New Challenge for Zika Virus Infection: Human Reservoirs?

Introduction

Zika virus (ZIKV) causes an acute self-limited infection in adults. When acquired in utero during pregnancy, however, it may lead to the development of congenital Zika syndrome (CZS) (6). In both circumstances, it is believed that there is complete viral clearance without viral persistency. However, the Flaviviridae family, which ZIKV belongs to, has well-documented registry of persistent infection and/or prolonged shedding for several viruses (13). Persistence of infection may be influenced by the individual immunological response and genetic factors (7,10,14), which substantiate the observation that some individuals become healthy viral carriers. Little is known about ZIKV prolonged shedding and persistent infection, except that it has been reported for longer periods in body fluids such as saliva, urine, and semen (5,18,19,22). To this respect, our work studied 77 subjects and shows the persistence of ZIKV from 18 months to 3 years in serum of five subjects, four of them belonging to the same family, including a child with microcephaly, and another unrelated child also with microcephaly. The viremia has persisted for up to 3 years after symptom onset. The presence of the virus and its infectious competency show that ZIKV infection could evolve to the occurrence of healthy viral carriers. The findings of this work have major consequence and implication for our understanding of ZIKV infection in humans, considering the existence of potential viral reservoirs.

Methods

Results

RT-qPCR assay demonstrated that five of the 77 subjects included in the study were positive for ZIKV up to 3 years after the initial onset of symptoms: four members of the same family (S1, S2, S3, and S4), and one other child with microcephaly (S5). For all five subjects, the results showed a cycle threshold below 38.5, which is the indicated limit of detection for the assay used (Table 1) (12). These results were validated by viral isolation. S1, S3, S4, and S5 were positive for anti-ZIKV IgG. S5 were also found positive for anti-ZIKV IgM, while S2 were negative for both. All five subjects were negative for anti-DENV IgM and IgG (Table 1). We have not tested for the presence of IgM and IgG antibodies against other viruses of the Flaviviridae family, since there is no report of other virus of this virus family circulating in the region.

The family is composed of four members with one pair of microcephalic dizygotic twins (S1). The mother became pregnant at 39 years of age, and at 2 months of pregnancy, she experienced a sudden onset of a low-grade fever, accompanied by pruritic rash, generalized malaise, and arthralgia in multiple joints. Four days after the start of her clinical manifestations, her husband, a 49-year-old man, experienced similar symptoms. They were attended by a primary care physician, who made a clinical diagnosis of a “light Dengue-like illness.” At that time, ZIKV had not yet been reported in Brazil. They were treated symptomatically and the symptoms disappeared in a few days. During her pregnancy, she was submitted to serological routine tests for hepatitis B, hepatitis C, syphilis, human immunodeficiency virus, human T leukemia virus, anti-cytomegalovirus (CMV) IgG/IgM, and anti-toxoplasmosis IgG/IgM. Except for anti-CMV IgG and anti-toxoplasmosis IgG, all other tests were negative (results not shown). Until the delivery, she performed multiple obstetric ultrasounds. Table 2 presents the results of those ultrasounds, where underdevelopment of fetus 1 (S1) can be observed.

The twins were born on October 13, 2015 (35 weeks), by cesarean surgery. The male twin (S2) was born with 2.065 g and the female (S1) with 1.335 g (low birth weight, below third percentile). At birth, cranial circumference of S2 was 31.5 cm, and S1 was 28.5 cm. They were both submitted to neonatal cranial ultrasonography (NCU). NCU for S2 showed normal results, while NCU for S1 revealed agenesis of corpus callosum. Computerized tomography scan presented thinning of the frontal, parietal, occipital, and temporal cortex; cerebral calcifications in the left cerebral hemisphere and at basal ganglia; and also dilation of the lateral ventricle. All these findings resemble CZS.

Between 2015 and end of 2016, the children were subjected to several medical tests, including antibodies detection using immunoassay methodology (Euroimmun anti-Zika ELISA anti-IgM and IgG). Test results demonstrated that (S2) is negative for anti-Zika IgG and anti-Zika IgM, while (S1) was positive for anti-Zika IgG, with negative result for anti-Zika IgM (Table 1).

Discussion

A new challenge has arisen in Brazil with the emergence of ZIKV and its co-circulation with other arboviruses (i.e., DENV and CHIKV). The clinical presentation of the disease brings considerable diagnostic difficulties on differentiating it from other arboviruses, and an increased concern regarding infection in pregnant women for being associated with development of microcephaly in fetus. ZIKV belongs to Flaviviridae family, which includes also the West Nile virus (WNV), Japanese encephalitis virus, and hepatitis C virus, all of them viruses that can cause persistent infection and/or prolonged shedding (7,14,20).

Virus transmitted mainly by biological vectors has been reported to have also interhost transmission, such as sexual route. For ZIKV, although the primary transmission occurs through mosquito bite, sexual and vertical transmission (across placenta or through breastmilk) have been previously reported (5,17,18,22), reinforcing the possibility of interhost transmission.

ZIKV was first identified in Brazil in April 2015, during a massive outbreak affecting large parts of the country (4), and now we described an unusual situation after Zika acute infection, leading to a new turning point with respect to ZIKV disease.

The results reported in this study show that ZIKV can persist in asymptomatic infected individuals and potentially transmit or perpetuate the virus as viral reservoirs. It is well documented that ZIKV can persist in human body fluids with RNA detection in serum, urine, saliva, and cerebrospinal fluids, and for longer periods in semen (188 days) (2,16). However, no study reported long-term viral persistence for up to 3 years after symptom onset/acute phase, as we report in this study. The persistence of ZIKV shedding in asymptomatic adults has so far not been reported, and poses intriguing questions.

Previous studies in rhesus monkeys infected with ZIKV demonstrated that central nervous system, lymphoid system, and gastrointestinal or genital tracts can act as anatomic sanctuaries reservoirs (9). The viral persistence in those anatomic sites suggests the involvement of an upregulation of mechanistic target of rapamycin and proinflammatory and antiapoptotic signaling pathways, among other immune mechanisms (1,11). These findings raise the possibility that viral replication could persist in these sanctuaries and lead to an asymptomatic or even a chronic infection.

The behavior of ZIKV infection resembles that of WNV or some members of the genus Pestivirus (bovine viral diarrhea virus [BVDV]) and genus Hepacivirus (hepatitis C virus) from Flaviviridae family, which have been implicated in chronic and persistence viral infections (3). Serological studies in WNV have identified persistent IgM antibodies at 6 months to 1 year postinfection in both serum and cerebrospinal fluid of infected patients (7). Another study reported IgM persistence for up to 8 years in subjects after the acute phase of infection, in parallel with a decline in IgG. The authors postulate a persistence of WNV through a still unknown mechanism (15).

Our study has limitations related to the diagnosis of the acute episode of the disease in mothers at the time of early pregnancy, as well as in their husbands. However, the pattern of the acute clinical presentation of both, during the most intense period of the ZIKV outbreak, is very much compatible with ZIKV, and nowadays both are IgG positive. The negative serology screening for the mother during pregnancy, and the undergrowth of one of her fetus in utero, associated with the neurological malformations compatible with CZS, leaves little ground for other possibilities other than ZIKV. There was also serological evidence of ZIKV infection, after birth, of the child with congenital syndrome of microcephaly.

The persistence of a viral infection implies a certain degree of immunological complacency. The clinical presentation of the S2 twin resembles the persistent infections in the genus Pestivirus (BVDV) (3). In BVDV, when the infection occurs in the third trimester of pregnancy, the individuals can be born asymptomatic, immune tolerant (serologically negative) with viremia (8,21). This early infection normally compromises the future development of the individual.

Diverse immune mechanisms may be responsible for a much larger panel of clinical presentations of ZIKV infection in humans, such as asymptomatic carriers, chronically infected infants born with CZS, chronically symptomatic patients with nonspecific symptoms, such as arthralgia and asthenia, or patients who, after the acute infection episode, have complete clearance of the virus with total resolution of the disease.

More studies are needed for a better understanding of the mechanisms involved in the persistence of ZIKV. Investigation of the host immune response, viral molecular mechanisms and the presence of anatomical sanctuary sites may give insights to elucidate the role of ZIKV viral persistence.