BackgroundHepatitis E virus (HEV) is an emerging public health concern in high-income countries and can cause acute and chronic hepatitis. Reported numbers of indigenously acquired HEV infection have increased in the past decade in many European countries. Since 2010, the National Reference Centre (NRC) for Hepatitis Viruses has been testing samples of suspected hepatitis E cases in Belgium.AimIn this surveillance report, we present the epidemiological trends of symptomatic HEV infections in Belgium, from the distribution by age, sex and geography to the molecular characterisation of the viral strains.MethodSerum samples of suspected cases sent to the NRC between 2010 and 2017 were analysed for the presence of HEV-specific IgM and RNA. Virus was sequenced for genotyping and phylogenetic analysis in all samples containing sufficient viral RNA.ResultsThe NRC reported an increase in the number of samples from suspected cases (from 309 to 2,663 per year) and in the number of laboratory-confirmed hepatitis E cases (from 25 to 117 per year). Among 217 sequenced samples, 92.6% were genotype 3 (HEV-3), followed by 6.5% of genotype 1 and 0.9% of genotype 4. HEV-3 subtype viruses were mainly 3f, 3c and 3e. HEV-3f was the most common subtype until 2015, while HEV-3c became the most common subtype in 2016 and 2017.ConclusionThe increasing trend of HEV diagnoses in Belgium may be largely explained by increased awareness and testing.

Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.

Some European countries recently reported an increase in hepatitis E virus genotype 3 (HEV-3) of the subtype 3c. No link between HEV-3 subtypes and severity is established to date. Here, we report that patients infected with HEV-3c were at lower risk of hospitalisation, compared to those infected with HEV-3f, the other main subtype circulating in Belgium.

Rabies virus is a highly neurovirulent RNA virus, which causes about 59000 deaths in humans each year. Previously, we described macrophage cytotoxicity upon infection with rabies virus. Here we examined the type of cell death and the role of specific caspases in cell death and disease development upon infection with two laboratory strains of rabies virus: Challenge Virus Standard strain-11 (CVS-11) is highly neurotropic and lethal for mice, while the attenuated Evelyn–Rotnycki–Abelseth (ERA) strain has a broader cell tropism, is non-lethal and has been used as an oral vaccine for animals. Infection of Mf4/4 macrophages with both strains led to caspase-1 activation and IL-1β and IL-18 production, as well as activation of caspases-3, -7, -8, and -9. Moreover, absence of caspase-3, but not of caspase-1 and -11 or -7, partially inhibited virus-induced cell death of bone marrow-derived macrophages. Intranasal inoculation with CVS-11 of mice deficient for either caspase-1 and -11 or -7 or both IL-1β and IL-18 led to general brain infection and lethal disease similar to wild-type mice. Deficiency of caspase-3, on the other hand, significantly delayed the onset of disease, but did not prevent final lethal outcome. Interestingly, deficiency of caspase-1/11, the key executioner of pyroptosis, aggravated disease severity caused by ERA virus, whereas wild-type mice or mice deficient for either caspase-3, -7, or both IL-1β and IL-18 presented the typical mild symptoms associated with ERA virus. In conclusion, rabies virus infection of macrophages induces caspase-1- and caspase-3-dependent cell death. In vivo caspase-1/11 and caspase-3 differently affect disease development in response to infection with the attenuated ERA strain or the virulent CVS-11 strain, respectively. Inflammatory caspases seem to control attenuated rabies virus infection, while caspase-3 aggravates virulent rabies virus infection.

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

In the frame of a Flemish wildlife surveillance in 2013, a serological screening was performed on sera from wild boar (Sus scrofa; n=238) in order to detect tick-borne encephalitis virus (TBEV)-specific antibodies. Neutralising antibodies were titrated with a seroneutralisation test (SNT), using two cut-off titres (1/10–1/15). Seven wild boars were found TBEV-seropositive and showed moderate (>1/15) to high (>1/125) SNT-titres; three individuals had borderline results (1/10–1/15). This study demonstrated the presence of TBEV-specific antibodies in wild boar and highlighted potential TBEV-foci in Flanders. Additional surveillance including direct virus testing is now recommended.

The risk of tick-borne encephalitis virus (TBEV) introduction into Belgium remains high, and the presence of infected wildlife in Belgium is suspected. Domestic animals can serve as excellent sentinels for TBEV surveillance to install an early warning surveillance component for this emerging zoonotic disease of public health importance. In a targeted, risk-based and cross-sectional sampling design, serological screening was performed on Belgian cattle (n=650), selected from the 2010 Belgian national cattle surveillance serum bank. All samples were subjected to a gold standard TBEV seroneutralization test (SNT), based on the rapid fluorescent focus inhibition test (RFFIT) protocol. Seventeen bovines were seropositive (titer >1/15) and six had borderline results (1/10 < titer < 1/15). The accuracy of the RFFIT-SNT was confirmed in a mouse inoculation test. The overall bovine TBEV seroprevalence in the targeted area was estimated between 2.61% and 4.29%. This confirms for the first time the presence of infected foci in Belgium. Further surveillance in cattle, other sentinels, ticks, and humans at risk is recommended to further determine the location and size of endemic foci and the risk for public health.

A 27-year-old previously healthy woman was admitted to the hospital with recurrent seizures. Status epilepticus developed that became refractory to third-line therapy with propofol and barbiturates. The patient had a very extensive diagnostic workup including autoimmune, viral and genetic investigations. A tentative immune therapy was proposed with high doses of steroids and plasma exchanges. Our patient had an inherited heterozygous single nucleotide variant in the sequence c.1280A>G [p.Lys427Arg] of the SMC3 gene that was insufficient to explain the seizures. Surprisingly, IgM antibodies against Japanese encephalitis virus were positive on the serum drawn 11 days after symptom onset, as detected by ELISA and the immunofluorescence antibody (IFA) technique. IgG antibodies were also positive using the IFA technique, but not with ELISA. The same investigations as well as the detection of the viral genome by the q-RT-PCR technique were negative on cerebrospinal fluid. Despite the suspicion of a viral infection, we concluded that our patient had a new-onset refractory status epilepticus of cryptogenic origin. Termination of the status epilepticus was obtained after 47 days, with a possible benefit from the introduction of ketamine.