3D Genomic Regions Research Articles

Iterative Excretion of Human Cosaviruses from Different Genotypes Associated with Combined Immunodeficiency Disorder

Background: Human cosaviruses (HCoSVs) are newly discovered enteric viruses in the Picornaviridae family. They have been described in non-polio acute flaccid paralysis, diarrheal patients, and healthy individuals. They remain rarely documented in immunodeficient patients. Objectives: This study reports iterative excretion of HCoSVs in a patient with major histocompatibility complex (MHC) class II combined immunodeficiency, a relatively common primary immunodeficiency in consanguineous settings. Methods: A total of 35 samples were collected from a patient followed for oral polio vaccine strains detection in stool samples during a 57-month period. Detection of HCoSVs in stools was performed by nested RT-PCR in the 5′ noncoding region. The genotype identification and screening for recombinant strains was performed by sequencing in the VP1 and 3D genomic regions followed by phylogenetic analysis. Results: The patient was infected with HCoSVs twice at a 3-year interval. The excreted viruses belonged to 2 different genotypes with 2 probable recombinant viruses. During HCoSV infections, the patient was also excreting Sabin-related polioviruses. Conclusions: This study describes excretion kinetics and genetic characteristics of HCoSVs in a patient with combined immunodeficiency due to MHC class II expression defect. The patient did not have concomitant symptoms related to the HCoSV infection.

Close genetic relatedness of picornaviruses from European and Asian bats.

Our investigation of 1004 faecal specimens from European bats for picornaviruses by broadly reactive nested reverse transcription-PCR found picornaviral RNA in 28 samples (2.8 %). Phylogenetic analysis of the partial 3D genomic region suggested that one bat virus belonged to the species Enterovirus G (EV-G, formerly Porcine enterovirus B). Bat infection was supported by relatively high EV-G concentrations of 1.1×106 RNA copies per gram of faeces. All other bat viruses belonged either to the bat-associated genus Mischivirus, or to an unclassified Picornaviridae group distantly related to the genus Sapelovirus. Members of this unclassified sapelovirus-related group had RNA secondary structures in their 3'-nontranslated regions that were typical of enteroviruses and that resembled structures that occur in bat-associated coronaviruses, suggesting ancient recombination events. Based on sequence distances, several picornaviruses from European and Chinese bats were likely conspecific, suggesting connectivity of virus populations. Due to their high mutation rates and their diversity, picornaviruses may be useful tools for studies of bat and virus ecology.

Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses.

Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains.

PCR assays for the identification of rare recombination types from VP1 to 3D genomic region of vaccine derived poliovirus strains

Poliomyelitis has been effectively controlled by the use of inactivated poliovirus vaccine (IPV) or trivalent live attenuated oral poliovirus vaccine (OPV). Since 1964, the use of OPV in mass vaccinations has resulted in drastic reductions of the number of poliomyelitis cases caused by wild-type polioviruses. However, the characterization of OPV derivatives with increased neurovirulence, constituted a real problem with respect to OPV safety. Mutations at attenuating sites of the genome and recombination events between Sabin strains of the trivalent OPV vaccine have been correlated with the loss of the attenuated phenotype of OPV strains and the acquisition of traits characteristic of wild polioviruses. In consequence, early detection and characterization of recombinant evolved derivatives of vaccine strains is highly important. In this report, ten PCR assays are described which allow for the identification of rare recombination events located in VP1, 2A, 2C, 3A, 3C and 3D genomic regions and predominant recombination events located in 2C and 3D genomic regions of OPV derivatives. These assays could be readily implemented in diagnostics laboratories lacking sequencing facilities as a first approach for the early detection and characterization of recombinant OPV derivatives.

Genetic analysis of poliovirus strains isolated from sewage in Poland

The study describes genetic characterization of poliovirus (PV) strains isolated from sewage samples in Poland. The analyses were performed for the detection of any putative polio revertants and recombinants in three genomic regions by sequencing analysis. Thirty-six strains were analyzed. The analyzed strains were identified by neutralization assay as 7 strains of serotype P1, 10 strains of serotype P2, and 19 strains of serotype P3. Sewage isolates were sequenced in 5'UTR, VP1, and 3D genomic regions. All detected PVs were classified as vaccine strains on the basis of VP1 sequence. Mutational differences in the VP1 sequences of isolated viruses ranged from 0.0% to 0.4%, indicating a limited replication period. The genetic analysis of the 3D region showed that some strains have recombinant genomes. Nine strains were found as dipartite recombinants (seven strains--S3/S2, one strain--S2/S1, one strain--S3/S1), while one strain was found as tripartite recombinant (S3/S2/S1). No recombinants with non-PV enteroviruses were identified. None of wild-type PVs or vaccine-derived polioviruses (VDPVs) were detected. This study showed the absence of wild or VDPV circulation in the country and demonstrated the usefulness of environmental surveillance in addition to acute flaccid paralysis (AFP) surveillance in support of polio eradication initiatives.

1457 Characterization of Recombinant Sabin Poliovirus Isolated From Children with Acute Flaccid Paralysis

Background and aims: Oral poliovirus vaccine (OPV) proved to be very effective vaccine for interrupting wild poliovirus transmission. However, due to its genetic instability OPV is the potential source of vaccine derived polio virus (VDPVs). Therefore, we studied to detect recombination in Sabin poliovirus (SPV) isolated from children with acute flaccid paralysis (AFP). Methods: Virus isolation was carried out from 800 consecutively collected stool samples from 400 patients with AFP enrolled during August- December 2009 in northern India. RT-PCR and sequencing was performed to detect recombination and characterization of SPV. Reproductive capacity temperature assay was done to study the phenotypic characteristics of recombinant Sabin PV. Results: Of 80 SPV, 5 (6.25%) were confirmed as recombinant SPV. Three strains (3.75%) were found to be bi-recombinant (Sabin3/Sabin2); two strains (2.50%) were characterized as multirecombinant (Sabin3/Sabin2/Sabin1). There was a common recombination junction in the 2C genomic region for three isolates and in 3D genomic region for two isolates. Four recombinant strains (80.0%) have lost temperature sensitivity and clinically behaved as wild on 60 days follow up of the patients. Conclusions: Our study underlines the role of specific positions and motifs of the poliovirus genomic sequences involved in recombination process. In the last stage of poliovirus eradication there is a need of careful monitoring and characterization of Sabin poliovirus to achieve the goal of poliomyelitis eradication world wide.

Correlation between recombination junctions and RNA secondary structure elements in poliovirus Sabin strains.

In order to test the hypothesis that RNA structural elements promote the distribution of certain types of recombination junctions in each one of the 2C and 3D poliovirus genomic regions (Sabin 3/Sabin 2 or Sabin 1 in 2C and Sabin 2/Sabin 1 or Sabin 3 in 3D), we searched in 2C and 3D regions of reference Sabin strains for high probability RNA structural elements that could promote recombination. Recombination junctions that were identified in clinical strains of this study, as well as in clinical strains of previous studies, were superimposed on RNA secondary structure models of 2C and 3D genomic regions. Furthermore, we created an in vitro model, based on double infection of cell-culture with two poliovirus strains, for the production and identification of recombinant Sabin strains in 2C and 3D regions. Our intention was to compare the results that refer to the correlation of recombination junctions and RNA secondary structures in 2C and 3D regions of clinical strains, with the respective results of the in vitro model. Most of the recombination junctions of the clinical strains were correlated with RNA secondary structure elements, which were identical between recombining Sabin strains, and also presented high predictive value. In consensus were, the respective results originated from the in vitro model. We propose that the distribution of specific types of recombination junctions in certain regions of Sabin strains is not fortuitous and is correlated with RNA secondary structure elements identical to both recombination partners. Furthermore, results of this study highlight an important role for the stem region of the RNA structure elements in promoting recombination.Electronic supplementary materialThe online version of this article (doi:10.1007/s11262-010-0512-5) contains supplementary material, which is available to authorized users.

Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece

Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties.

A new RT-PCR assay for the identification of the predominant recombination types in 2C and 3D genomic regions of vaccine-derived poliovirus strains

In the post-eradication era of wild polioviruses, the only remaining sources of poliovirus infection worldwide would be the vaccine-derived polioviruses (VDPVs). As the preponderance of countries certified to be polio-free has switched from OPV (oral poliovirus vaccine) to IPV (inactivated poliovirus vaccine), importation of recombinant evolved derivatives of vaccinal strains would have serious implication for public health. To test the robustness of the proposed RT-PCR screening analysis, eleven recombinant vaccine-derived polioviruses that were characterized previously by sequencing by our group, in addition to three recently identified recombinant environmental isolates were assayed. Although the most definitive characterization of VDPVs is by genomic sequencing, in this study we describe a new, inexpensive and broadly applicable RT-PCR assay for the identification of the predominant recombination types S3/Sx in 2C and S2/Sx in 3D genomic regions respectively of VDPVs, that can be readily implemented in laboratories lacking sequencing facilities as a first approach for the early detection of vaccine-derived poliovirus (VDPVs).

Vaccine derived bi- and multi-recombinant Sabin strains

A retrospective analysis of five Sabin intertypic recombinant strains, isolated from human feacal specimens during the time period 1978-1985 in Greece, was performed by RT-PCR, Restriction Fragment Length Polymorphism (R.F.L.P.) and sequencing. Of the studied strains, three (EPA, EPB, EPC) were found to be bi-recombinant Sabin3/Sabin2/Sabin3 (S3/S2/S3), one strain was characterized as a probable S3/S2- CAV18 or CAV21-S2/S1 multi-recombinant (EDP11) and one was identified as a tripartite one S3/S2/S1 (EDP12). Samples EPA, EPB and EPC presented a common recombination junction in the 2C genomic region. Moreover, strains EPA and EPB shared also the second recombination site in the 3D genomic region, whereas the second recombination of EPC was also determined in 3D but in a different nucleotide position. Strains EDP11 and EDP12 presented both identical recombination motifs and recombination sites. The first was detected in the 2C genomic region and the second in the 3D region. Strain EDP11 presented an interesting feature since a sequence of 120 nucleotides seems to have derived from a member of human enteroviruses species C (CAV18 or CAV21). This finding is of great importance, considering that this strain (EDP11) was isolated from an area and time period, where no Coxsackie A virus or poliovirus epidemics occurred. Our study underlines the role of specific positions and motifs of the poliovirus genomic sequences involved in recombination events and prompts that Coxsackie A viruses belonging to human enterovirus species C (genetically closely related to PV) are considered as the possible counterparts of the recombination.

Sequence analysis of the RNA polymerase gene of foot-and-mouth disease virus serotype Asia1.

The complete nucleotide (nt.) sequence of the RNA polymerase (3D) gene and 81 nt. in the 3'-untranslated region of foot-and-mouth disease virus (FMDV) serotype Asial (IND63/72) was determined and compared with the sequence of other FMDV serotypes. The 3D genomic region was 1410 nt. long encoding 470 amino acids with an inframe stop codon (TAA) at nt. position 1411-1413. The deduced amino acid sequence of the protein showed 8 conserved motifs as reported in other picornaviruses, 2 of which are 100% identical across the serotypes. Antigenic regions in the polymerase protein were predicted and found to be located at the N-terminus of the protein. The phylogenetic analysis showed that the FMD viruses were segregated into different clusters based on geographical origin; the Asia1 virus did not cluster tightly with any of the geographical groups.