Venezuela is still polio-free

AFP surveillance in Pakistan

AFP surveillance in Pakistan. Image credit.

In early June it was widely reported that the first case of poliomyelitis in 30 years had been identified in Venezuela (see this Tech Times report as an example). Fortunately these reports were incorrect, and Venezuela remains free of polio. Let’s unpack exactly what happened.

In early June the Pan-American Health Organization reported that on 29 April 2018 a 34 month old Venezuelan child developed acute flaccid paralysis (AFP). AFP is defined as a sudden onset of paralysis/weakness in any part of the body of a child less than 15 years of age.

AFP has many causes, only one of which is poliovirus infection. About 50,000 cases of AFP are reported each year in India, even though polio was declared eradicated from that country in 2014.

AFP surveillance is used as part of the poliovirus eradication effort to identify cases of polio. When cases of AFP are detected, a stool sample is taken to determine if poliovirus is present. In the case of the Venezuelan child, Sabin poliovirus type 3 was isolated from the stool.

This child had not been previously immunized with Sabin vaccine, so why was the virus present in the stool? Sabin’s poliovirus vaccines are taken orally – hence the name oral poliovirus vaccine (OPV). As in a natural poliovirus infection, Sabin’s vaccines replicate in the intestinal tract and induce protective immunity there and in the bloodstream. Sabin produced these vaccine strains by passaging polioviruses in different animals and cells until viruses were obtained that no longer cause paralysis.

We now understand that recipients of OPV may excrete, within a few days, viruses that are more neurovirulent that the vaccine strains. During replication of the OPV strains in the human intestine, the viral genome undergoes mutation and recombination that eliminate the attenuating mutations that Sabin so carefully selected by passage in different hosts.

Such reversion to neurovirulence of the Sabin OPV strains can cause polio in vaccine recipients or their contacts. For example, from 1961 to 1989 in the US there were an average of 9 cases (range, 1-25 cases) of vaccine-associated paralytic poliomyelitis (VAPP), or 1 VAPP case per 2.9 million doses of OPV distributed.

As wild type polioviruses are eliminated, most of the cases of polio are caused by the vaccine: in 2017, there were 96 cases of VAPP and 22 caused by wild type poliovirus.

Here is the crux of the matter: vaccine-derived polioviruses can circulate in humans for many years undetected. When polio immunization coverage drops, these circulating vaccine-derived polioviruses can cause outbreaks of poliomyelitis.

The combination of lack of immunization of the child, the finding of AFP and Sabin type 3 OPV in the stool led to the erroneous conclusion by many (including me!) that this was a case of VAPP.

Determination of the nucleotide sequence of the Sabin type 3 poliovirus isolated from the child’s stool revealed it did not have the mutations known to cause VAPP.

How can this conclusion be made from the viral genome sequence?

As Sabin polioviruses replicate in the human intestine, are excreted, and spread in the population, they sustain genome mutations at a rate of about 1% per year. This mutation rate makes it possible to determine how long the viruses have been circulating in humans. Some of these mutations are known to restore the ability of the virus to cause paralysis. Examination of the genome of the virus isolated from the child indicated that is was very much like Sabin 3 poliovirus, and does not have the capacity to cause polio (I’ve not seen the sequence myself, so I have to take the word of the Global Polio Eradication Initiative).

The Venezuelan child had never received any type of poliovaccine, and lived in an under-immunized community. A mass vaccination campaign, using Sabin types 1 and 3 poliovaccine, had been done a few weeks before the onset of AFP. The child likely acquired Sabin 3 poliovirus from that immunization campaign, which coincided with the development of AFP. Sabin polioviruses are isolated from thousands of individuals each year who have AFP, but they are not the causative agents. The actual cause of AFP will likely never be known.

Had this been a case of poliovirus type 3 VAPP in an under-immunized area, it would have been bad news. How could we ever stop vaccinating against polio if infections can occur 30 years after the declaration of eradication?

The fact that immunization rates have fallen in parts of Venezuela is the real story here. As long as we are using Sabin OPV, immunization rates must remain high, to protect against VAPP. Meanwhile, the transition to the use of inactivated poliovaccine must be done to eliminate the threat of VAPP. As long as we use Sabin OPV, we cannot eradicate poliovirus.

TWiV 497: Europic 2018

At Europic 2018, a meeting on picornaviruses in the Netherlands, Vincent speaks with Sasha Gorbalenya, Jim Hogle, Ann Palmenberg and Frank van Kuppeveld about their careers and their research.

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TWiV 483: Every infection is unhappy in its own way

Vincent and the Virals review undermining of antiviral effectiveness by genital inflammation, and heterogeneity of influenza virus infection in single cells.

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TWiV 459: Polio turns over a new leaf

The TWiV team reviews the first FDA approved gene therapy, accidental exposure to poliovirus type 2 in a manufacturing plant, and production of a candidate poliovirus vaccine in plants.

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TWiV 435: Two virus particles walk into a cell

The TWiVome discuss the blood virome of 8,420 humans, and thoroughly geek out on a paper about the number of parental viruses in a plaque.

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The purity of plaques

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.

 

TWiV 425: All picornaviruses, all the time

The TWiVaniellos discuss a thermostable poliovirus empty capsid vaccine, and two cell genes that act as a switch between entry and clearance of picornavirus infection.

You can find TWiV #425 at microbe.tv/twiv, or listen below.

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Three countries endemic for poliovirus

poliovirusI cannot let September pass without noting that 34 years ago this month, I arrived at Columbia University to start my laboratory to do research on poliovirus (pictured). That virus is no longer the sole object of our attention – we are wrapping up some work on poliovirus and our attention has shifted elsewhere. But this is a good month to think about the status of the poliovirus eradication effort.

So far this year 26 cases of poliomyelitis have been recorded – 23 caused by wild type virus, and three caused by vaccine-derived virus. At the same time in 2015 there were 44 reported cases of polio – small progress, but, in the words of Bill Gates, the last one percent is the hardest.

One of the disappointments this year is Nigeria. It was on the verge of being polio-free for one year – the last case of type 1 poliovirus in Nigeria had been recorded in July of 2014. In August the government reported that 2 children developed polio in the Borno State. The genome sequence of the virus revealed that it had been circulating undetected in this region since 2011. Due to threats from militant extremists, it has not been possible for vaccination teams to properly cover this area, and surveillance for polioviruses has also been inefficient. The virus can circulate freely in a poorly immunized population, and as only 1% of infections lead to paralysis, cases of polio might have been missed.

The conclusion from this incident is that the declaration that poliovirus is no longer present in any region is only as good as the surveillance for the virus, which can never be perfect as all sources of infection cannot be covered.

Of the 26 cases of polio recorded so far in 2016, most have been in Afghanistan and Pakistan (9 and 14, respectively). It is quite clear that conflict has prevented vaccination teams from immunizing the population: in Pakistan, militants have attacked polio teams during vaccination campaigns.

Recently 5 of 27 sewage samples taken from different parts of the province of Balochistan in Pakistan have tested positive for poliovirus. Nucleotide sequence analysis revealed that the viruses originated in Afghanistan. The fact that such viruses are present in sewage means that there are still individuals without intestinal immunity to poliovirus in these regions. In response to this finding, a massive polio immunization campaign was planned for the end of September in Pakistan. This effort would involve 6000 teams to reach 2.4 million children. Apparently police will be deployed to protect immunization teams (source: ProMedMail).

The success of the polio eradication program so far has made it clear that if vaccines can be deployed, circulation of the virus can be curtailed. If immunization could proceed unfettered, I suspect the virus would be gone in five years. But can anyone predict whether it will be possible to curtail the violence in Pakistan, Afghanistan, and Nigeria that has limited polio vaccination efforts?

TWiV 403: It’s not easy being vaccine

The TWiV team takes on an experimental plant-based poliovirus vaccine, contradictory findings on the efficacy of Flumist, waning protection conferred by Zostavax, and a new adjuvanted subunit zoster vaccine.

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Whole plant cells producing viral capsid protein as a poliovirus vaccine candidate

poliovirusAlthough the use of the live, attenuated (Sabin) poliovirus vaccines has been instrumental in nearly eradicating the virus from the planet, the rare reversion to virulence of these strains has lead to the World Health Organization to recommend their replacement with inactivated poliovirus vaccine (IPV). Unfortunately IPV is also not without shortcomings, including high cost, failure to induce intestinal immunity, and the need to keep the vaccine at low temperatures. An experimental poliovirus vaccine produced in plants could overcome these problems.

A new vaccine candidate was made by producing the poliovirus capsid protein VP1 in the chloroplast of tobacco plants (nuclear-directed antigen synthesis is often inefficient). VP1 was fused to the cholera toxin B (CTB) subunit which allows good transmucosal delivery of the protein. Leaves were freeze dried, ground to a powder, mixed with saline and fed to mice after subcutaneous inoculation with IPV. The results show that boosting with the plant-derived VP1-CTB protein lead to higher antibody neutralizing titers (against all three poliovirus serotypes) both in the blood and in fecal extracts, compared with mice inoculated with IPV alone.

The VP1-CTP protein within lyophilized plant cells was stable for 8 months at ambient temperatures. If immunogenicity is maintained under these conditions, it would eliminate the need for a cold chain to maintain vaccine potency, an important achievement.

The authors propose that plant-produced VP1-CTP protein could substitute for IPV once the use of OPV is discontinued. Whether this suggestion is true depends on confirmation, by clinical trial, of these findings in humans. Furthermore, oral administration of VP1-CTP plant cells alone produces no serum neutralizing antibodies, and whether VP1-CTP boosts immunity in OPV recipients remains to be determined. Because VP1-CTP does not provide protection in children who have never received IPV or OPV, it cannot be used if poliovirus circulation continues indefinitely in the face of a growing cohort that has not been immunized with IPV or OPV. Nevertheless the technology has promise for the development of other vaccines that are inexpensive and do not need low temperature storage.