Majid Laassri

Microarray analysis of evolution of RNA viruses: Evidence of circulation of virulent highly divergent vaccine-derived polioviruses

Two approaches based on hybridization of viral probes with oligonucleotide microarrays were developed for rapid analysis of genetic variations during microevolution of RNA viruses. Microarray analysis of viral recombination and microarray for resequencing and heterogeneity analysis were able to generate instant genetic maps of vaccine-derived polioviruses (VDPVs) and reveal the degree of their evolutionary divergence. Unlike conventional methods based on cDNA sequencing and restriction fragment length polymorphism, the microarray approaches are better suited for analysis of heterogeneous populations and mixtures of different strains. The microarray hybridization profile is very sensitive to the cumulative presence of small quantities of different mutations, including those that cannot be revealed by sequencing, making this approach useful for characterization of profiles of nucleotide sequence diversity in viral populations. By using these methods, we identified a type-3 VDPV isolated from a healthy person and missed by conventional methods of screening. The mutational profile of the polio strain was consistent with >1 yr of circulation in human population and was highly virulent in transgenic mice, confirming the ability of VDPV to persist in communities despite high levels of immunity. The proposed methods for fine genotyping of heterogeneous viral populations can also have utility for a variety of other applications in studies of genetic changes in viruses, bacteria, and genes of higher organisms.

Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides

Variola virus (VARV), causing smallpox, is a potential biological weapon. Methods to detect VARV rapidly and to differentiate it from other viruses causing similar clinical syndromes are needed urgently. We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples.

Effect of Different Vaccination Schedules on Excretion of Oral Poliovirus Vaccine Strains

Inactivated poliovirus vaccine (IPV) is believed to induce significantly lower mucosal immunity than oral poliovirus vaccine (OPV). Most of the data supporting this were generated before enhanced IPV (eIPV) was introduced. Excretion of poliovirus by OPV recipients can be used to assess intestinal immunity. We studied polymerase chain reaction amplification of viral complementary DNA from the stool of children vaccinated with either OPV alone or eIPV. Of first-time OPV recipients, 92% excreted virus after 1 week, and 81% excreted virus after 3 weeks. Prior vaccination with OPV reduced the number to 22% and shortened the duration of virus excretion (to 5% after 3 weeks). Two doses of IPV reduced the number of poliovirus-positive 1-week samples (to 76%), the duration of shedding (to 37% at 3 weeks), and the quantity of excreted virus. This suggests that IPV-vaccinated communities are partially protected from the spread of poliovirus. Further enhancement of IPV potency may lead to even higher levels of mucosal immunity.

Randomized Trial of Inactivated and Live Polio Vaccine Schedules in Guatemalan Infants

BACKGROUND The immunogenicity of inactivated poliovirus vaccine (IPV) in developing countries is not well documented. This study compared the immune response to IPV with that to oral poliovirus vaccine (OPV) in Guatemalan infants. METHODS This was an open-label, randomized comparison of IPV only, OPV only, or IPV followed by OPV in Guatemalan public health clinics. Serum samples were tested for neutralizing antibodies, and stool samples were tested for Sabin strain polioviruses. RESULTS Seropositivity rates 2 months after 2 doses of IPV were 98%-100% for polio types 1, 2, and 3 and were 97.1%, 99.3%, and 92.1% for OPV-only recipients (P<.001 for the response to type 3). One month after the third dose, 100% of IPV-only recipients had protective antibodies against all 3 types, compared with 99%, 100%, and 97% against polio types 1, 2, and 3 respectively, among recipients of OPV only. Infants who received IPV only had higher geometric mean titers than infants who received OPV only. Maternal antibodies lowered the final antibody responses to IPV but did not prevent the development of protective levels of antibody. Of 191 stool samples from infants who received IPV only, 5 (2.6%) were positive for poliovirus vaccine strains. CONCLUSIONS IPV alone and IPV followed by OPV are safe and effective for Guatemalan infants.

Genomic Analysis of Vaccine-Derived Poliovirus Strains in Stool Specimens by Combination of Full-Length PCR and Oligonucleotide Microarray Hybridization

ABSTRACT Sabin strains of poliovirus used in the manufacture of oral poliovirus vaccine (OPV) are prone to genetic variations that occur during growth in cell cultures and the organisms of vaccine recipients. Such derivative viruses often have increased neurovirulence and transmissibility, and in some cases they can reestablish chains of transmission in human populations. Monitoring for vaccine-derived polioviruses is an important part of the worldwide campaign to eradicate poliomyelitis. Analysis of vaccine-derived polioviruses requires, as a first step, their isolation in cell cultures, which takes significant time and may yield viral stocks that are not fully representative of the strains present in the original sample. Here we demonstrate that full-length viral cDNA can be PCR amplified directly from stool samples and immediately subjected to genomic analysis by oligonucleotide microarray hybridization and nucleotide sequencing. Most fecal samples from healthy children who received OPV were found to contain variants of Sabin vaccine viruses. Sequence changes in the 5′ untranslated region were common, as were changes in the VP1-coding region, including changes in a major antigenic site. Analysis of stool samples taken from cases of acute flaccid paralysis revealed the presence of mixtures of recombinant polioviruses, in addition to the emergence of new sequence variants. Avoiding the need for cell culture isolation dramatically shortened the time needed for identification and analysis of vaccine-derived polioviruses and could be useful for preliminary screening of clinical samples. The amplified full-length viral cDNA can be archived and used to recover live virus for further virological studies.

Microarray-based assay for the detection of genetic variations of structural genes of West Nile virus.

Adaptation through fixation of spontaneous mutations in the viral genome is considered to be one of the important factors that enable recurrent West Nile virus (WNV) outbreaks in the U.S. Genetic variations can alter viral phenotype and virulence, and degrade the performance of diagnostic and screening assays, vaccines, and potential therapeutic agents. A microarray assay was developed and optimized for the simultaneous detection of any nucleotide mutations in the entire structural region of WNV in order to facilitate public health surveillance of genetic variation of WNV. The DNA microarray consists of 263 oligonucleotide probes overlapping at half of their lengths which have been immobilized on an amine-binding glass slide. The assay was validated using 23 WNV isolates from the 2002-2005 U.S. epidemics. Oligonucleotide-based WNV arrays detected unambiguously all mutations in the structural region of each one of the isolates identified previously by sequencing analysis, serving as a rapid and effective approach for the identification of mutations in the WNV genome.

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre‐membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH‐SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non‐structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH‐SY5Y cells shared two common mutations: G2337C (Met457Ile) in the E gene and A6751G (Lys125Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture‐passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains. J. Med. Virol. 83:910–920, 2011.

Quantitative one-step RT-PCR assay for rapid and sensitive identification and titration of polioviruses in clinical specimens.

Rapid identification and quantitation of polioviruses in clinical specimens is important for surveillance and analysis of virus shedding by vaccine recipients, which could be used to assess the level of mucosal immunity. A quantitative one step RT-PCR was developed for identification and titration of all three poliovirus serotypes. The assay could be an alternative to the traditional procedure based on cell culture isolation and subsequent determination of poliovirus serotype and virus titration. The method is based on quantitative PCR performed with reverse transcription reaction in the same tube. The multiplex assay that quantifies all three serotypes of poliovirus was found to be highly specific, sensitive, and takes only one day to complete.

Environmental surveillance of viruses by tangential flow filtration and metagenomic reconstruction

An approach is proposed for environmental surveillance of poliovirus by concentrating sewage samples with tangential flow filtration (TFF) followed by deep sequencing of viral RNA. Subsequent to testing the method with samples from Finland, samples from Pakistan, a country endemic for poliovirus, were investigated. Genomic sequencing was either performed directly, for unbiased identification of viruses regardless of their ability to grow in cell cultures, or after virus enrichment by cell culture or immunoprecipitation. Bioinformatics enabled separation and determination of individual consensus sequences. Overall, deep sequencing of the entire viral population identified polioviruses, non-polio enteroviruses, and other viruses. In Pakistani sewage samples, adeno-associated virus, unable to replicate autonomously in cell cultures, was the most abundant human virus. The presence of recombinants of wild polioviruses of serotype 1 (WPV1) was also inferred, whereby currently circulating WPV1 of south-Asian (SOAS) lineage comprised two sub-lineages depending on their non-capsid region origin. Complete genome analyses additionally identified point mutants and intertypic recombinants between attenuated Sabin strains in the Pakistani samples, and in one Finnish sample. The approach could allow rapid environmental surveillance of viruses causing human infections. It creates a permanent digital repository of the entire virome potentially useful for retrospective screening of future discovered viruses.

Deep sequencing approach for genetic stability evaluation of influenza A viruses

Assessment of genetic stability of viruses could be used to monitor manufacturing process of both live and inactivated viral vaccines. Until recently such studies were limited by the difficulty of detecting and quantifying mutations in heterogeneous viral populations. High-throughput sequencing technologies (deep sequencing) can generate massive amounts of genetic information and could be used to reveal and quantify mutations. Comparison of different approaches for deep sequencing of the complete influenza A genome was performed to determine the best way to detect and quantify mutants in attenuated influenza reassortant strain A/Brisbane/59/2007 (H1N1) and its passages in different cell substrates. Full-length amplicons of influenza A virus segments as well as multiple overlapping amplicons covering the entire viral genome were subjected to several ways of DNA library preparation followed by deep sequencing using Solexa (Illumina) and pyrosequencing (454 Life Science) technologies. Sequencing coverage (the number of times each nucleotide was determined) of mutational profiles generated after 454-pyrosequencing of individually synthesized overlapping amplicons were relatively low and insufficiently uniform. Amplification of the entire genome of influenza virus followed by its enzymatic fragmentation, library construction, and Illumina sequencing resulted in high and uniform sequencing coverage enabling sensitive quantitation of mutations. A new bioinformatic procedure was developed to improve the post-alignment quality control for deep-sequencing data analysis.