Introduction
Chikungunya virus (CHIKV) is an arbovirus of the Togaviridae family and Alphavirus genus (Schuffenecker et al., 2006), which was first isolated in 1953 in Tanzania. It is endemic in Africa and Asia (Lumsden, 1955); however in 2014, it was introduced in the Americas, causing major epidemics (Escobar et al., 2016). CHIKV is a positive-strand RNA virus, enveloped, and spherical with a 60–70 nm-diameter (Higashi et al., 1967; Simizu et al., 1984; Powers et al., 2001). Its name is derived from a Makonde word meaning “the one which bends up” due to the posture some infected individuals assume as a result of arthralgia (Mavalankar et al., 2008). Other common symptoms are fever, rash, headache and myalgia (Silva and Dermody, 2017).
The principal mode of CHIKV transmission is by the bite of infected Aedes mosquitoes. However, although rare, CHIKV vertical transmission has already been reported, with the first evidence in June 2005, during an outbreak on La Reunion Island (Robillard et al., 2006). As consequence, symptomatic neonates may develop classic chikungunya similar to adults, which resolves spontaneously within 2 weeks (Ramful et al., 2007). Nevertheless, complicated or severe forms of chikungunya, such as hemorrhagic, cardiac, and neurologic manifestations have also been reported in neonates (Gérardin et al., 2008; Senanayake et al., 2009), requiring admission to intensive care units, intubation, and mechanical ventilation (Ramful et al., 2007). The rate of infected newborn was up to 49%, in mothers with intrapartum viremia (within 2 days of delivery; Ramful et al., 2007), thus, the intrapartum period seems to be the most critical one for transmission to the neonate. Most authors suggest transplacental transmission shortly before delivery is the most likely mode of transmission via mother-to-child through microtransfusions when the mother is experiencing CHIKV viremia (Ramful et al., 2007). Early maternal-fetal transmission of CHIKV has also been reported. Spontaneous abortion was observed in three cases of CHIKV infection before 16 weeks of gestation, and the viral genome was detected in amniotic fluid, chorionic villi, and in the fetal brain (Lenglet et al., 2006; Touret et al., 2006). There have also been reports of spontaneous abortion associated with CHIKV infection in which the virus was detected and placental samples exhibited malformations (Salomão et al., 2021). The presence of CHIKV has been reported in the placenta (Prata-Barbosa et al., 2018), newborn cerebrospinal fluid, amniotic fluids (Grivard et al., 2007), serum (Shenoy and Pradeep, 2012; Lyra et al., 2016), and urine (Lyra et al., 2016) along with Chikungunya IgM antibody in cerebrospinal fluid.
Here, we aimed to analyze placentas of five women who had confirmed Chikungunya during pregnancy, focusing on histopathology, investigation of viral antigens, as well as cells and cytokines that may have contributed to placental alterations.
Materials and methods
Ethical procedures
The study was approved by the Ethics Committee of the Oswaldo Cruz Foundation (CAEE: 92728218.5.0000.5248). All the mothers provided written informed consent and authorized publication of the results.
Case descriptions
The pregnant women (PW) became infected between 2018 and 2019 during Chikungunya epidemics in the cities of Rio de Janeiro and Campos dos Goytacazes. In 2019, incidence was 374.05 cases per 100,000 inhabitants in Rio de Janeiro (Superintendência de Vigilância em Saúde, 2018; CGVS, 2019). In 2018, incidence was 918.9 cases per 100,000 in Campos dos Goytacazes and rose to 942.5 cases per 100,000 in the first 6 months of 2019 (Secretaria de estado de saúde do Rio de Janeiro, 2019). The cases are described below:
– Case 1: A 40-year-old patient at 36 weeks of gestation (3rd trimester). She attended the Regional Center for Infectious Diseases (CRDI) of the Plantadores de Cana Hospital (Campos dos Goytacazes) in May 2018, reporting symptoms such as headache, severe arthralgia and exanthema. Three days after the onset of symptoms, she was hospitalized with oligohydramnios. Serological tests were performed during 3rd trimester. The placenta was collected at the time of onset (10 days after symptoms) for investigation of CHIKV infection. Newborn serum was nonreactive IgG and IgM on day 10 (result provided by hospital).
– Case 2: 26-year-old patient, 7 weeks pregnant (1st trimester). In July 2018, she attended the Regional Center for Infectious Diseases (CRDI) at the Plantadores de Cana Hospital (Campos dos Goytacazes) reporting that he had started symptoms such as arthralgia, rash, fever and nausea for 4 days. At the time of consultation, she was afebrile, anicteric and acyanotic, with blood pressure of 120 × 80 mmHg. Serum was collected for serology tests (1st sample was collected on July 17, 2018—IgM reactive; 2nd sample was collected 10 days later—IgG reactive; both during 1st trimester). The placenta was collected at delivery (40 weeks). The newborn was asymptomatic newborn, and not tested.
– Case 3: Patient aged 19 years. She attended the Gafrée and Guinle University Hospital (HUGG), Rio de Janeiro, at 12 weeks of gestation, reporting symptoms such as arthralgia, rash and fever, when the serum was positive for CHIKV (PCR and serology—IgM). At 20 weeks, she was admitted to the ICU with urinary sepsis. The placenta was collected later at delivery at 40 weeks’ gestation. There is no information about the serum of the newborn.
– Case 4: A 37-year-old patient compared to the Regional Center for Infectious Diseases (CRDI) at the Plantadores de Cana Hospital (Campos dos Goytacazes). She had symptoms at the time of delivery, in September 2018, when the placenta was collected (38 weeks). The newborn had non-reactive anti-CHIKV IgG and IgM on the first day of life (result provided by hospital).
– Case 5: A 28-year-old patient in the third trimester of pregnancy. Attended the Hospital Dr. Beda (Campos dos Goytacazes) in June 2019 for delivery (38 weeks and 4 days), in which serum was collected for serological tests (IgM reactive; IgG non-reactive). She reported symptoms such as severe arthralgia, rash, fever and headache 2 days before delivery.
Sample collection
Blood samples from the pregnant women were collected upon onset of symptoms. Placenta samples were collected and fixed in formalin (10%) upon delivery. Samples were collected at the Plantadores de Cana Hospital, Gaffrée and Guinle University Hospital, and the Doctor Beda General Hospital. Samples of term placentas from healthy donors served as controls.
Serological diagnosis of chikungunya
Serological diagnosis was performed on serum samples from PW (controls and cases) using commercial kits, according to the manufacturer’s protocol. Anti-CHIKV IgM and IgG antibodies were detected using Anti-CHIKV IgM and IgG ELISA kits, respectively (Euroimmun, Lubeck, Germany).
Viral RNA extraction and RT-qPCR for chikungunya virus in placental samples
RNA was extracted from formalin-fixed paraffin-embedded (FFPE) placental samples (collected at term—from the 37th week of pregnancy—during the delivery) using the PureLink™ FFPE RNA Isolation Kit (Invitrogen, Carlsbad California, USA) following the manufacturer’s instructions and stored at-70°C. RT-qPCR of CHIKV RNA was performed as described elsewhere (Lanciotti et al., 2007) using the ABI Prism® 7,500 Sequence Detection System (Applied Biosystems, Foster City, California, USA).
Histopathological analysis
Placental samples fixed in formalin (10%) were sectioned, dehydrated in ethanol, clarified in xylene-impregnated paraffin, and embedded in paraffin resin. Tissue was then sectioned into 5 μm slides and incubated for 90 min at 60°C to melt the paraffin. Slides were then deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol (100 to 70%). The slides were subsequently stained with hematoxylin and eosin and visualized by light microscopy (Olympus BX 53F, Japan). Digital images of main histological features were obtained using Image-Pro Plus software version 4.5.
Immunohistochemistry procedure
The paraffin-embedded tissues (4 μm thick) were incubated for 90 min at 60°C, deparaffinized in xylene, and rehydrated with ethanol. Antigen retrieval was performed by heating the tissue in the presence of citrate buffer (pH 6.0). Tissues sections were then incubated with 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase and then rinsed in Tris–HCl (pH 7.4). Slides were then rinsed in Protein Blocker solution (ScyTek, Logan, Utah, USA) for 10 min to reduce non-specific binding. Samples were incubated with the following primary antibodies: polyclonal anti-CHIKV mouse hyperimmune ascites fluids diluted 1:700; and monoclonal anti-human against: CD8 (DAKO Cytomation, USA, diluted 1:100); CD163 (Abcam, United Kingdom, 1:200); IFN-γ (R&D Systems, USA, diluted 1:30); TNF-α (Abbiotec, USA, diluted 1:200); TGF-β (Santa Cruz, USA, diluted 1:300) and IL-10 (R&D Systems, USA, diluted 1:100) and at 4°C overnight. Subsequent incubations were performed with the Two-Step Polymer Immunohistoprobe Plus (Redwood, California, USA): Amplifier for Mouse & Rabbit IgG for 15 min and HRP Polymer Detector at room temperature for 15 min. Samples were exposed to diaminobenzidine (ScyTek, Logan, Utah, United States), and Mayer’s hematoxylin (Dako, Palo Alto, California, USA) was used to counterstain. Sections were analyzed with an Olympus BX 53 microscope and images were acquired using a coupled Olympus DP72 camera.
Quantification analysis
To quantify positive cells, images from 20 random fields for each specific primary antibody (3 negative controls and 5 positive CHIKV cases) were acquired at 1,000× magnification, in an Olympus BX 53F microscope, using the software Image Pro version 7. The number of positive cells was quantified (manually counted) in each of the 20 fields, and the mean per placenta was calculated. Representative images are provided on the boards. Three placenta controls (negative for CHIKV) collected between the 28th and 40th week of pregnancy were included.
Statistical analysis
Data were analyzed with GraphPad Prism software v 6.0 (GraphPad Software, San Diego, California, USA). Cases and controls were compared using a Mann–Whitney test with a significance level of 0.05.
Discussion
The exact mechanism of CHIKV transmission to the fetus and the possible role of the placenta in this process remain unknown. Herein we analyzed placental tissues from five cases of CHIKV infection during the 1st, 2nd and 3rd trimesters. FFPE placental tissues were positive for CHIKV by real time RT-PCR in cases 1, 2, 3, and 4. In case 5, real time RT-PCR was performed only on the serum (mother and newborn) with positives results, evidencing the presence of CHIKV genome.
The serology was performed to analyze the presence of anti-CHIKV antibodies. Usually, IgM antibody is detected during the acute phase (≤7 days post symptom onset) of CHIKV infection, indicating recent infection; however could persist up to 3 months (Andrew et al., 2022). IgG antibodies are used to indicate late infection, since these antibodies are detected mainly in samples of convalescent phase (>7 days post symptom onset; Andrew et al., 2022). For example, the serum of case 2 was collected at delivery and evidenced the presence of IgG antibodies only, displaying a late infection, during the first trimester of gestation.
Regarding histopathological alterations, we found features of fetal vascular malperfusion: such as (1) stem vessel obliteration, with intramural fibrinoid deposition and also fibromuscular sclerosis, defined as thickening of the vessel wall, that results in partial or complete obliteration of the vascular lumen (Khong et al., 2016); and (2) occlusive thrombosis, a blood clot in the fetal villi vessel which blocks the blood passage, impairing exchanges between mother and fetus (Kraus, 2013). These changes were also reported in placental samples in cases of maternal hypertension and diabetes mellitus (Aldahmash et al., 2021), SARS-CoV-2 (Rebutini et al., 2021), dengue virus (DENV) (Fernandes Ribeiro et al., 2017) and other congenital infections (Stanek et al., 2011; Heider, 2017; Roberts and Torous, 2022).
We also noted villous edema and fibrinoid deposition, which were already described by Sandoval (Sandoval, 2016), and in other arboviruses infections such as DENV (Nunes et al., 2016, 2019; Fernandes Ribeiro et al., 2017) and zika virus (ZIKV; Rabelo et al., 2018, 2020). Villous edema is a pathological change related to hypoxia (Fernandes Ribeiro et al., 2017). Hypoxia in early pregnancy can alter trophoblastic differentiation and placental establishment; and in late gestational hypoxia includes changes in mitochondrial functions, endoplasmic reticulum stress, hormone production, nutrition and secretion of angiogenic factors; which depends on the extent of hypoxia (Colson et al., 2021). The increase of perivillous fibrin seems to occur in cases of trophoblastic necrosis (Chen and Roberts, 2018), which was observed in case 2. Depending on the extent of the fibrin deposition, obstruction of maternal flow may be observed, causing fetal consequences (Chen and Roberts, 2018). Mononuclear inflammatory cell infiltration in the decidua (deciduitis) and in the intervillous space (intervillositis) are chronic inflammatory lesions and have also observed as in other viral infections, such as ZIKV (Chimelli et al., 2017; Zanluca et al., 2018), DENV (Fernandes Ribeiro et al., 2017) and SARS-CoV-2 (Vivanti et al., 2020; Leal et al., 2021; Marton et al., 2021). In addition, placental exhibited dystrophic calcification, a deposition of calcium phosphate crystals in damaged areas, with necrotic portions or decreased circulation. This type of observation was noted also in ZIKV-infected placenta (Azevedo et al., 2018; Rabelo et al., 2020).
The use of the IHC technique facilitated immunolocalization of CHIKV antigen in decidual cells, Hofbauer cells, endothelial cells, stroma of chorionic villi, trophoblast cells and in cells of the intervillous space. Here, we have representative images of CHIKV+ cells in each case; however, we cannot relate the period of infection to the cell type positive for the virus; furthermore, the sample size is small to define it. These target cells are also positive in other arboviruses infections, such as DENV (Nunes et al., 2016; Fernandes Ribeiro et al., 2017) and ZIKV (Rabelo et al., 2017, 2020).
CD8+ cell expression was increased in infected placentas compared to controls, particularly in cases 1 and 2, consistent with the inflammatory infiltrate areas observed in histopathological findings. The recruitment of CD8+ T cells to tissue typically occurs due to pathogenic infection and contributes to IFN-γ and TNF-α production (Halle et al., 2017). Increase of these cells were reported also in cases of pregnancies complicated by pre-eclampsia, fetal growth restriction and small-for-gestational age (Lager et al., 2020).
CD163, which served as a macrophage activation marker, and Hofbauer cells (fetal-placental macrophages) were increased in infected placental tissues compared to the control. Hyperplasia of Hofbauer cells was also reported in ZIKV-infected placenta (Schwartz, 2017; De Noronha et al., 2018; Rabelo et al., 2020), gestational diabetes mellitus (Barke et al., 2018) and glucocorticoid administration (Tang et al., 2013). Hyperplasia of Hofbauer cells is a pathologic condition reported in ascending infections, TORCH infections, and villitis of unknown etiology (Schwartz, 2017). It contributes to the immunopathogenesis of the disease, since it plays a role in the production of inflammatory mediators; in addition to being a site of viral replication. In ZIKV studies, authors suggest that these cells could play a role in the dissemination of ZIKV to the fetus, as they are mobile and occur in perivascular locations (Simoni et al., 2017). Similarly, we found that CHIKV was able to infect Hofbauer cells, so it could be a pathway through which the virus reaches the fetus; however, this mechanism needs further investigation.
As well as histopathological changes, the infiltration of CD8+ T cells, fetal macrophages (Hofbauer cells) and several inflammatory cells into the chorionic villi may happen at any stage of pregnancy; which will culminate in complicated gestation or bad outcomes depending on the extension.
In addition to cellularity, cytokine profiles were analyzed through the immunohistochemistry. IFN-γ and TNF-α are pro-inflammatory cytokines, expression of which was increased in infected placentas compared to controls, and was expressed mainly by trophoblast cells, Hofbauer cells and inflammatory cells (in intervillous space and inside fetal capillaries); contributing to a pro-inflammatory environment. High expression of TNF-α in these cell types was also reported in DENV (Nunes et al., 2019) and ZIKV (Rabelo et al., 2020) infections.
In addition to the increase of pro-inflammatory cytokines, we also found increase of anti-inflammatory cytokines; perhaps as an attempt to contain the local inflammatory process without threatening the pregnancy. Anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF-β) are typically increased primarily in the second trimester, and decrease during the first and third trimesters (Bränn et al., 2019; Meyyazhagan et al., 2022). For the two cytokines we noted statistical difference between controls and cases. This increase has also been reported in cases of pre-eclampsia (Lyall et al., 2001) and molar lesions (Xuan et al., 2007); which could result in inhibition of trophoblast cell migration and invasion (Yang et al., 2021). High IL-10 expression was also reported in ZIKV-infected HTR8 (Luo et al., 2018). Such an increase was also observed in placental cytotrophoblast in Cytomegalovirus infection, associated with reduced matrix metalloproteinase 9 activity, which contributes to impaired cytotrophoblast remodeling of the uterine vasculature and consequently restricted fetal growth (Yamamoto-Tabata et al., 2004). Moreover, TGF-β and IL-10 are associated with tissue repair, healing and angiogenesis (Schliefsteiner et al., 2020).
CHIKV vertical transmission is known to occur, although is a rare event. Here, in all placentas we found histopathological and immunological changes, in addition to CHIKV antigen detection. However, vertical transmission was confirmed in only one case. Our findings highlight the importance of avoiding infections during pregnancy − in this case, Chikungunya − since modifications of the placenta can interfere with the functioning of this organ, and culminate in unfavorable outcomes for the pregnant women and fetus.