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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Evidence 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.
Data 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.
Three reports have been published that together make a compelling case that Zika virus is causing microcephaly in Brazil.
An epidemic of Zika virus infection began in Brazil in April 2015, and by the end of the year the virus had spread through 19 states, many in the northeastern part of the country. Six months after the start of the outbreak, there was a surge in the number of infants born with microcephaly. It was not known if most of the mothers had been infected with Zika virus, as results of serological tests, virus isolation, or PCR were not available.
An initial report of 35 Brazilian infants with microcephaly born to women who either resided in or traveled to areas where Zika virus is circulating revealed that 74% of mothers had a rash (one sign of Zika virus infection) in the first or second trimester. At the time of this study no laboratory confirmation of Zika infection was available, but the infants did not have other infections associated with birth defects, including syphilis, toxoplasmosis, rubella, cytomegalovirus or herpes simplex virus.
Yesterday the CDC reported on the analysis of tissues from two infants with microcephaly who died within 20 hours of birth, and two miscarriages, all from the state of Rio Grande do Norte in Brazil. The mothers all had rashes typical of Zika virus infection in the first trimester of pregnancy, but were not tested for infection.
All four specimens were positive for Zika virus RNA by polymerase chain reaction (PCR) done with primers from two different regions of the viral RNA. Staining of tissues with anti-viral antibodies revealed the presence of viral antigens in two of the four samples, in the brain of one newborn and in the placenta from one of the miscarriages.
A second report from the University of Sao Paulo documents ocular abnormalities in Brazilian infants (from the state of Bahia) with microcephaly and presumed Zika virus infection. The mothers of 23 of 29 infants (79.3%) with microcephaly reported signs of Zika virus infection (rash, fever, joint pain, headache, itch, malaise). Of these, 18 (78.3%) had symptoms during the first trimester of pregnancy, 4 (17.4%) during the second trimester, and 1 (4.3%) during the third trimester.
No laboratory results were available to confirm Zika virus infections, but toxoplasmosis, rubella, cytomegalovirus, herpes simplex virus, syphilis, and HIV were ruled out.
Abnormalities of the eye were found in 10 of 29 (34.5%) of infants with microcephaly. These included focal pigment mottling, chorioretinal atrophy, optic nerve abnormalities, displacement of the lens, or a hole in the iris.
These observations suggest that Zika virus infection may also cause lesions of the eye, although confirmation of infection needs to be done to prove causation. This uncertainty is reflected in the title of the article: “Ocular findings in infants with microcephaly associated with presumed Zika virus congenital infection in Salvador, Brazil” (italics mine).
The final paper is, in my opinion, the blockbuster. In this single case report, a 25 year old European woman working in Natal, Brazil, became pregnant in February 2015. In the 13th week of gestation she had fever, muscle and eye pain, and rash. Ultrasound in Slovenia at 14 and 20 weeks revealed a normal fetus.
At 28 weeks of gestation fetal abnormalities were detected, including microcephaly, and the pregnancy was aborted. Autopsy revealed severe brain defects, and 42 to 54 nm virus particles were detected in the brain by electron microscopy.
Infection with a variety of microbes was ruled out, but Zika virus RNA was subsequently detected in brain tissue by PCR.
Here is the clincher – the entire Zika virus genome was identified in brain tissue by next-generation sequencing! Analysis of the sequence revealed 99.7% nucleotide identity with a Zika virus strain isolated from a patient from French Polynesia in 2013, and a strain from Sao Paulo from 2015. These findings agree with the hypothesis that the current Brazilian outbreak was triggered by a virus from Asia.
Up to now there have been few data that strongly link Zika virus infection to congenital birth defects. Of these three new studies, the recovery of a full length Zika virus genome from an infant with microcephaly is the most convincing. Given the rapidity by which new data are emerging, it seems likely that additional evidence demonstrating that Zika virus can cause microcephaly will soon be forthcoming.
I’m amazed that a flavivirus can cause birth defects – when no flavivirus has done so before*. This is a virus spread by mosquitoes, and to which most of the world is not immune. The Zika virus outbreak will surely test our ability to respond rapidly with substantial mosquito control, diagnostics, antivirals, and a vaccine.
Update 2/11/16: A second paper has been published documenting ocular abnormalities in ten infants born to mothers in Brazil who had symptoms consistent with Zika virus infection.
Update 2/12/16: *Japanese encephalitis virus and West Nile virus have been shown to cross the placenta and infect the fetus. Such events must be rare because a larger association with birth defects has not been reported.
On episode #375 of the science show This Week in Virology, the TWiVziks present everything you want to know about Zika virus, including association of infection with microcephaly and Guillain-Barré syndrome, transmission, epidemiology, and much more.
You can find TWiV #375 at microbe.tv/twiv.
The rapid spread of Zika virus through the Americas, together with the association of infection with microcephaly and Guillain-Barré syndrome, have propelled this previously ignored virus into the limelight. What is this virus and where did it come from?
Zika virus was first identified in 1947 in a sentinel monkey that was being used to monitor for the presence of yellow fever virus in the Zika Forest of Uganda. At this time cell lines were not available for studying viruses, so serum from the febrile monkey was inoculated intracerebrally into mice. All the mice became sick, and the virus isolated from their brains was called Zika virus. The same virus was subsequently isolated from Aedes africanus mosquitoes in the Zika forest.
Serological studies done in the 1950s showed that humans carried antibodies against Zika virus, and the virus was isolated from humans in Nigeria in 1968. Subsequent serological studies revealed evidence of infection in other African countries, including Uganda, Tanzania, Egypt, Central African Republic, Sierra Leone, and Gabon, as well as Asia (India, Malaysia, Philippines, Thailand, Vietnam, Indonesia).
Zika virus moved outside of Africa and Asia in 2007 and 2013 with outbreaks in Yap Island and French Polynesia, respectively. The first cases in the Americas were detected in Brazil in May 2015. The virus circulating in Brazil is an Asian genotype, possibly imported during the World Cup of 2014. As of this writing Zika virus has spread to 23 countries in the Americas.
Zika virus is a member of the flavivirus family, which also includes yellow fever virus, dengue virus, Japanese encephalitis virus, and West Nile virus. The genome is a ~10.8 kilobase, positive strand RNA enclosed in a capsid and surrounded by a membrane (illustrated; image copyright ASM Press, 2015). The envelope (E) glycoprotein, embedded in the membrane, allows attachment of the virus particle to the host cell receptor to initiate infection. As for other flaviviruses, antibodies against the E glycoprotein are likely important for protection against infection.
Zika virus is transmitted among humans by mosquito bites. The virus has been found in various mosquitoes of the Aedes genus, including Aedes africanus, Aedes apicoargenteus, Aedes leuteocephalus, Aedes aegypti, Aedes vitattus, and Aedes furcifer. Aedes albopictus was identified as the primary vector for Zika virus transmission in the Gabon outbreak of 2007. Whether there are non-human reservoirs for Zika virus has not been established.
Signs and Symptoms
Most individuals infected with Zika virus experience mild or no symptoms. About 25% of infected people develop symptoms 2-10 days after infection, including rash, fever, joint pain, red eyes, and headache. Recovery is usually complete and fatalities are rare.
Two conditions associated with Zika virus infection have made the outbreak potentially more serious. The first is development of Guillain-Barré syndrome, which is progressive muscle weakness due to damage of the peripheral nervous system. The association of Guillain-Barré was first noted in French Polynesia during a 2013 outbreak.
Congenital microcephaly has been associated with Zika virus infection in Brazil. While there are other causes of microcephaly, there has been a surge in the number of cases during the Zika virus outbreak in that country. Whether or not Zika virus infection is responsible for this birth defect is not known. One report has questioned the surge in microcephaly, suggesting that it is largely attributed to an ‘awareness’ effect. Current epidemiological data are insufficient to prove a link of microcephaly with Zika virus infection. Needed are studies in which pregnant women are monitored to see if Zika virus infection leads to microcephaly.
Given the serious nature of Guillain-Barré and microcephaly, it is prudent for pregnant women to either avoid travel to areas that are endemic for Zika virus infection, or to take measures to reduce exposure to mosquitoes.
There are currently no antiviral drugs or vaccines that can be used to treat or prevent infection with Zika virus. We do have a safe and effective vaccine against another flavivirus, yellow fever virus. Substituting the gene encoding the yellow fever E glycoprotein with that from Zika virus might be a good approach to quickly making a Zika vaccine. However testing of such a vaccine candidate might require several years.
Mosquito control is the only option for restricting Zika virus infection. Measures such as wearing clothes that cover much of the body, sleeping under a bed net, and making sure that breeding sites for mosquitoes (standing water in pots and used tires) are eliminated are examples. Reducing mosquito populations with insecticides may also help to reduce the risk of infection.
It is not surprising that Zika virus has spread extensively throughout the Americas. This area not only harbors mosquito species that can transmit the virus, but there is little population immunity to infection. Infections are likely to continue in these areas, hence it is important to determine whether or not Zika virus infection has serious consequences.
Recently Zika virus was identified in multiple states, including Texas, New York, and New Jersey, in international travelers returning to the US . Such isolations are likely to continue as long as infections occur elsewhere. Whether or not the virus becomes established in the US is a matter of conjecture. West Nile virus, which is spread by culecine mosquitoes, entered the US in 1999 and rapidly spread across the country. In contrast, Dengue virus, which is spread by Aedes mosquitoes, has not become endemic in the US.
We recently discussed Zika virus on episode #368 of the science show This Week in Virology. You can be sure that we will revisit this topic very soon.
Added 1/28/16 9:30 PM: The letter below to TWiV provides more detail on the situation in Brazil.
I hope this email finds you all well and free of pathogenic viruses.
My name is Esper Kallas, an ID specialist and Professor at the Division of Clinical Immunology and Allergy, University of São Paulo, Brazil.
I have been addicted to TWIV since a friend from U. Wisconsin participated in the GBV-C episode (David O’Connor, episode #260). Since then, never missed one episode. After long silent listening, I decided to write for the first time, motivated by the ongoing events in my country, potentially related to the Zika virus.
In the last episode, Emma wrote about events taking place in the small town of Itapetim, State of Pernambuco, Northeastern Brazil, which I will describe a bit later in this email. Before, let me bring some background information on the current situation.
Most believed Zika was a largely benign virus, causing a self-limited disease, clearly described in episode #368. Its circulation was documented after an outbreak became noticed in the State of Bahia (NE Brazil) by a group led by Guilherme Ribeiro, a talented young Infectious Diseases Scientist from Fiocruz (PMID: 26584464, Emerg Infect Dis. 2015 Dec;21(12):2274-6, free access)
However, things started to get awkward around October 2015, when a single hospital in Recife (NE Brazil) and some other practicing Obstetricians and Pediatricians from the region started reporting a mounting number of microcephaly cases in newborns, later confirmed by the national registry of newborns. The numbers are astonishing. The graph below depicts the number of cases per year prior to the surge in 2015. Only this year, 2,975 cases were reported by December 26, the vast majority in the second semester of the year. Cases are concentrated in the Northeast (map), with 2,608 cases, including 40 stillbirths or short living newborns.
In response to the situation, the Brazilian Ministry of Health has declared a national public health emergency (http://portalsaude.saude.gov.br/index.php/cidadao/principal/agenciasaude/20629-ministerio-da-saude-investiga-aumento-de-casos-de-microcefalia-em-pernambuco).
The Brazilian Ministry of Health has been presenting updates every week (see link: http://portalsaude.saude.gov.br/index.php/o-ministerio/principal/leia-mais-o-ministerio/197-secretaria-svs/20799-microcefalia). It is important to observe some imperfections in these numbers: 1. There may be an over reporting after the news made to the big media, suggesting an association between microcephaly and Zika virus. 2. The criterion to consider a microcephaly case has been changed after the current epidemic from 33cm to 32cm; this is because 33cm of head circumference is sitting in the 10th percentile of newborns at 40 weeks of pregnancy and the adjustment would bring the limit to the 3rd percentile, increasing the specificity to detect a true microcephaly case (this may result in an over reporting in the beginning of the epidemic).
The association between Zika virus infection and microcephaly was suspected since the beginning, when Brazilian health authorities ruled out other potential causes, together with the fact that the microcephaly epidemic followed Zika virus spread. Further evidences were the two positive RT-PCR for Zika RNA in two amniotic fluids obtained from two pregnancies of microcephalic fetuses and a stillborn microcephaly case with positive tissues for Zika RNA. In fact, French Polynesia went back to their records and also noticed an increase of microcephaly case reporting, following their epidemic by the same virus strain in 2013 and 2014.
Now, Zika virus transmission has been detected in several countries in the Americas (http://www.paho.org/hq/index.php?option=com_topics&view=article&id=427&Itemid=41484&lang=en).
Although strong epidemiological data suggest the association between Zika virus and the microcephaly epidemic, a causal link between the virus and the disease is still lacking and is limited to few case reports. Many questions still remain. Does the virus damage embryonic neural tissue? What is the percentage of fetuses getting infected when the mother acquires Zika virus during pregnancy? Does the stage of pregnancy interfere with virus ability to be transmitted to the fetus and the development of neurologic effects? Are there other neurological defects related to Zika virus infection? Is there another cofactor involved, such as malnutrition or other concurrent infection? All these questions are exceedingly important to provide counseling to pregnant women and those who are planning to become pregnant, especially in Northeastern Brazil. In fact, Brazilian authorities have been recommending avoiding pregnancy until this situation is further clarified.
The microcephaly epidemic impact is unimaginable. It is a tragedy. These children are compromised for life and the impact on their families is beyond any prediction.
Back to the story sent by Emma. A small town in the North of Pernambuco State, named Itapetim, has almost 14 thousand inhabitants and has reported 11 cases of microcephaly in the past 3 months. This very same town has been suffering from a prolonged drought, since September 2013 when the last reservoir went dry. Perhaps the storage of clean water or the limited resources has led to the best environment for arbovirus spread and the development of microcephaly.
But the Zika virus’s impact may be reaching further. An increase in Guillain-Barré syndrome cases has also been noticed in the Northeast of Brazil, possibly related to the epidemic.
Several groups have been trying to establish animal models to study the interaction of Zika virus with neural tissue. The forthcoming developments are critical to better understand the virus immunopathology and confirm (or refute) the association between the virus infection and neurologic damage in fetuses and in the infected host developing Guillain-Barré syndrome. Many things still shrouded in mystery.
Keep on the good work. I will keep on listening!
On episode #365 of the science show This Week in Virology, Vincent, Alan, and Kathy trace the feud over genome editing, a new virus discovered in human blood, and the origins of hepatitis A virus.
You can find TWiV #365 at www.microbe.tv/twiv.
Episode #344 of the science show This Week in Virology was recorded at the Glasgow Science Festival microTALKS, where Vincent spoke with Ruth, Glen, and Esther about their research on viruses and Hodgkin lymphoma, adenovirus structure and entry into cells, and interactions between arthropod borne viruses and their hosts.
You can find TWiV #344 at www.microbe.tv/twiv.