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.


TWiVOn episode #379 of the science show This Week in Virology, Scott Tibbetts joins the TWiVirate to describe his work on the role of a herpesviral nocoding RNA in establishment of peripheral latency, and then we visit two last minute additions to the Zika virus literature.

You can find TWiV #379 at microbe.tv/twiv, or listen below.

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ATP EBOV antiviralA small molecule antiviral compound has been shown to protect rhesus monkeys against lethal Ebolavirus disease, even when given up to three days after virus inoculation.

The compound, called GS-5734, is a nucleoside analog. After uptake into cells, GS-5734 is converted to a nucleoside triphosphate (illustrated, bottom panel) which is incorporated by the viral RNA dependent RNA polymerase as it copies the viral genome. However, the nucleoside is chemically different from ATP (illustrated, top) and no further nucleotides can be incorporated into the growing RNA strand. RNA synthesis ceases, blocking production of infectious virus particles.

In cell culture GS-5734 inhibits viral replication at micromolar concentrations, in a variety of human cell types including monocyte-derived macrophages, primary macrophages, endothelial cells, and a liver cell line. The drug inhibits replication of several strains of Zaire ebolavirus, including Kikwit and Makona (from the West African outbreak); Bundibugyo ebolavirus, and Sudan ebolavirus. It also inhibits replication of another filovirus, Marburg virus, as well as viruses of different families, including respiratory syncytial virus, Junin virus, Lassa fever virus, and MERS-coronavirus, but not chikungunya virus, Venezuelan equine encephalitis virus, or HIV-1.

The RNA dependent RNA polymerase of Ebolaviruses has not yet been produced in active form, so the authors determined whether GS-5734 inhibits a related polymerase from respiratory syncytial virus. As predicted, the compound was incorporated into growing RNA chains by the enzyme, and caused premature termination.

Typically tests of antiviral candidates begin in a small animal, and if the results are promising, proceed to nonhuman primates. While a mouse model of Ebolavirus infection is available, the serum from these animals degrades GS-5374. Consequently a rhesus monkey model of infection was used to test the compound.

After intravenous administration of GS-5374, the NTP derived from it was detected in peripheral blood mononuclear cells, testes, epididymis, eyes, and brain within 4 hours. All 12 monkeys inoculated intramuscularly with Zaire ebolavirus died by 9 days post-infection. In contrast, all animals survived after administration of GS-5374 2 or 3 days after virus inoculation. These animals also had reduced virus associated pathology as measured by liver enzymes in the blood and blod clotting. Viral RNA in serum reaches 109 copies per milliliter on days 5 and 7 in untreated animals, and was undetectable in 4 of 6 treated animals.

It is likely that resistant viruses can be obtained by passage in the presence of GS-5734; whether such mutant viruses emerge early in infection, and at high frequency, is an important question that will impact clinical efficacy of the drug. The authors did not detect changes in the viral RNA polymerase gene that might be assoicated with resistance, but further work is needed to address how readily such mutants arise.

These promising results have lead to the initiation of a phase I clinical trial to determine whether GS-5734 is safe to administer to humans, and if the drug reaches sites where Ebolaviruses are known to replicate. However, determining the efficacy of the compound requires treatment of acutely Ebolavirus infected humans, of which there are none. It might be of interest to determine the ability of GS-5734 to clear persistent virus from previously infected individuals.

You can bet that GS-5734 has already been tested for activity against Zika virus.

TWiVOn episode #378 of the science show This Week in Virology, Greg Smith joins the TWiVirate to reveal how his lab discovered a switch that controls herpesvirus neuroinvasion, and then we visit the week’s news about Zika virus.

You can find TWiV #378 at microbe.tv/twiv, or you may listen below.

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FlavivirusThe title of a Eurosurveillance article, “An autochthonous case of Zika due to possible sexual transmission, Florence, Italy, 2014” was written to make the headlines. The title should be “An autochthonous case of Zika due to person to person contact, Florence, Italy, 2014.”

An Italian man returns from a 10 day holiday in Thailand and a day later develops a rash with fever and headache. Within 6 days the rash has subsided. About two weeks later his girlfriend develops a similar disease. As this was 2014 no one looked for Zika virus and both were presumed to have dengue virus infection.

The serum samples taken from the patients were pulled from the freezer after Zika virus becomes a household word in 2015. Both patients’ sera are shown to contain neutralizing antibodies against Zika virus, with a clear rise between samples taken early in illness and after recovery.

Apparently the couple had sex between the time the man’s rash subsided, and the onset of the girlfriend’s symptoms. The authors of the paper conclude that transmission by semen is suggested.

Inexplicably, the authors write:

Other transmission modalities (i.e. direct contact with other bodily fluids) are unlikely to play a role but may not be completely ruled out.

Why is it unlikely that the man had a residual rash, possibly leaking virus, which he then transferred to the woman, perhaps on one or more mucus membranes? This mode of transmission is also known as ‘close contact’ between individuals. I am waiting for a similar case report in which the couple used condoms, yet Zika virus infection was still transmitted.

Like everyone else, the authors are seduced by the possibility of sexual transmission of Zika virus. I have yet to see any clear, convincing evidence of sexual transmission of Zika virus. At worst, the risk is extremely low, although probably not zero, given that Zika virus RNA (not virus) has been found in semen of one individual. Consider these facts and act accordingly.

Zika virus and the fetus

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.

TWiVOn episode #377 of the science show This Week in Virology, the TWiVniks review the past week’s findings on Zika virus and microcephaly, and reveal a chicken protein that provides insight on the restriction of transmission of avian influenza viruses to humans.

You can find TWiV #377 at microbe.tv/twiv, or you can listen below.

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TWiEVOOn episode #5 of the science show This Week in Evolution, Sara Sawyer and Kartik Chandran join Nels and Vincent to talk about how the filovirus receptor NPC1 regulates Ebolavirus susceptibility in bats.

You can find TWiEVO #5 at microbe.tv/twievo, or you can listen below.

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TogavirusAmidst the fear surrounding Zika virus, remember that there are over 100,000 children born each year with birth defects caused by infection with rubella virus.

The virus
Rubella virus is a member of the Togaviridae family, which also includes chikungunya virus. The genome is a 9.7 kilobase, positive strand RNA enclosed in a capsid and surrounded by a membrane (illustrated; image from ViralZone).

Humans are the only natural host and reservoir of rubella virus. The virus is transmitted from human to human by respiratory aerosols.  Upon entry into the upper respiratory tract, the virus replicates in the mucosa and local lymph nodes. Virus then enters the blood and spreads to regional lymph nodes, where it replicates and a second viremia ensues. The incubation period is approximately 14 days, after which virus is shed by respiratory secretions, allowing transmission to other hosts. The second viremia brings virus to the skin, where a rash appears after 14-21 days.

Signs and Symptoms
Rubella is a mild disease associated with low grade fever, swollen lymph nodes and a morbilliform rash. Before a vaccine was available, infection typically occurred between 5-9 years of age. In 1942 the opthalmologist Norman Gregg found that many children with cataracts also had other serious congenital defects. He noticed that an epidemic of congenital cataracts was preceded by a rubella outbreak, and proposed that cataracts and other abnormalities were caused by maternal infection during pregnancy. Eventually other investigators confirmed that rubella virus could cause fetal defects when infection of the mother occurred in the first trimester of pregnancy.

Congenital rubella syndrome (CRS) is the name give to fetal defects caused by rubella virus infection. These include eye manifestations (cataracts, glaucoma, retinitis), congenital heart defects, hearing loss, microcephaly, bone disease, mental retardation, and diabetes. When a pregnant mother is infected early in pregnancy, the virus crosses the placenta and infects most fetal organ systems. No animal models are available to study how the virus causes tissue damage.

In the US during the pre-vaccine era, outbreaks of rubella were routinely accompanied by congenital rubella syndrome. An example is the 1962-65 epidemic of 12.5 million cases of rubella and 20,000 children with congenital abnormalities. The incidence of CRS during rubella outbreaks is 1-2 per 1,000 live births

Rubella infection may also lead to encephalopathy or encephalomyelitis in one case per 6,000 infections. In these cases the virus can be found in cerebrospinal fluid and in the central nervous system.

Rubella virus was the first virus shown to be teratogenic in humans. This discovery hastened development of an infectious, attenuated vaccine, which was licensed in the US in 1969. It is now given to children as part of the MMR vaccine – measles, mumps, and rubella. Its use in the US, and in many other countries, has controlled outbreaks of rubella and eliminated congenital rubella syndrome. The Centers for Disease Control declared in 2005 that endemic congenital rubella syndrome had been eliminated from the US.

Many countries, including much of African, India, Afghanistan, and Pakistan do not include rubella vaccine in routine immunization schedules. As of 2009 less than 40% of the global birth cohort was protected from rubella virus infection. The consequence is that women of childbearing age are susceptible to rubella. In nonepidemic years there are more than 100,000 infants born with CRS every year (source: WHO)

Closing Thoughts
Rubella is an eradicable disease, because the vaccine produces durable immunity, and humans are the only reservoir of the virus. In contrast, Zika virus probably cannot be eradicated, because there is a non-human reservoir of the virus – possibly non-human primates.

Before immunization, rubella was endemic worldwide, with epidemics taking place every 6-9 years, as pools of susceptibles reached a threshold. I wonder if we will see similar behavior with Zika virus, once the initial wave of spread subsides.

Like the outbreak of microcephaly in Brazil, the 100,000 children born annually with congenital rubella syndrome is a tragedy. It’s important to remember that having an effective vaccine does not guarantee control of disease – the vaccine has to be distributed to all who need it.

TWiVOn episode #376 of the science show This Week in Virology, the TWiV team discusses the latest data on Zika virus, including ocular defects in infants with microcephaly, and isolation of the entire viral genome from fetal brain tissue.

You can find TWiV #376 at microbe.tv/twiv.