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recombination

A genetically stable attenuated poliovirus vaccine

13 May 2021 by Vincent Racaniello

png_polio_gpei_310

Eradication of poliomyelitis appears to be on track: types 2 and 3 polioviruses have been declared eradicated, and in the past 12 months there have been just 338 cases of type 1 polio in Afghanistan and Pakistan. But there have also been 491 cases of polio caused by the type 2 Sabin vaccine. The development of a modified version of the type 2 vaccine component could improve this situation.

The oral poliovirus vaccines (OPV) developed by Albert Sabin have played a huge role in reducing cases of polio globally from 400,000 a year in 1980 to the current numbers. Their success, however, comes with a cost: they may in rare cases cause the disease they are designed to prevent. The three serotypes of OPV are taken orally and then reproduce in the intestines where they confer effective immunity to polio. During reproduction of the viruses in the intestine, the mutations originally selected by Sabin to eliminate the neurovirulence of the viruses are lost. Most immunized children shed vaccine revertants, and about 1 in 1.4 million vaccine recipients contract polio.

These vaccine revertants also circulate extensively throughout the human population, and may cause outbreaks of polio in areas where vaccine coverage drops. To address this problem, in 2016 WHO removed the type 2 component of poliovirus from OPV, which is responsible for most of the vaccine-associated cases. However vaccine-derived type 2 polioviruses continue to circulate even after this vaccine withdrawal and have caused a number of outbreaks. The response to control these outbreaks is to conduct mass immunizations with OPV type 2 – which re-introduces vaccine-derived polioviruses into the environment.

The solution might be to develop a more genetically stable strain of type 2 OPV. Such strains have been developed by leveraging advances in basic research on polioviruses that have been carried out since the 1980s. A new OPV2 strain (nOPV2) was developed by introducing three different types of changes in the OPV2 genome. First, mutations were introduced in the 5’-noncoding region of the viral RNA in the area of a single base that is a major attenuating mutation in OPV. These changes were designed to stabilize this region against reversion. Second, an RNA stem loop structure called the cre element, which is essential for viral RNA synthesis, was relocated from its original position in the genome to the 5’-noncoding region. This alteration should prevent RNA recombination that would replace the viral 5’-end with that of other enteroviruses, thereby removing the stabilizing changes. Finally, the RNA polymerase was modified so that it made fewer copying errors and had reduced recombination frequency.

The resulting nOPV2 was tested extensively in cells in culture and in experimental animals to demonstrate that the virus did not revert within the 5’-noncoding region, did not recombine with other enteroviruses, and maintained an attenuation phenotype in animals.

Based on these findings nOPV2 and another redesigned strain produced by codon-deoptimization were tested in a phase I trial. The adult volunteers, previously immunized with poliovirus vaccine, were housed in a containment facility to prevent environmental release of nOPV2. After oral administration of either vaccine, adults were monitored for symptoms, induction of immunity, and reversion of the virus to neurovirulence. The results indicated that the nOPV2s are safe, immunogenic, and do not revert to neurovirulence, while maintaining a stable 5’-noncoding region.

Pending ongoing phase 2 trials, nOPV2 is likely to be licensed for use in quelling outbreaks of type 2 vaccine-derived polio. It cannot be tested for efficacy because there are insufficient cases of polio anywhere to allow such a study. It is hoped that the excreted vaccines will not revert to neurovirulence and will circulate for a limited time in humans, as suggested by the preclinical data, thereby eliminating type 2 vaccine-induced polio. However, the numbers of subjects in the clinical trial have been small, and the selection pressure imposed by thousands of human guts might change this outcome. Viruses have been known before to defy our expectations.

Filed Under: Basic virology, Information Tagged With: attenuated vaccine, genetic stability, neurovirulence, OPV, poliovirus, recombination, reversion, Sabin, viral, virology, virus, viruses

TWiV 619: Recombination led to emergence of SARS-CoV-2

27 May 2020 by Vincent Racaniello

Raul Rabadan joins TWiV to explain the use of computational biology to demonstrate how recombination and mutation led to emergence of SARS-CoV-2.

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Filed Under: Uncategorized Tagged With: bat CoV, computational biology, coronavirus, CoV, COVID-19, mutation, pandemic, recombination, SARS-CoV-2, spillover, viral, virology, virus, viruses, zoonosis

TWiEVO 55: Coronavirus evolution from soup to nuts

7 May 2020 by Vincent Racaniello

Nels and Vincent continue their discussion of SARS-CoV-2 evolution, with a report that the coronavirus proofreading enzyme stimulates RNA recombination, and debunking the conclusion that a change in the viral spike glycoprotein is associated with increased human to human transmission.

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Filed Under: This Week in Evolution Tagged With: coronavirus, CoV, COVID-19, error correction, evolution, exonuclease, founder effect, recombination, SARS-CoV-2, selection, spike glycoprotein, viral, virology, virus

TWiV 512: Flexuous SUMO wrestlers

23 September 2018 by Vincent Racaniello

Anne Simon joins the TWiV team to talk about plant viruses, including plum pox virus that devastates nut and stone fruit trees, and a geminivirus protein that regulates viral DNA synthesis.

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Filed Under: This Week in Virology Tagged With: dna replication, geminivirus, papaya ringspot virus, PCNA, plant virus, plum pox virus, potyvirus, recombination, rep protein, SUMO, viral, virology, virus, viruses

The purity of plaques

30 March 2017 by Vincent Racaniello

dose-response-plaque-assayThe plaque assay – my favorite assay in the world – is a time-honored procedure to determine the number of viruses in a sample, and to establish clonal virus stocks. The  linear relationship between the number of infectious particles and the plaque count (illustrated; image credit) shows that one infectious particle is sufficient to initiate infection. Despite the one-hit kinetics of plaque formation, could more than one virus contribute to a plaque?

To answer this question, ten genetically marked polioviruses were mixed and subjected to plaque assay. Of 123 plaques, 6 (4.9%) contained more than one virus. Similar results were found when polioviruses with phenotypic markers were studied.

Examination of poliovirus stocks by electron microscopy revealed both single particles and aggregates of 2 to 10 particles. Increasing particle aggregation by treatment of viruses with low pH increased co-infection frequency, indicating that aggregation of particles leads to multiply infected cells.

When these experiments were repeated with mutagenized polioviruses, the co-infection frequency increased – probably because recombination and complementation between two defective genomes leads to rescue of the defects.

Do these findings indicate that poliovirus plaque formation does not follow one-hit kinetics? The results do not prove that, in unmutagenized virus stocks, more than one poliovirus is needed to form a plaque. They only show that a small percentage of plaques contain more than one poliovirus. The presence of more than one poliovirus in 5-7% of plaques is likely a consequence of virion aggregation. It would be informative to prepare poliovirus stocks with no aggregates, and determine if co-infected plaques are still observed.

Some viruses of plants and fungi follow two-hit kinetics: two virus particles, with two different genomes, are needed for infection (illustrated). Assuming that 4-7% of poliovirus plaques are initiated by multiple viruses, the resulting plots deviate only slightly from a straight line, and do not resemble the curves of two-hit kinetics.

What are the implications of these findings for the use of plaque assays to produce clonal virus stocks? Even though the frequence of multiply infected plaques is low, the possibility of producing a mixed population is still possible, if only one plaque purification is done. In our laboratory we have always repeated the plaque purification three times, which should ensure that no multiply infected plaques are isolated.

Update 3/31/17: I would like to see similar experiments done with other viruses, to see how often multiple viruses can be found in a plaque. Examples included hepatitis A virus, which is released from cells in membranous vesicles containing multiple virus particles; and enveloped viruses, which might aggregate more frequently than naked viruses.

I looked back at the 1953 publication in which Dulbecco and Vogt first described the plaque assay for poliovirus, and demonstrated one hit kinetics. The dose-response curve clearly shows one-hit kinetics with little deviation of the individual data points from a straight line.

plaque dose response
Linear relationship between the number of plaques and the virus concentration. Image credit.

 

Filed Under: Basic virology, Information Tagged With: complementation, one hit kinetics, plaque assay, poliovirus, recombination, two hit kinetics, viral, virology, virus, virus aggregation, viruses

Genome recombination across viral families

27 October 2016 by Vincent Racaniello

coronavirus and reovirus A novel coronavirus isolated from bats in China is unusual because the genome contains a gene from a virus in a completely different family, the Reoviridae (link to paper). The finding suggests that recombination occurred between a (+) strand RNA virus and a virus with a segmented, double-stranded RNA genome.

The unusual recombinant virus was identified in rectal swabs from Rousettus leschenaulti bats in Yunnan Province, China. Sequence analysis revealed a typical coronavirus genome with the exception of a small region near the 3′-end of the viral RNA with homology to a bat reovirus. This sequence, called p10, was also detected in viral mRNAs from infected bats, further demonstrating the presence of the reovirus-like gene in the coronavirus genome.

In bat reoviruses, the p10 gene is known to encode a protein that causes cell fusion. When the p10 gene from the bat coronavirus was expressed in cells, formation of syncytia (fused cells) was observed. Furthemore, the p10 protein was detected by western blot analysis of feces from infected bats. These results indicate that the p10 protein is produced from the viral genome and that the protein is functional.

This report is not the first suggesting recombination between viruses of different families – we discussed one example here previously (link to article), and there are a handful of other examples. The important question is how such inter-family recombinants arise. It must begin with co-infection of a host with two different viruses – in this case, likely a bat – but the precise molecular events are unknown. It might be useful to attempt to isolate such recombinants in cell culture to understand the underlying mechanisms.

Filed Under: Basic virology, Information Tagged With: bat, coronavirus, MERS, recombination, reovirus, SARS, viral, virology, virus, viruses

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