A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment.

Hepatitis E virus is a prominent cause of viral hepatitis worldwide. In Western countries, most infections are asymptomatic. However, acute self-limiting hepatitis and chronic cases in immunocompromised individuals can occur. Studying HEV is challenging due to its difficulty to grow in cell culture. Consequently, the detection of the virus mainly relies on RT-qPCR, which cannot differentiate between infectious and non-infectious particles. To overcome this problem, methods assessing viral integrity offer a possible solution to differentiate between intact and damaged viruses. This study aims at optimizing existing HEV cell culture models and RT-qPCR-based assays for selectively detecting intact virions to establish a reliable model for assessing HEV infectivity. In conclusion, these newly developed methods hold promise for enhancing food safety by identifying approaches for inactivating HEV in food processing, thereby increasing food safety measures.

Hepatitis E virus in pork meat products and exposure assessment in Belgium.

Zoonotic hepatitis E virus (HEV) genotype 3 infections are the predominant cause of acute viral hepatitis in Europe, mostly associated with the consumption of HEV contaminated pork meat. In this study we looked at the HEV RNA positivity rate of pork meat products readily available from Belgian supermarkets and evaluated the overall HEV consumer exposure in a Belgian context. Two basic assessments were performed in a 'worst-case' scenario setting: one solely focusing on the contamination level of the product itself (ingredients and processing parameters) and another estimating the overall consumer exposure, taking into account consumption habits in Belgium. Non-thermal-processed ready-to-eat (i.e. ready for consumption without additional cooking step by consumer) pork meat products (e.g. raw dried sausages), had a high estimated HEV contamination level, while thermal-processed ready-to-eat pork meat products (e.g. pork liver pâté) had the highest overall consumer exposure estimates. Following these assessments, pork liver pâtés, raw dried hams and raw dried sausages (n = 54) were purchased from Belgian supermarkets (n = 3) and analyzed for HEV RNA by RT-PCR. In total, 31 % (n = 17) products tested positive. HEV RNA was found in 65 % of the pork liver pâtés, 15 % of raw dried hams and 0 % of raw dried sausages. Phylogenetic analysis of four isolates (all gt3c) from pork liver pâté samples showed similarities with human clinical cases from Germany and Belgium.

Subtype-specific differences in the risk of hospitalisation among patients infected with hepatitis E virus genotype 3 in Belgium, 2010-2018.

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.

Epidemiology and genotype 3 subtype dynamics of hepatitis E virus in Belgium, 2010 to 2017.

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.

New-Onset Refractory Status Epilepticus: More Investigations, More Questions.

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.

First TBEV serological screening in Flemish wild boar.

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.

Protective effect of different anti-rabies virus VHH constructs against rabies disease in mice.

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.

Autochthonous tick-borne encephalitis virus-seropositive cattle in Belgium: a risk-based targeted serological survey.

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 two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

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.

Infectivity of rabies virus-exposed macrophages.

Rabies virus distributes widely in infected mice, including lymphoid tissues and spleen macrophages. The infection characteristics in murine macrophages and the infectivity of virus-exposed macrophages were examined upon inoculation in mice. In vitro, Mf4/4 spleen macrophages supported mild virus production (10(4)-fold less than neuroblastoma), with formation of typical virions. Bone marrow-derived macrophages (BMM) were most efficient to capture virus, but new virus production was not detected. Virus-induced cell death was significantly stronger in BMM, which might have eliminated BMM with productive infection. Still, viral RNA remained detectable in the remaining BMM for at least 4 weeks. Injection of in vitro-infected Mf4/4 in the nose or brain proved efficient to propagate infection in mice, even when cells were pre-incubated with neutralizing antibodies. Surprisingly, injection of ex-vivo-infected BMM in the brain also led to lethal infection in 8 out of 12 mice. Injection of infected Mf4/4 in the muscle mostly favoured a protective antibody response. Despite that macrophages are less fit to support virus production, they can still act as a source of infectious virus upon transfer in mice. This may be relevant for screening donor organs/cells, for which RT-PCR should be preferred over the traditional antigen or virus isolation assays.