virology blog
About viruses and viral disease
The TWiVians present an imported case of yellow fever in New York City, and explain how a dengue virus subgenomic RNA disrupts immunity in mosquito salivary glands to increase virus replication.
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Show notes at microbe.tv/twiv.
A new breed of vaccines is on the horizon: they replicate in one type of cell, allowing for their production, but will not replicate in humans. Two different examples have recently been described for influenza and chikungunya viruses.
The influenza virus vaccine is produced by introducing multiple amber (UAG) translation stop codons in multiple viral genes. Cloned DNA copies of the mutated viral RNAs are not infectious in normal cells. However, when introduced into specially engineered ‘suppressor’ cells that can insert an amino acid at each amber stop codon, infectious viruses can be produced. These viruses will only replicate in the suppressor cells, not in normal cells, because the stop codons lead to the production of short proteins which do not function properly.
When inoculated into mice, the stop-codon containing influenza viruses infect cells, and although they do not replicate, a strong and protective immune response is induced. Because the viral genomes contain multiple mutations, the viruses are far less likely than traditional infectious, attenuated vaccines to sustain mutations that allow them to replicate in normal cells. It’s a clever approach to designing an infectious, but replication-incompetent vaccine (for more discussion, listen to TWiV #420).
Another approach is exemplified by an experimental vaccine against chikungunya virus. The authors utilize Eilat virus, a virus that only replicates in insects. The genes encoding the structural proteins of Eilat virus were replaced with those of chikungunya virus. The recombinant virus replicates in insect cells, but not in mammalian cells. The virus enters the latter cells, and some viral proteins are produced, but genome replication does not take place.
When the Eilat-Chikungunya recombinant virus in inoculated into mice, there is no genome replication, but a strong and protective immune response is induced. The block to replication – viral RNA synthesis does not occur – is not overcome by multiple passages in mice. Like the stop-codon containing influenza viruses, the Eilat recombinant virus is a replication-incompetent vaccine.
These are two different approaches to making viruses that replicate in specific cells in culture – the suppressor cells for influenza virus, and insect cells for Eilat virus. When inoculated into non-suppressor cells (influenza virus) or non-insect cells (Eilat virus), a strong immune response is initiated. Neither virus should replicate in humans, but clinical trials have to be done to determine if they are immunogenic and protective.
The advantage of these vaccine candidates compared with inactivated vaccines is that they enter cells and produce some viral proteins, likely resulting in a stronger immune response. Compared with infectious, attenuated vaccines, they are far less likely to revert to virulence, and are easier to isolate.
These two potential vaccine technologies have been demonstrated with influenza and chikungunya viruses, but they can be used for other virus. The stop-codon approach is more universally applicable, because the mutations can be introduced into the genome of any virus. The Eilat virus approach can only be used with viruses whose structural proteins are compatible with the vector – probably only togaviruses and flaviviruses. A similar approach might be used with insect-specific viruses in other virus families.
Why do I call these vaccines ‘paradoxical’? Because they are infectious and non-infectious, depending on the host cell that is used.
Note: The illustration is from a t-shirt, and the single letter code of the protein spells out a message. However the title, ‘the gene stops here’, is wrong. It should be ‘the protein stops here. The 3’-untranslated region, which continues beyond the stop codon, is considered part of the gene.
There are many examples of viruses with segmented genomes – like influenza viruses – but these genomes segments are packaged in  one virus particle. Sometimes the genome segments are separately packaged in virus particles. Such multicomponent viruses are commonly found to infect plants and fungi, but only recently have examples of such viruses that infect animals been discovered (paper link).
Sequence analysis of viruses isolated from Culex mosquitoes in Central and South American Countries revealed six new viruses with segmented RNA genomes, which was confirmed by gel electrophoresis of RNA extracted from virus particles.
Some of the virus isolates appear to lack the fifth RNA segment, and the results of RNA transfection experiments indicated that this RNA is not needed for viral infectivity.
RNA viruses with segmented genomes are common, but in this case, the surprise came when it was found that the dose-response curve of infection for these viruses was not linear. In other words, one virus particle was not sufficient to infect a cell (illustrated). In this case, between 3 and 4 particles were needed to establish an infection. These findings indicate that the viral RNA segments are separately packaged, and must enter a cell together to initiation infection.
These novel viruses, called Guaico Culex virus (GCXV) are distantly related to flaviviruses, a family of non-segmented, + strand RNA viruses. They are part of a clade of RNA viruses with segmented genomes called the Jingmenvirus, which includes a novel tick-borne virus isolated in China (previously discussed on this blog), and a variant isolated from a red colobus monkey in Uganda. These viruses are also likely to have genomes that are separately packaged.
An interesting question is to identify the selection that lead to the emergence of multicomponent viruses that require multiple particles to initiate an infection. Perhaps transmission of these types of viruses by insect vectors facilitates the introduction of multiple virus particles into a cell. How such viral genomes emerged and persisted remains a mystery that might be solved by the analysis of other viruses with similar genome architectures.
[Read more…] about Animal viruses with separately packaged RNA segments
Flaviviruses are unusual because antibodies that cross-react with different viruses can enhance infection and disease. This property, called antibody-dependent enhancement or ADE, has been documented to occur among the four serotypes of dengue virus. It has implications for infection with or vaccination against Zika virus or dengue virus.
Earlier this year (virology blog link) it was shown that antibodies to dengue virus – in the form of serum from infected patients, or two human monoclonal antibodies – bind to Zika virus and can enhance infection of Fc-receptor bearing cells (Fc receptors bind the antibody molecule, allowing uptake into cells – illustrated). When the antibodies to dengue virus were omitted, Zika virus barely infected these cells. The conclusion is that dengue antibodies enhance infection of cells in culture by Zika virus.
This early work was first published as a preprint on the bioRxiv server – which lead some to criticize me for discussing the work before peer review. However, I subjected the paper to my own peer review, of which I am entirely capable, and decided it was worthy of discussion on this blog.
The results have now been confirmed by an independent group (paper link). Sera from patients that were infected with dengue virus, as well as dengue virus specific human monoclonal antibodies, were shown to bind Zika virus and enhance infection of Fc receptor bearing cells. These are the same findings of the group who first published on bioRxiv. That paper still has not been published – apparently it is mired in peer review, with many new experiments requested. I do hope that none of the authors of the second paper are involved in delaying its publication – something that happens all too often in science. As a colleague once remarked, ‘the main function of peer review is to prevent your competitors from publishing their work’.
Whether or not antibodies to dengue virus enhance Zika virus disease in humans is an important unanswered question.
If you are wondering whether antibodies to Zika virus can enhance dengue virus infection, the answer is yes (paper link). Monoclonal antibodies were isolated from four Zika virus-infected patients, and shown to enhance infection of Fc receptor bearing cells with either Zika virus or dengue virus. Furthermore, administration of these antibodies to mice before infection with dengue virus led to severe disease and lethality, a demonstration of antibody-dependent enhancement in an animal model.
Of interest is the finding that ADE mediated lethality in this mouse model can be completely prevented by co-administering the same antibody that has been modified to block binding to Fc receptors on cells. This result suggests a modality for treating patients with enhanced disease caused by either dengue virus or Zika virus.
These observations suggest that we need to be careful when deploying vaccines against Zika virus or dengue virus – it is possible that the antibody response could enhance disease. Recently a dengue virus vaccine called Dengvaxxia was approved for use in Brazil, Mexico, and the Philippines. However, the vaccine is not licensed for use in children less than 9 years of age because in clinical trials, immunization lead to more severe disease after infection compared with non-immunized controls. Analysis of the clinical trial data (paper link) indicates that seronegative individuals of all ages were at increased risk for developing severe disease that requires hospitalization. The authors suggest that severe disease is a consequence of enhancement of infection caused by antibodies induced by the vaccine (see CIDRAP article for more information).
These observations lead to the question of whether immunization against dengue and Zika viruses might enhance disease caused by either virus. Could a solution to this potential problem be to use a vaccine that combines the four serotypes of dengue virus with Zika virus? If so, the dengue virus component should not be Dengvaxia, but possibly another vaccine (e.g. TV003 – virology blog link) that does not induce disease enhancing antibodies.
Possible sexual transmission of Zika virus, and a cell protein that allows hepatitis C virus replication in cell culture by enhancing vitamin E mediated protection against lipid peroxidation, are the subjects discussed by the TWiVerati on this week’s episode of the science show This Week in Virology.
You can find TWiV #393 at microbe.tv/twiv, or listen below.
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