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Zika

Paradoxical vaccines

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gene stops hereA 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.

Zika virus in Brazilian non-human primates

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Callithrix jacchusZika virus RNA has been detected in New World monkeys from the Northeast region of Brazil. This finding suggests that primates may serve as a reservoir host for the virus, as occurs in Africa.

The results of numerous serological surveys have shown that different Old World monkeys in Africa and Asia, including Rhesus macaques, Grivets, Redtail monkeys, and others, have antibodies that react with Zika virus. In these areas Zika virus is probably transmitted among monkeys in what is called a sylvatic cycle. Periodic outbreaks (epizootics) of Zika virus infections in nonhuman primates have been documented.

Where monkey reservoirs of Zika virus are present, humans may be infected with virus transmitted from a monkey. When non-human primates are absent, as on Yap Island, where an outbreak occurred in 2007, mosquitoes transmit the virus from human to human.

The Zika virus outbreak in Brazil has been thought to have been mainly transmitted between humans by mosquitoes. However, the results of this new study suggests that nonhuman primates could also be involved. The authors used polymerase chain reaction (PCR) to detect Zika virus RNA in sera or oral swabs from 15 marmosets and 9 capuchin monkeys in Ceará State where the virus is currently circulating. Four marmosets and three capuchins tested positive for Zika virus in this test.

Nucleotide sequence analysis of the PCR products from one marmoset and one capuchin monkey showed 100% identity with the strain of Zika virus that is circulating in Brazil.

The sampled animals were obtained from distant regions of the State. The marmosets were all free-ranging but had contact with humans, while 8 capuchins were pets and one was kept in a screening center for wild animals.

If these findings are confirmed and extended to other parts of Brazil, they would suggest that Zika virus might be spreading through non-human primates in that country. If so, they could serve as a reservoir for infection of humans via mosquito vectors.

An interesting question is when Zika virus entered monkeys in Brazil. It has been suggested that the virus entered Brazil in 2013 or 2014, and might have spread first in monkeys, first in humans, or both at the same time. I also wonder whether monkey to human transmission leads to a different disease than when virus circulates among humans.

TWiV 383: A zillion Zika papers and a Brazilian

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TWiVEsper Kallas and the Merry TWiXters analyze the latest data on Zika virus and microcephaly in Brazil, and discuss publications on a mouse model for disease, infection of a fetus, mosquito vector competence, and the cryo-EM structure of the virus particle. All on episode #383 of the science show This Week in Virology.

Audio and full show notes for TWiV #383 at microbe.tv/twiv or listen below.

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Zika virus infection of the nervous system

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FlavivirusEvidence is mounting that Zika virus is neurotropic (able to infect cells of the nervous system) and neurovirulent (causes disease of the nervous system) in humans.

The most recent evidence comes from a case report of an 81 year old French man who developed meninogoencephalitis 10 days after returning from a 4 week cruise to New Caledonia, Vanuatu, Solomon Islands, and New Zealand (meningoencephalitis is infection of the meninges - the membranes that cover the brain - and the brain). His symptoms included fever, coma, paralysis, and a transient rash. A PCR test revealed Zika virus genomes in the cerebrospinal fluid, and infectious virus was recovered after applying the CSF to Vero cells in culture.

A second case report concerns a 15 year old girl in Guadeloupe who developed left hemiparesis (weakness of one side of the body), left arm pain, frontal headache, and acute lower back pain. After admission she developed dysuria (difficulty urinating) that required catheterization. PCR revealed the presence of Zika virus genomes in her serum, urine, and cerebrospinal fluid; other bacterial and viral infections were ruled out.

Until very recently Zika virus was believed to cause a benign infection comprising rash, fever, joint pain, red eyes, and headache. There is now strong evidence that the virus can cause congential birth defects, and growing evidence that the virus is neurotropic and neurovirulent. Previously the entire Zika virus genome was recovered from brain tissue of an aborted fetus.

Zika virus is classified in the family Flaviviridae, and other members are known to be neurotropic, including West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus. West Nile virus infection may lead to acute flaccid paralysis, meningitis, encephalitis, and ocular manifestations. Examination of additional cases of Zika virus infection will be needed to document the full spectrum of illness caused by this virus.

Update: Neurotropism of Zika virus is also indicated by the findings that the virus infects human cortical neural progenitors.

Congenital Zika Syndrome

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FlavivirusData from several clinical studies in Brazil establish a strong link between infection of pregnant women with Zika virus and a variety of birth defects collectively called congenital Zika syndrome.

In the latest study conducted in Rio de Janeiro, the authors enrolled 88 pregnant women who had a rash in the previous 5 days. Of the 88 subjects, 72 tested positive for Zika virus by PCR. Fetal ultrasound was performed in 42 of the Zika virus positive women, and in all the Zika virus negative women.

The results are convincing: fetal abnormalities were detected in 12 of the 42 Zika virus positive women (29%) and in none of the Zika virus negative women.

The abnormalities include fetal death (2), microcephaly (5), ventricular calcification or other central nervous system lesions (7), and abnormal amniotic fluid volume or cerebral or umbilical artery flow (7). These observations show that Zika virus infection may lead to birth defects other than microcephaly.

The infections of these pregnant women with Zika virus took place throughout pregnancy, from week 8 to week 35. This window of susceptibility is in contrast to rubella virus which is more likely to cause birth defects when infection occurs in the first trimester.

Not all Zika virus infections seem to cause birth defects – 29% in this study. If this number holds outside of Rio de Janeiro, then birth defects should also be observed in other countries with high rates of infection. Only 20% of Zika virus infections are symptomatic, and it will be important to determine if these also lead to congenital Zika syndrome.

The increase in microcephaly associated with Zika virus infection was first noted in the northeast of Brazil. This study was done with women who live in Rio de Janeiro, in the southeast of Brazil, showing that the association is not geographically limited.

It has been suggested that fetal defects might be partly due to the presence of antibodies to dengue virus that cross-react with Zika virus and cause immune-mediated enhancement of disease. Thirty-one percent of the Zika virus positive women in this study were also positive for antibodies to dengue virus, but the paper does not report how these correlate with fetal defects.

These findings, together with results of previous studies showing recovery of the entire Zika virus genome from amniotic fluid or from fetal brain, demonstrate that this fast spreading and newly emerging virus infection is clearly a threat to the developing fetus.

We should not be surprised that a virus that had until recently only infected several thousand individuals, and which we believed caused a mild, self-limiting rash, suddenly is found to be extremely dangerous to the developing fetus. The potential for fetal damage was likely always present, but unobserved until the virus was introduced into a large population of susceptible individuals and hundreds of thousands of individuals were infected. The lesson to be learned, often easily forgotten, is that we should always expect more from viruses than we initially observe. Such was certainly the case for HIV-1; immunodeficiency was only the tip of the clinical syndrome caused by infection.

Given the pace at which Zika virus is racing through susceptible humans, it is likely to generate enough population immunity in the next five years to curtail this outbreak. However as susceptible individuals are born and accumulate, regular outbreaks will likely occur. Similarly, outbreaks of rubella virus in the US occurred every 5-6 years in the pre-vaccine era.

Not only do rubella and Zika viruses cause similar fetal and placental abnormalities, in the mother they both lead to rash, joint pain, skin itching, and lymphadenopathy without high fever.

Hopefully the similarities between rubella virus and Zika virus will stop there: it took nearly 30 years to develop a rubella virus vaccine after the discovery that infection caused birth defects.

 

Zika virus and the fetus

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FlavivirusAn epidemic of Zika virus infection began in Brazil in April 2015, and six months later there was a surge in the number of infants born with microcephaly. Confirming that Zika virus causes microcephaly will require much more information than is currently available. So far there have been few isolations of Zika virus RNA from microcephalic fetuses or amniotic fluid.

A single case report revealed the entire Zika virus genome in fetal brain tissue from a 25 year old who developed fever, muscle and eye pain, and rash during the 13th week of gestation in Natal, Brazil. The fetus was aborted at 28 weeks of gestation when fetal abnormalities, including microcephaly, were detected. Virus particles 42 to 54 nm in diameter were detected in the brain by electron microscopy.

It is probably not normal to have Zika virus in the fetal brain. However, its presence there might be a consequence of microcephaly, not a cause. As Dr. Steven Seligman writes at ProMedMail, “It is possible that brain tissue in cases of microcephaly become susceptible to Zika virus infection by a mechanism such as diminution of he blood-brain barrier.”

The entire Zika virus genome has also been detected in amniotic fluid, which surrounds the developing fetus. Two pregnant women from the state of Paraíba in Brazil reported clinical symptoms early in pregnancy consistent with Zika virus infection (fever, myalgia, rash). Microcephaly was diagnosed at 21 weeks gestation by ultrasound, and 7 weeks later samples of amniotic fluid were obtained by amniocentesis.

Amniotic fluid was centrifuged to purify virus particles, and RNA was extracted, copied into DNA by reverse transcriptase, amplified by polymerase chain reaction and subjected to deep sequencing.

The complete Zika virus genome sequence was obtained from one sample, and two smaller genome fragments from the second. Sequence analyses revealed 97-100% similarity with Zika viruses isolated from French Polynesia in 2013.

IgM antibodies to Zika virus were detected in both amniotic fluid samples, indicating that the fetus was likely infected and mounting an immune response against the virus (this antibody does not cross the placenta). In contrast, serum and urine from both mothers was negative for Zika virus IgM. This antibody appears first during infection, then subsides as levels of IgG antibody rise. It is possible that the mothers were infected with Zika virus early in pregnancy and cleared the infection, but the virus entered the fetus where it persisted.

Even if Zika virus does cause birth defects, a vaccine will likely not be available for another two years. In the meantime it would be highly advisable to practice mosquito avoidance and control.

Update 2/24/16: I asked Carolyn Coyne how a virus might reach the amniotic fluid. Her reply:

Amniotic fluid is mainly urine from the baby (after month 4ish) so if the virus is being shed in the urine, that is one way. Cells from the baby are also shed into the fluid (these are usually skin cells, but I imagine could also be from the mouth as the baby is usually drinking amniotic fluid at later stages of gestation). I will note that this is usually in a normal pregnancy and I imagine is the fetus were dying/dead (a fetus can die in utero and not be miscarried for a shockingly long time sometimes), the virus might easily enter the amniotic fluid as the fetus begins to decompose (which also happens in utero).

The two routes of entry are hematogenous or ascending. In hematogenous infections, virus present in the maternal blood would have to cross the placenta across the villous trees. In an ascending infection, the virus would be introduced into the vagina, then would have to bypass the cervix and still have to cross the placenta to access the fetus. Usually ascending infections are associated with bacteria (from UTIs mainly, but can be other). In either case, the placenta is there and would have to be crossed.

Update 2/25/16: A report in PLoS Neglected Tropical Diseases describes finding Zika virus RNA by RT-PCR in neuronal tissues but not in heart, lung, liver or placenta, in a stillborn infant with microcephaly and hydrops fetalis. The 20 year old woman from Salvador, Brazil denied having any symptoms consistent with Zika virus infection, but only one in five infections are symptomatic. As in the case described above, we do not know if Zika virus caused the fetal defects, or if virus was able to invade the fetus as a consequence of severe developmental damage.