NS3 helicase specific antibodies and viral RNA detection of a BovHepV positive

Besides the discovery of BovHepV in Germany (167) and Africa (155), presence of BovHepV has recently been described in calves from USA dairy herds (288), in cattle serum samples from Brazil dairy herds (169) and China (289). These findings demonstrate a wide distribution of BovHepV on several continents. Apart from the existence and prevalence of BovHepV the possibility of BovHepV causing persistent infections in cows, like HCV does in humans, was addressed in this thesis. For this, 25 cows in a German BovHepV positive herd were investigated over one year in two to three month intervals, which allowed a clear differentiation of acute and persistent infections under natural conditions. In a first study, it was suggested that the bovine hepacivirus is able to establish persistent infections (167). For further evaluation of the biological properties of BovHepV, including course of infection, antibody response, and host tropism, a qRT-PCR with primers and probe binding in a highly conserved region in the viral 5` NTR was established to detect viral RNA and a LIPS assay using the NS3 helicase domain as antigen was developed and used to analyze the animals’

immune responses. With regard to the course of infection the animals could be allocated into three groups depending on the presence of BovHepV RNA in the serum.

The first group contained five animals that were tested positive at five consecutive sampling points and were thus classified as persistently infected. 10 cattle in group 2 were at least once positive on RNA level and the remaining 10 animals in group 3 were negative at all sampling points. Interestingly, all five animals in group 1 showed high RNA genome copy numbers between 10⁴ and more than 10⁸ copies per ml serum, but specific antibodies against NS3 helicase were not detectable. Especially animals 3, 6, 25 and 35 in group 1 showed

consistently high virus copies, which is typical for persistent infections. In addition, these four cows were tested negative for antibodies at each sampling point. One possible explanation for the antibody absence might be the production of antibodies against other viral proteins than NS3, like the envelope protein E2, as immune response. Another possibility could be a stressed immune system by different pathogens like viruses or bacteria weak antibody production occured. By using a third-generation anti-HCV kit (core, NS3, NS4 and NS5) or an enzyme immunoassay (E2) it was shown that immunocompromised HCV infected patients can exhibit detectable antibodies against the envelope protein 2 whereas antibodies against the core, NS3, NS4 and NS5 were below the detection limit and some patients were tested positive on HCV RNA and E2 antibodies (290).

Three cows [no. 19, 31 and 39] in group 2 showed a low RNA level at only one sampling point. The remaining animals of group 2 [no. 11, 12, 17, 21, 24, 26 and 40] were tested positive at least twice in succession. An immune response by production of antibodies against NS3 could be observed in five cattle [no. 17, 21, 24, 26 and 40]. In animals 11 and 12 BovHepV RNA was detectable in the serum at four and five sampling points respectively, but no antibodies were identified [no. 12] or the antibody level dropped from the first to the last time point of sampling [no. 11]. The remaining cows, which were PCR positive only once [no. 19, 31 and 39], showed an immune response, although BovHepV RNA was not detectable at an earlier time point [no. 19], showed an increased antibody response at the last sampling [no. 31] or showed no immune response over the whole range [no. 39].

Additionally, high antibody titers could be detected at the sampling dates after viral RNA was detected [no. 17, 21 and 24], which is typical for an acute infection and virus clearance, whereas cattle 26 and 40 displayed a reinfection after the clearance. Moreover, the antibody levels decreased under the detection level in the LIPS assay after virus elimination.

Surprisingly, animal 11 had NS3 antibodies that dropped during the sampling periods while the RNA copies increased. Perhaps the infection was cleared at the first sampling time point in the presence of NS3 specific antibodies, whereas after re-infection antibodies against other BovHepV proteins, like envelope proteins, were produced by the immune system, but could not be detected by the NS3 specific LIPS assay. Furthermore, after the clearance at the fifth visit a re-infection occurred in this cow. Animal 12 showed relatively high copy numbers at visits four to seven but the immune response was absent with the exception of sampling time

point seven, whereas the immune system seemed to produce antibodies against BovHepV NS3. A possible reason could be a suppressed immune system caused by other infectious agents or negative environmental influences leading to delayed immunological reactions. The antibody production of animal 31 was missing despite infection with BovHepV, while in animal 39 it was initiated at late time points after genome detection. One explanation could be that a very low copy number of about 10² copies per ml found in animal 39 might be insufficient to activate the immune system. Furthermore, animal 31 displayed a higher copy number than animal 39 (about 10⁴) but nevertheless lower than the other animals with a fast immune response. Perhaps the virus load was not high enough to stimulate the immune system immediately. Surprisingly, animal 19 exhibited a high antibody level at time point five although at earlier time points virus detection was negative. Reasons could be a cross reactivity with other related hepaciviruses or with an unknown virus or a short infection with low virus load between time point four and five. Group 3 was tested negative on genome level as well as on antibody level, which showed the absence of an active infection and a specific immunological response to BovHepV.

Burbelo et al. investigated herd independent horse serum samples at a single time point on presence of EHcV NS3 specific antibodies and viral genomes. Some horses cleared the EHcV infection because no EHcV viral RNA but antibodies were observed at this time point or the virus loud was under the detection level. Other horses displayed an infection with EHcV as the samples were tested positive on virus genome RNA and antibodies. All negative tested samples on EHcV virus genome were also negative for NS3 specific antibodies, which indicated that these animals had never been in contact with EHcV so far or that the infection occurred a long time ago (148). A second working group analyzed an equine cohort for the presence of viral RNA and antibodies for at least 6 months. As found in our study, some animals were acutely or persistently infected and some cleared the EHcV infection because no viral RNA but antibodies were still detectable, or the virus load was under the detection level (106). Moreover, Reichert et al. tested equine serum samples for viral RNA and immune response and, in accordance with our and other working groups studies, they found acute, persistent, as well as cleared infections and horses that were never infected with EHcV (271).

The hepatitis C virus is a blood-borne virus and transmission occurs mainly via parenteral route (291). Gather et al. could show that EHcV can be transmitted vertically in horses (280).

Some authors envisaged the possibility that HCV evolved from a horse-to-human transmission event because EHcV is closely related to HCV (105, 160), while others suggested that HCV originated in relatively recent times from one or multiple cross-species transmission events from a still to be defined species (160, 292, 293). However, the strict species-specificity as well as the ability of HCV to persist lifelong in humans led to the alternative hypothesis according to which HCV-related viruses have been infecting humans and other primates throughout their evolutionary history (160, 294). Whereas the hepaciviruses of the so far known species are far clustered and no cross-transmission could be observed so far, it seems that the hepaciviruses are species specific (167). In our study it could only be speculated how the virus was introduced into the cattle herd and how it was transmitted between the animals, but mass vaccination without changing needles can be one possibility. So far, there is no evidence for a transmission via milk, feces, urine or on a respiratory route because such samples of BovHepV positive animals were tested negative in a previous study (167).