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

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

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

A multifaceted approach for evaluating Hepatitis E virus infectivity in vitro : cell culture and innovative molecular methods for integrity assessment

Abstract: 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

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

Mean brain/serum concentration ratio.

<p>Mean brain/serum concentration ratio over time for HLE Rab-E8/H7 and Rab-E8/H7 upon intraperitoneal injection of 5 mg HLE Rab-E8/H7 or 10 mg Rab-E8/H7 in mice.</p

Co-administration of anti-rabies Rab-E8/H7 and virus directly in the brain efficiently inhibits virus infection.

<p>Mice were inoculated intracerebrally with a mix of rabies virus and 0.12 µg (1 IU) anti-rabies Rab-E8/H7 (A) or irrelevant VHH (B) and euthanized 7 days later. The anti-rabies VHH-treated mice were protected from disease, whereas all the mock-treated mice developed severe nervous disease. The pictures represent an immunofluorescence staining for viral nucleocapsid in the brain tissue. No viral antigens were visible in the brain of anti-rabies VHH-treated mice (A), whereas green fluorescent spots indicate the abundant spread of virus in the brain of mock-treated mice (B). The graph (C) presents the viral RNA load in the brains of different groups. Viral loads were significantly different between groups treated with Rab-E8/H7 and irrelevant control VHH, between Rab-E8/H7 and uninfected controls and between irrelevant control VHH and uninfected controls (*** p<0.01).</p

Post-exposure treatment with human anti-rabies immunoglobulins (Imogam).

<p>Mice were treated intraperitoneally with 65 mg (111 IU, 1 ml) of human rabies immunoglobulins at 24 hours after intranasal virus inoculation in two independent experiments (A and B). The median survival time was prolonged by 2 days, but all mice developed serious nervous disease, requiring euthanasia.</p

Comparison of the neutralizing potency of different anti-rabies VHH constructs <i>in vitro</i> and <i>in vivo</i> following pre-incubation with rabies virus.

<p>Comparison of the neutralizing potency of different anti-rabies VHH constructs <i>in vitro</i> and <i>in vivo</i> following pre-incubation with rabies virus.</p