Since Burbelo et al. [1
] first reported the infection of horses by an equine hepacivirus (EqHV) in 2012, the presence of this non-primate hepacivirus (NPHV) has been described in the equine populations of the five continents [1
]. Depending on the geographic locations, the prevalence of EqHV measured by quantitative reverse transcriptase PCR (RT-PCR) varies from less than 1 to over 10 percent [2
]. Among all hepaciviruses recently discovered in different animal species, EqHV displays the highest genomic homology to hepatitis C virus, which chronically infects humans in the liver. The study of hepaciviral infections in equids could shed some light on the physiopathology of HCV [10
]; conversely, knowledge on HCV could also help investigating EqHV pathogenicity and elucidating its transmission route(s) (Figure 1
A recent study performed in an area where EqHV is endemic in horses failed to detect the virus in a sample of over 5000 mosquitoes, making the latter an unlikely vector of this virus [12
]. Other studies have reported the detections of EqHV-specific antibodies and/or of viral genome in serum, tissue samples and, lastly, in cerebrospinal fluid [13
]. Detection and replication of EqHV genome in different organs of adult horses, such as liver, spleen, cerebellum, and lungs were also reported following the experimental infections [9
]. Despite recent progress, EqHV tissue tropism remains largely uncharacterized in non-experimental conditions and little is known about the presence of the EqHV in equine fetuses. By comparison, most HCV infections in young children result from vertical transmission [16
]. The contamination could take place during pregnancy or upon delivery [17
], but its exact timing and mechanisms are not fully understood. Vertical transmission of EqHV in horses has started to be investigated only recently. In one study, the presence of EqHV viral RNA was measured in serum samples from 20 mare-foal pairs [19
]. Evidence of transmission to the foal was reported for only one mare, and no potential route of transmission of the virus, such as intrauterine or postpartum transfer was identified [19
The purpose of the present study is, by measuring the amount of viral genome in samples of a large population of foals deceased during the perinatal period (aborted fetuses, stillborn foals, death occurring during the first week of life), to evaluate the incidence of EqHV vertical transmission by the intrauterine route. Genomic sequence analyses showed that infections of paired mare and dead foal were caused by identical EqHV strains, demonstrating the existence of an in utero vertical transmission.
From a phylogenetic point of view, EqHV (or NPHV) is very closely related to HCV. Therefore, transmission paths reported for HCV in humans—or in chimpanzees prior to the international ban on experiments involving great apes—have also been partially investigated in horses (Figure 1
). The parenteral transmission of NPHV was experimentally demonstrated by Ramsay et al. and by Scheel et al. [14
]. The works of Postel et al. and, more recently, of Lu et al. described the presence of equine hepacivirus in biological products from horses [26
]. These observations highlight the risk of contamination when these products are injected as is in horses, as observed until the end of the 1980s for HCV with human blood transfusion, leading to an epidemic that has infected up to 3% of the world’s human population [29
]. EqHV is also believed to be transmitted directly by blood, like reported for two more-distantly-related equine pegiviruses, TDAV (Theiler’s disease-associated virus), and EPgV (equine pegivirus) [30
]. Other hypotheses, previously described for other viruses, cannot be totally excluded, such as infections transmitted by mosquitos or, less unlikely, medical treatment with contaminated blood products or instruments [1
]. Yet, plasma and antitoxin inoculations are unlikely to account for the high seroprevalence of NPHV and EPgV in horses [32
], suggesting that other modes of transmission may exist.
In addition to the parenteral route, a mother-to-child (vertical) transmission has been observed in 5% of human hepatitis C cases [16
]. In spite a high prevalence of EqHV infections worldwide [1
], such occurrence appears infrequent in horses. One case of vertical transmission from a mare to her colt was reported in 2015 [19
]. This study was carried out on 20 gestating mares, 4 of whom were infected with EqHV at the time of delivery. The presence of EqHV genome was also detected in umbilical cord blood and in the serum of one of the foals. To the authors’ knowledge, this was the first report suggesting the possibility of in utero infection by EqHV. The rate of transversal infections was surprisingly high in the stud farm where this study took place; hence, we sought to further evaluate the prevalence of EqHV vertical transmissions in samples collected between 2013 and 2016 from several stud farms in France.
Among almost 400 cases of foal perinatal death, for which samples had been harvested in our laboratory, only three new cases of possible vertical transmission were identified, confirming an anticipated low incidence of new EqHV infections by this route. In Case #2, the presence of EqHV genome in three different biological compartments: organs of the foal (liver + lungs), the allantochorion, and the serum from the mare, could support this interpretation. The same 5′UTR sequences were obtained from organs of the foal and allantochorion, while only one-base difference was identified between NS5B sequences (FR-Eq69). The viral load in the allantochorion was very low and no NS3 sequence was obtained, but in our hands NS5B sequence is the most discriminating between strains [2
]. Unfortunately, no blood sample was drawn from the mare at the time of delivery, but serum had been obtained 10 months later (FR-Eq74).
In HCV-infected individuals, quasispecies are defined as a group of similar-yet-not-identical viral genetic variants evolving at a rate between 0.8 and 2 milli-substitutions per nucleotide per site per year and overall presenting less than 5% nucleotide difference between genomes [20
]. However, Gather et al. found only one-nucleotide change between EqHV genomes in maternal serum, umbilical cord blood and serum from her foal [19
]. This could result from a relatively short delay between EqHV transmission and foal delivery. Alternatively, EqHV quasispecies in horses could drift at a slower pace than HCV quasispecies in humans. Therefore, in Case #2, it is far from clear whether the delay in the mare’s sampling would entirely explain a 5-nucleotide difference with her foal’s samples.
However, the most compelling argument for in utero transmission comes from the high viral loads detected in tissue samples from the neonate foal. First, it is highly improbable that a transmission upon delivery would produce so many genome copies in only two days [14
]. Second, a simple contamination of the foal samples by maternal blood is also very unlikely, both during gestation and upon delivery. Thus, in the latter case, the virion concentration of maternal origin should be much more diluted than observed. In the former case, allantochorion anatomy is such that antibodies barely cross the chorionic barrier, if at all [33
]; let alone viral particles of probably 50–70 nm in diameter. This comes in contrast to women, whose syncytiochorial placenta is bathed in maternal blood with potentially easy transfer of related HCV (and virus specific antibodies) from the maternal circulation to the syncytiotrophoblast. On the contrary, the equine epitheliochorial placenta has six layers of maternal and fetal tissues between the two blood circulations; hence, EqHV would be unlikely crossing this barrier, unless a receptor required for viral entry or acting as virion carrier was expressed on the allantochorion [34
]. Were it nevertheless the case, given the worldwide prevalence of EqHV infections in mares, the number of neonate foals contaminated should be much higher than what is observed. Therefore, unless a hypothetical receptor isoform is involved, variants of the virus could promote EqHV in utero transmission. Finally, if the virus replicated within the allantochorion itself, as our results suggest, viral loads in the fetus would not any longer result from a contamination by maternal virions. Instead, the allantochorion would become a likely source of transmission to the fetus. The most likely explanation for Case #2 is, therefore, an in utero transmission of EqHV to the fetus.
In utero transmission to the fetus is also supported by the results obtained in Case #1. As no maternal sample was available, a direct link could not be established between EqHV identified in the three animals. Yet, the sequences of two EqHV-positive samples, one from a dead foal (liver + lung), born in 2013, and one from an apparently-healthy foal (serum), born later in 2015, were genetically clustered (FR-Eq73 and FR-Eq84). A 100% sequence homology was even found between the two isolates, which strongly suggested a similar source of transmission over a two-year period, here from the mare to her two offspring. In Case #3, the presence of a low EqHV viral load in allantochorion was detected, which displayed a 100% sequence homology with that identified in the mare’s serum (5’UTR and NS5B region). A serum sample from her foal could be analyzed only two months after foaling, with a negative result. As the virus load in allantochorion was low, a hypothesis is that the foal cleared its infection within two months. The mare also presenting with a weak viral load upon delivering was perhaps clearing her own infection; if so, her colostrum probably contributed to the foal’s quick recovery. In the absence of serological data, this cannot be confirmed, but a link between virus load and the risk of fetal infection has recently been discussed for HCV [35
Our data are in agreement with Gather et al. [19
], who suggested for the first time the possibility of in utero transmission of EqHV. Yet, in their study, all four placentas recovered from the EqHV-positive mares were negative by quantitative RT-PCR. The lack of EqHV RNA detection in placenta could result from a very low viremia, a low test sensitivity or the region of sampling given the anatomical heterogeneity of horse allantochorion [33
]. In previous studies, we have developed a new EqHV quantitative RT-PCR method with specific primers designed on the basis of the first available equine NPHV genome [2
]. Similarly, most studies, including that of Gather et al. [19
], use primers from Burbelo et al. [1
], initially designed to detect NPHV from different species. At parturition, placentitis was observed in one case, for which no EqHV genome was detected in the foal, suggesting to the authors that vertical transmission of NPHV occurred without an infection and inflammation of the placental tissue itself. In our study, the absence of histological inflammation in two placentas of EqHV-positive foals is in agreement with this interpretation, with the difference that it also establishes for the first time the presence of the virus genome in placenta. These findings suggest that, in the fetus like in adult horses, the presence of the virus is not associated to overt macroscopic or histological lesions, independently of the viral load. Nevertheless it takes sometimes several years before the consequences of what is at first considered a benign infection are identified. This outlines the importance of identifying as many transmission routes as possible.
Routes of transmission identified for other members of the Flaviviridæ
family infecting horses and humans account for most observed infections. Thus, West Nile virus (WNV) is transmitted by mosquitoes; even if in rare occurrences, additional routes of transmission have been reported (for a review, see [36
]). For example, the first case of in utero WNV transmission was reported in a woman in 2002 [37
]. Abortion cases because of Japanese Encephalitis virus and severe Dengue infections were also reported after in utero transmission [38
]. Mother-to-child transmission of WNV via breast milk has also been described (for a review, see [36
]). Lastly, an epidemic of fetal microcephaly developing during Zika virus infections of pregnant women has raised serious concerns in several parts of the world [40
]. Occupational exposure were also reported by different studies, which led to important safety implications for persons who work in these area.
Recently, our team completed a study on nasopharyngeal swabs and detected the presence of EqHV genomes in 4 of the 93 samples analyzed [41
]. It did not enable establishing whether the transmission involved the respiratory tract, yet pointed at a possible role of the oropharyngeal sphere, as recently suggested by Altan et al. with the detection of EqHV genomes in a pool of four swabs [29
]. Other works aim to elucidate the chain of transmission, such as a vector (e.g., mosquitoes) or, even if not yet reported, sperm during insemination or natural breeding. Additional studies are necessary to confirm or refute an involvement of these pathways during the transmission of the virus.