Tonate Virus and Fetal Abnormalities, French Guiana, 2019

We report a case of vertical transmission of Tonate virus in a pregnant woman from French Guiana. The fetus showed severe necrotic and hemorrhagic lesions of the brain and spinal cord. Clinicians should be made aware of possible adverse fetal outcomes in pregnant women infected with Tonate virus.

To detect serum TONV IgM, the Arbovirus National Reference Center in French Guiana used an inhouse IgM capture ELISA test that used whole virusbased antigens obtained from the brains of newborn mice and hyperimmune ascitic fluids. We calculated the ratio of the optical density obtained from the patient's serum to the TONV antigen divided by the optical density of the same serum on a TONV-negative antigen. We set a ratio of >3 to define the presence of TONV IgM. Evolution of the test ratio from 1.1 (negative) to 19 (strongly positive) between the 2 samples with a threshold of positivity defined by a ratio >3 suggested maternal seroconversion during early pregnancy. We obtained additional molecular amplifications from amniotic fluid using primers targeting different regions of the TONV genome (Appendix Table) and sequenced the amplicons, which yielded partial genome sequences of 256 bp corresponding to the 5′NC/nonstructural protein 1 genomic region, 176 bp to the nonstructural protein 1 region, and 374 bp to the E3/E2. We compared phylogenetic analysis results of the sequences against available VEE complex sequences in GenBank, which showed that the virus was very closely related to TONV (accession no. AF075254); the considered genome sequences shared 96.8%-98.9% nt sequence identity and 98.7%-100% aa sequence identity with TONV ( Figure 1; Appendix). The rarity of molecular detection of TONV and its divergence from the only strain previously available at our laboratory ruled out contamination as a possible cause of these results.
Fetal autopsy identified a male fetus, small for 22 weeks of gestation, with dysmorphism and fetal akinesia deformation sequence ( Figure 2, panel A). Neuropathologic examination discovered a notable meningeal hemorrhage and confirmed mild hydrocephaly ( Figure 2, panel B). Histologic examination found neuronal migration disorders (overmigration and nodular heterotopia), microglial reaction, and subarachnoidal hemorrhage ( Figure 2, panel D). The spinal cord was depleted of motor neurons ( Figure 2, panel C). We detected multiple calcifications in the grey matter of the brain, cerebellum, upper cervical spine, and mesencephalon ( Figure 2, panel B). The retina was dysplastic. In addition, the viscera revealed stigmata of ingestion of inflammatory fluid, rich in polynuclear cells. We found calcification in the liver. Because of the unavailability of a commercial probe and a positive control slide, reading the immunostaining TONV antibody test results was difficult. The high level of background suggested that the positivity of the anti-TONV signal in the cortical mantle should be interpreted with caution ( Figure 2, panel E).
We report a detailed description of fetal anomalies, mainly neurological, associated with vertical transmission of TONV in the first half of an asymptomatic pregnancy. Despite a wide prevalence in the Guianese population (52.9% in the Bas Maroni region in 2001), human infections with TONV remain poorly documented, unsurprising given the scarcity of diagnostic tools in French Guiana. TONV often involves signs and symptoms described as dengue fever-like and in rare cases, encephalitis, which attest to the neurotropism of the virus (9,10). The present diagnosis became possible only through the recent implementation of real-time RT-PCR for VEE detection at the Arbovirus National Reference Center. The evidence of vertical transmission of TONV we present could be an exception or could be more common, its occurrence having gone undetected mainly because of a lack of testing facilities. Documenting the possibility of vertical transmission of TONV by partially sequencing the viral genome in the amniotic fluid is a substantial finding indicating that the virus should be considered for public health monitoring (11,12) even though no previous cases of fetal abnormalities related to this virus have been re-ported. The presence of TONV in the amniotic fluid of a pregnant woman with a fetus with severe anomalies raises questions about a possible causal link that require special attention.
First, the co-occurrence of several histological features (presence of polynuclear cells in the digestive tract, intense glial reactions observed in the nervous system, and cellular calcifications) indicates a potential fetal infection with immunological reaction and cellular deaths. Viral encephalitis is a major cause of microglial activation and microglial nodules. Second, the spectrum of fetal lesions, particularly those observed in the central and peripheral nervous systems, has been observed with other neuroteratogenic viruses (11)(12)(13)(14). Thus, microcephaly, which received broad public attention during the Zika epidemic, appears to be the common outcome of first-trimester infections with a wide range of neuroteratogens (12). In our observation, although the hypothesis of a genetic with the presence of siderophages (sign of premortem meningeal hemorrhage, arrow). Scale bar = 1 mm. D) CD68 immunohistochemistry demonstrating microglial activation and small clusters of microglia and macrophages in the brain (hematoxylin counterstain). Scale bar = 1 mm. E-G) Immunohistochemistry, using anti-TONV mouse serum, of patient (E), control (F), and negative control (G) brains. Note the strong staining of many positive cells in the patient (arrows and inset in panel E), compared to the control brain, where a moderately diffuse background signal is shown but without strong positive cells such as in the patient. In the negative control (without anti-TONV mouse serum antibody), there is a very slight staining (arrowheads and inset in panel G) in the same cells compared with those in the patient, indicating the background signal (color trapping) in these cells. Scale bars = 300 µm; insets in panels E and G = 20 µm. cause cannot be eliminated, the fact that the patient had a normal karyotype, plus results from an array of comparative genomic hybridization and 3-generation pedigree, suggest low risk that the condition resulted from a genetic disorder. Moreover, a study providing a historical description of 8 fetuses during a 1962 VEE virus outbreak observed a hemorrhagic component in VEE virus-related fetal brain damage (2), in line with observations of the fetus in our study, indicating stigmata of hemorrhages, both old and recent, supporting the hypothesis of an infectious origin. On the basis of findings from a series of autopsies, the VEE virus study describes a case of a first-trimester maternal infection in which the fetus manifested the same spectrum of lesions, including microcephaly, arthrogryposis, and ocular anomalies (2). However, although immunostaining did not yield any strong evidence for the presence of TONV in the brain, we believe that these anomalies associated with confirmed maternal seroconversion should be reported. As experienced during the 2015-2016 Zika epidemic, any delay in identifying teratogens can have serious consequences (13).
In conclusion, our findings illustrate the possibility of vertical transmissibility of TONV and strongly suggest its neuroteratogenic effects, even in asymptomatic women. The virus's potential ability to spread beyond current endemic areas makes it critical that diagnostic tools become widely available to strengthen epidemiological surveillance and to provide more data about the potential danger of TONV for pregnant women.