have been classified primarily into three genogroups (A to C) on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D) and 2 African ones (E and F). Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied.
The epitope of the VP1 protein of porcine parvovirus
Hong-ling Xie, Zhao Wang, Shang-jin Cui, Chao-fan Zhang, Yu-dong Cui
Virology Journal , 2010, DOI: 10.1186/1743-422x-7-161
Abstract: Porcine parvovirus (PPV), which was first isolated from sows in Germany by Mayr et al. [1] and which belongs to the genus Parvovirus in the family Parvoviridae, is the major causative virus in a syndrome of reproductive failure in swine. This syndrome is referred to as SEDI and includes stillbirths, mummified fetuses, early embryonic death, and infertility [2]. Recent studies indicate that, in addition to inducing reproductive failure, PPV also causes dermatitis, diarrhea, and respiratory system disease [3-10].PPV is composed of structural protein and non-structural protein, and the structural protein is the virus's main immunological antigen. Using SDS-PAGE, Moliter et al. [11] identified three kinds of structural proteins: VP1, VP2, and VP3. All three of these proteins can induce hemagglutination inhibition (HI) antibodies and neutralization antibodies in rabbits; induction of HI antibodies was greatest with VP3, intermediate with VP2, and least with VP1.VP1 has an important biological function for PPV: its intranuclear signal sequence, which is similar to that of the VP1 proteins of Simian virus 40 and human papillomavirus [12], is very important for PPV positioning within the host nucleus. The N-terminal of VP1 is rich in alkaline amino acids, which enhance binding with host DNA to stabilize viral single-strand genome DNA; the binding is required for the initiation of viral DNA reproduction and for the packaging of the viral genome [13].The structural proteins of a related virus, canine parvovirus (CPV), have three antigen epitope regions In the case of CPV, the epitope located in region B1 possesses good antigenicity and induces the host to produce neutralization antibody [14,15]. Unfortunately, such information about antigen epitope is unavailable for PPV.Phage display is a powerful tool for the study of the interaction between antigen and antibody. This molecular biology technique can directly select simulated antigen epitopes that can combine to antibody in
Characterization of outbreak hepatitis a isolates in five Tunisian childcare centers
Gharbi-Khelifi, Hakima;Abid, Nabil Ben Salem;Sdiri, Khira;Harrath, Rafik;Beji, Abir;Bhiri, Leila;Billaudel, Sylviane;Ferre, Virginie;Aouni, Mahjoub;
Brazilian Journal of Microbiology , 2011, DOI: 10.1590/S1517-83822011000300046
Abstract: in the present study, epidemiological survey and molecular characterization of hepatitis a virus during an outbreak in five tunisian childcare centers in el-mahres during october and november 2006 were carried out. five well-water and five drinking water samples were included in the present study. serological investigation and molecular characterization were carried out. all patients were igm seropositive and the viral genome was detected in all clinical and well-water samples whereas it was not detected in drinking water from the five childcare centers. sequence analysis showed that all tunisian strains belong to sub-genotype ia. the genetic profile of the vp1/2a junction showed that the outbreak isolates underwent an amino acid substitution which was absent in virus's strains detected previously in tunisia. further studies need to be conducted to evaluate the emergence of the virus's strains in clinical and water samples and more epidemiological data need to be collected about the risk factors which may contribute to acute hepatitis.
Quasispecies and recombination  [PDF]
Martin Nilsson Jacobi,Mats Nordahl
Quantitative Biology , 2005,
Abstract: Recombination is introduced into Eigen's theory of quasispecies evolution. Comparing numerical simulations of the rate equations in the non-recombining and recombining cases show that recombination has a strong effect on the error threshold and, for a wide range of mutation rates, gives rise to two stable fixed points in the dynamics. This bi-stability results in the existence of two error thresholds. We prove that, under some assumptions on the fitness landscape but for general crossover probability, a fixed point localized about the sequence with superior fitness is globally stable for low mutation rates.
Influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C  [cached]
Deepak Kumar, Abdul Malik, Mohammad Asim, Anita Chakravarti, Rakha H Das, Premashis Kar
World Journal of Gastroenterology , 2008,
Abstract: AIM: To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C.METHODS: Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH), 9 with cirrhosis, and 6 with hepatocellular carcinoma (HCC)] were screened for the presence of quasispecies by single stranded conformational polymorphism (SSCP) analysis in the hypervariable region (HVR) and non-structural 5B (NS5B) viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients (11 non-responders and 12 showing a sustained virological response) was sequenced for the assessment of mutations.RESULTS: The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association (P < 0.05) with the non-responders, and leads to persistence of infection. Significant differences (P < 0.05) in viral load (log10 IU/mL) were observed among patients infected with complex quasispecies (CQS), those infected with simple quasispecies (SQS) and those with no quasispecies (NQS), after 12 wk (CQS-5.2 ± 2.3, SQS-3.2 ± 1.9, NQS-2.8 ± 2.4) and 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, NQS-2.1 ± 2.3) in the HVR region. However, a statistically significant difference (P < 0.05) was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, and NQS-2.1 ± 2.3) and 48 wk (CQS-3.1 ± 2.7, SQS-2.3 ± 2.4, NQS-2.0 ± 2.3) of therapy. Disease severity was significantly associated with viral load during therapy. The strains isolated from non-responders showed close pairing on phylogeny based on the NS5B gene, but dissimilar HVR regions. This revealed the possibility of the selection of resistant strains during the evolution of quasispecies in NS5B.CONCLUSION: Viral quasispecies may be an important predictor of virological responses to combination therapy in patients with chronic hepatitis C. Complex quasispecies and resistant strains may lead to high viral loads during therapy, with a concerted effect on disease severity.
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