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Through Metagenomics Analysis, Researchers Uncover Zika Virus Genome in Amniotic Fluid

NEW YORK (GenomeWeb) – A Brazilian research team has detected the Zika virus in the amniotic fluid of two women who'd contracted the virus early in their pregnancies. Their fetuses were subsequently found to have microcephaly.

As the Oswaldo Cruz Institute's Ana de Filippis and her colleagues reported yesterday in the Lancet Infectious Diseases, they used a metagenomics-based approach to search for viruses within amniotic fluid samples obtained from the pregnant women, coming up only with the Zika virus genome. This, the researchers noted, indicates that the virus can cross the placenta. 

"Previous studies have identified Zika virus in the saliva, breast milk, and urine of mothers and their newborn babies, after having given birth," de Filippis said in a statement. "This study reports details of the Zika virus being identified directly in the amniotic fluid of a woman during her pregnancy, suggesting that the virus could cross the placental barrier and potentially infect the fetus."

In the last year, there has been sharp rise in the number of new Zika virus cases in South and Central America as well as in the Caribbean. Brazil has been the most affected by the mosquito-borne disease, with an estimated 440,000 cases to 1.3 million cases there through the end of 2015. The most common symptoms of the virus are fever, rash, joint pain, and red eyes, according to the US Centers for Disease Control and Prevention.

At the same time as the outbreak, there has been dramatic, 20-fold increase in the number of infants born with microcephaly, suggesting a link between the two.

De Filippis and her colleagues examined amniotic fluid samples from two women who had Zika-like symptoms. The first patient, a 27-year-old woman from Paraíba in northeastern Brazil, developed a rash and then a fever and muscle pain at 18 weeks' gestation. At 21 weeks, a fetal ultrasound showed signs of microcephaly, a finding that was confirmed with a second ultrasound at 27 weeks. The baby was born at 40 weeks' gestation with a head circumference of 30 centimeters.

Meanwhile, the second patient, a 35-year-old woman also from Paraíba, developed mild Zika-like symptoms at 10 weeks of gestation. An ultrasound at 22 weeks showed mild underdevelopment of the cerebellum vermis and a follow-up ultrasound at 25 weeks indicated more severe underdevelopment and microcephaly. The baby was born with enlarged ventricles of the brain, abnormally small eyes, cataracts, and severely contracted arms and leg joints.

Both women were otherwise in good health, with no evidence of diabetes or blood pressure disorders, and both did not report taking any medications or drugs, nor drinking alcohol or smoking during their pregnancy.

An amniocentesis was performed on both women at gestational week 28 to investigate the cause of microcephaly.

Using a series of RT-PCR as well as ELISA tests, de Filippis and her colleagues reported that the amniotic fluid, urine, and serum samples from the two women were free of dengue and chikungunya viruses, while another test panel indicated they were also free of the toxoplasmosis parasite, the syphilis bacterium, and the HIV, measles, rubella, cytomegalovirus, and herpes simplex viruses.

RT-PCR testing, though, confirmed the presence of the Zika virus in the amniotic fluid of both women. Additionally, the researchers isolated the viral particles found in the amniotic fluid samples for metagenomic analysis. The sequences they obtained from the first patient covered 10,793 bases of the Zika genome to 19X coverage. No other viruses were detected by sequencing and Blast analysis.

This indicates, the researchers said, that the Zika virus could cross the placenta and possibly infect the fetus.

"This study cannot determine whether the Zika virus identified in these two cases was the cause of microcephaly in the babies," de Filippis noted. "Until we understand the biological mechanism linking Zika virus to microcephaly we cannot be certain that one causes the other, and further research is urgently needed."

A case report in the New England Journal of Medicine last week found that the virus does appear to make its way to fetuses' brain.

Using an RT-PCR assay, researchers from Slovenia found the virus within the brain of a fetus with microcephaly whose mother had been living in Brazil when she became pregnant.

The woman became ill with a high fever early in her pregnancy and while Zika virus infection was suspected, no diagnostic testing was performed. When she returned to Slovenia, sonograms at 29 weeks of gestation and 32 weeks showed fetal anomalies, including microcephaly.

After the pregnancy was terminated and a fetal autopsy was performed, the researchers collected samples for further analysis.

The virus wasn't found by RT-PCR in any other fetal organs, suggesting to the researchers that it becomes localized in neurons.

Both studies further sequenced the Zika virus samples they isolated using Illumina's MiSeq platform and Thermo Fisher Scientific's Ion Torrent platform, respectively, to find that they were most similar to ones previously uncovered in French Polynesia, which had an outbreak in 2013 to 2014. The Brazilian Zika virus genome, the Brazilian team reported, shared between 97 percent and all of its sequence identity with an isolate from French Polynesia.