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

Post-exposure Treatment with Anti-rabies VHH and Vaccine Significantly Improves Protection of Mice from Lethal Rabies Infection

<div><p>Post-exposure prophylaxis (PEP) against rabies infection consists of a combination of passive immunisation with plasma-derived human or equine immune globulins and active immunisation with vaccine delivered shortly after exposure. Since anti-rabies immune globulins are expensive and scarce, there is a need for cheaper alternatives that can be produced more consistently. Previously, we generated potent virus-neutralising VHH, also called Nanobodies, against the rabies glycoprotein that are effectively preventing lethal disease in an <i>in vivo</i> mouse model. The VHH domain is the smallest antigen-binding functional fragment of camelid heavy chain-only antibodies that can be manufactured in microbial expression systems. In the current study we evaluated the efficacy of half-life extended anti-rabies VHH in combination with vaccine for PEP in an intranasal rabies infection model in mice. The PEP combination therapy of systemic anti-rabies VHH and intramuscular vaccine significantly delayed the onset of disease compared to treatment with anti-rabies VHH alone, prolonged median survival time (35 versus 14 days) and decreased mortality (60% versus 19% survival rate), when treated 24 hours after rabies virus challenge. Vaccine alone was unable to rescue mice from lethal disease. As reported also for immune globulins, some interference of anti-rabies VHH with the antigenicity of the vaccine was observed, but this did not impede the synergistic effect. Post exposure treatment with vaccine and human anti-rabies immune globulins was unable to protect mice from lethal challenge. Anti-rabies VHH and vaccine act synergistically to protect mice after rabies virus exposure, which further validates the possible use of anti-rabies VHH for rabies PEP.</p></div

Set-up of the pre-exposure vaccination experiment and interventions in different treatment groups.

<p>Set-up of the pre-exposure vaccination experiment and interventions in different treatment groups.</p

Set-up of the post-exposure treatment experiment and interventions in different treatment groups.

<p>Set-up of the post-exposure treatment experiment and interventions in different treatment groups.</p

Post-exposure treatment with vaccine and anti-rabies VHH: Effect on the viral RNA load in the brain of mice.

<p>The viral load was determined at the peak of clinical symptoms in mice that developed disease (filled symbols) or at the end of the observation period (open symbols) in survivor (non-diseased) mice. The dashed line represents the limit of detection (= 5 ΔCq). Mice treated with vaccine + VHH had significantly lower viral RNA loads than naïve mice (p<0.0001), mice treated with vaccine only (p<0.001) or mice treated with VHH only (p<0.05). Viral loads of diseased mice were also lower (25.85 ΔCq) in mice that were treated with vaccine + VHH compared to naïve mice (29.58 ± 1.29 ΔCq) or treated with VHH only (29.59 ±0.76 ΔCq). Survivor mice (vaccine + VHH, VHH alone) had comparably low viral loads (3.3–11.3 ΔCq).</p

Effect of post-exposure prophylactic treatment with vaccine and human rabies immune globulins on survival in rabies mouse model.

<p>Mice were intranasally inoculated with rabies virus followed by treatment with human rabies immune globulins (HRIG) (IP) 24 hours later, either alone or in conjunction with vaccine (IM). Vaccinated mice received a second vaccine dose 3 days later. The control group consisted of mice that were not treated (virus only group). Combined treatment with vaccine and human rabies immune globulins did not differ significantly from treatment with human rabies immune globulins alone and was unable to rescue mice from lethal infection.</p

Effect of pre-exposure vaccination on survival in rabies mouse model.

<p>Mice received intramuscular (IM) vaccination at day -28 alone or in conjunction with intraperitoneal (IP) anti-rabies VHH (Rab-E8-H7-ALB11, 1.5 mg/mouse). Vaccinated mice received a booster vaccination at day -25. Control groups received a single dose of anti-rabies VHH or mock treatment (Saline) at day -28. Preventive vaccination could protect 50% of the animals from lethal infection whereas mice receiving vaccine simultaneously with anti-rabies VHH, or VHH alone, were significantly less protected from lethal disease (p<0.001).</p

No evidence of coronavirus infection by reverse transcriptase-PCR in bats in Belgium

No coronavirus was detected by PCR in lung and intestine samples of 100 bats, mostly common pipistrelles (Pipistrellus pipistrellus), collected dead between 2008 and 2013 for rabies surveillance in Belgium. The negative results contrast with the high prevalence of coronaviruses detected in fecal pellets from live-captured bats in some European countries