Geographic Disparities in Domestic Pig Population Exposure to Ebola Viruses, Guinea, 2017-2019.

Although pigs are naturally susceptible to Reston virus and experimentally to Ebola virus (EBOV), their role in Orthoebolavirus ecology remains unknown. We tested 888 serum samples collected from pigs in Guinea during 2017-2019 (between the 2013-16 epidemic and its resurgence in 2021) by indirect ELISA against the EBOV nucleoprotein. We identified 2 hotspots of possible pig exposure by IgG titer levels: the northern coast had 48.7% of positive serum samples (37/76), and Forest Guinea, bordering Sierra Leone and Liberia, where the virus emerged and reemerged, had 50% of positive serum samples (98/196). The multitarget Luminex approach confirms ELISA results against Ebola nucleoprotein and highlights cross-reactivities to glycoprotein of EBOV, Reston virus, and Bundibugyo virus. Those results are consistent with previous observations of the circulation of Orthoebolavirus species in pig farming regions in Sierra Leone and Ghana, suggesting potential risk for Ebola virus disease in humans, especially in Forest Guinea.

Epidemiologic Survey of Crimean-Congo Hemorrhagic Fever Virus in Suids, Spain.

We conducted a cross-sectional study in wild boar and extensively managed Iberian pig populations in a hotspot area of Crimean-Congo hemorrhagic fever virus (CCHFV) in Spain. We tested for antibodies against CCHFV by using 2 ELISAs in parallel. We assessed the presence of CCHFV RNA by means of reverse transcription quantitative PCR protocol, which detects all genotypes. A total of 113 (21.8%) of 518 suids sampled showed antibodies against CCHFV by ELISA. By species, 106 (39.7%) of 267 wild boars and 7 (2.8%) of 251 Iberian pigs analyzed were seropositive. Of the 231 Iberian pigs and 231 wild boars analyzed, none tested positive for CCHFV RNA. These findings indicate high CCHFV exposure in wild boar populations in endemic areas and confirm the susceptibility of extensively reared pigs to CCHFV, even though they may only play a limited role in the enzootic cycle.

Prion protein gene (PRNP) variation in German and Danish cervids.

The structure of cellular prion proteins encoded by the prion protein gene (PRNP) impacts susceptibility to transmissible spongiform encephalopathies, including chronic wasting disease (CWD) in deer. The recent emergence of CWD in Northern European reindeer (Rangifer tarandus), moose (Alces alces alces) and red deer (Cervus elaphus), in parallel with the outbreak in North America, gives reason to investigate PRNP variation in European deer, to implement risk assessments and adjust CWD management for deer populations under threat. We here report PRNP-sequence data from 911 samples of German red, roe (Capreolus capreolus), sika (Cervus nippon) and fallow deer (Dama dama) as well as additional data from 26 Danish red deer close to the German border and four zoo species not native to Germany. No PRNP sequence variation was observed in roe and fallow deer, as previously described for populations across Europe. In contrast, a broad PRNP variation was detected in red deer, with non-synonymous polymorphisms at codons 98, 226 and 247 as well as synonymous mutations at codons 21, 78, 136 and 185. Moreover, a novel 24 bp deletion within the octapeptide repeat was detected. In summary, 14 genotypes were seen in red deer with significant differences in their geographical distribution and frequencies, including geographical clustering of certain genotypes, suggesting "PRNP-linages" in this species. Based on data from North American CWD and the genotyping results of the European CWD cases, we would predict that large proportions of wild cervids in Europe might be susceptible to CWD once introduced to naive populations.

In silico identification of novel pre-microRNA genes in Rift valley fever virus suggest new pathomechanisms for embryo-fetal dysgenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the post-transcriptional expression of target genes. Virus-encoded miRNAs play an important role in the replication of viruses, modulate gene expression in both the virus and host, and affect their persistence and immune evasion in hosts. This renders viral miRNAs as potential targets for therapeutic applications, especially against pathogenic viruses that infect humans and animals. Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic RNA virus that causes severe disease in both humans and livestock. High mortality among newborn lambs and abortion storms are key characteristics of an RVF outbreak. To date, limited information is available on RVFV-derived miRNAs. In this study, computational methods were used to analyse the RVFV genome for putative pre-miRNA genes, which were then analysed for the presence of mature miRNAs. We detected 19 RVFV-encoded miRNAs and identified their potential mRNAs targets in sheep (Ovis aries), the most susceptible host. The identification of significantly enriched O. aries genes in association with RVFV miRNAs will help elucidate the molecular mechanisms underlying RVFV pathogenesis and potentially uncover novel drug targets for RVFV.

Hepatitis E Seroprevalence and Detection of Genotype 3 Strains in Domestic Pigs from Sierra Leone Collected in 2016 and 2017.

Hepatitis E virus (HEV) is the main cause of acute hepatitis in humans worldwide and is responsible for a large number of outbreaks especially in Africa. Human infections are mainly caused by genotypes 1 and 2 of the genus Paslahepevirus, which are exclusively associated with humans. In contrast, viruses of genotypes 3 and 4 are zoonotic and have their main reservoir in domestic and wild pigs, from which they can be transmitted to humans primarily through the consumption of meat products. Both genotypes 3 and 4 are widespread in Europe, Asia, and North America and lead to sporadic cases of hepatitis E. However, there is little information available on the prevalence of these genotypes and possible transmission routes from animal reservoirs to humans in African countries. We therefore analysed 1086 pig sera collected in 2016/2017 in four districts in Sierra Leone for antibodies against HEV using a newly designed in-house ELISA. In addition, the samples were also analysed for HEV RNA by quantitative real-time RT-PCR. The overall seroprevalence in Sierra Leone was low with only 44 positive sera and a prevalence of 4.0%. Two serum pools were RT-PCR-positive and recovered partial sequences clustered into the genotype 3 (HEV-3) of the order Paslahepevirus, species Paslahepevirus balayani. The results are the first evidence of HEV-3 infection in pigs from Sierra Leone and demonstrate a low circulation of the virus in these animals to date. Further studies should include an examination of humans, especially those with close contact with pigs and porcine products, as well as environmental sampling to evaluate public health effects within the framework of a One Health approach.

Approaching the complexity of Crimean-Congo hemorrhagic fever virus serology: A study in swine.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals. For the detection of anti-CCHFV antibodies in swine, we established a swine-specific in-house ELISA using a panel of swine sera from CCHFV-free regions and regions with reported CCHFV circulation. We initially screened more than 700 serum samples from wild boar and domestic pigs and observed a correlation of ≃67% between the commercial and the in-house test. From these sera, we selected a panel of 60 samples that were further analyzed in a newly established indirect immunofluorescence assay (iIFA) and virus neutralization test. ELISA-non-reactive samples tested negative. Interestingly, only a subset of samples reactive in both ELISA and iIFA displayed CCHFV-neutralizing antibodies. The observed partial discrepancy between the tests may be explained by different test sensitivities, antibody cross-reactivities or suggests that the immune response to CCHFV in swine is not necessarily associated with eliciting neutralizing antibodies. Overall, this study highlights that meaningful CCHFV serology in swine, and possibly other species, should involve the performance of multiple tests and careful interpretation of the results.

Evaluation of Non-Vector Transmission of Usutu Virus in Domestic Canaries (Serinus canaria).

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV "overwinters", the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>102 TCID50). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.

A Review on Crimean-Congo Hemorrhagic Fever Infections in Tunisia.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic tick-borne disease, caused by an arbovirus of the genus Orthonairovirus and the family Nairoviridae. Crimean-Congo hemorrhagic fever virus (CCHFV) is widespread in several regions of the world. While the virus is not pathogenic to all susceptible livestock and wild mammals, it can lead to severe hemorrhagic fever in humans. In this review, we compiled published data on CCHFV infections in humans, animals, and ticks in Tunisia. Based on that, we discussed the epidemiology and the distribution patterns of CCHFV infections highlighting the risk factors for this virus in the country. CCHF infection prevalence in humans was estimated to 2.76% (5/181) and 5% (2/38) in Tunisian febrile patients and Tunisian slaughterhouse workers, respectively. Concurrently, seroprevalence in domestic ungulates (sheep, goats, cattle, and dromedaries) ranged from zero to 89.7%, and only one Hyalomma impeltatum tick specimen collected from dromedaries in southern Tunisian was positive for CCHFV by reverse transcriptase-polymerase chain reaction (0.6%; 1/165). As Tunisian studies on CCHFV are geographically scattered and limited due to very small sample sizes, further studies are needed to improve the knowledge on the epidemiology of CCHF in Tunisia.

Immunisation of pigs with recombinant HEV vaccines does not protect from infection with HEV genotype 3.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide. Up to now, no approved treatment nor a globally licensed vaccine is available. Several recombinant HEV vaccines have been developed to protect against HEV infection in humans, including the commercially available Hecolin vaccine, which are mainly based on HEV genotype 1. However, the efficacy of these vaccines against other HEV genotypes, especially genotype 3 is unknown. In this study, we evaluated the protective efficacy of Hecolin® and a novel genotype 3-based vaccine p239(gt3) against HEV-3 in a pig infection model. Pigs were divided into three groups: one group was vaccinated with Hecolin®, the second group was vaccinated with p239(gt3), and the control group received no vaccine. All pigs were subsequently challenged with HEV genotype 3 to assess the effectiveness of the vaccines. Although all immunised animals developed a high titer of neutralizing antibodies, the results showed that both vaccine applications could not provide complete protection against HEV (gt3) infection: Two out of four animals of the Hecolin® group displayed even virus shedding, and viral RNA could be detected in bile and/or liver of three out of four animals in both vaccination groups. Only one out of four animals in each group was fully protected. Neither Hecolin® nor the novel p239(gt3) vaccine provided sufficient protection against genotype 3 infection. While Hecolin® only partial protected pigs from HEV shedding, the novel p239(gt3) vaccine was at least able to prevent infected pigs from virus shedding. The results highlight the need for further development of HEV vaccines that exhibit broad protection against multiple HEV genotypes and the use of appropriate animal infection models.

Seroprevalence and Phylogenetic Characterization of Hepatitis E Virus (Paslahepevirus balayani) in Guinean Pig Population.

Background: Hepatitis E virus (HEV) is transmitted by the fecal route, usually through contaminated water in humans and/or infected animals, especially pigs. The objective of this study was to evaluate the level of anti-HEV antibodies in a panel of pig sera and to identify HEV in pig feces in farms. Methodology: The presence of HEV antibodies was tested by an in-house ELISA and a commercial ELISA IDvet. HEV genome was assessed by nested RT-PCR, and then, genotype was identified by sequencing (MinION Nanopore technology). Results: In 2017-2019, the 43% seroprevalence found in Forest Guinea was significantly higher than the 7% found in the Lower region (p < 0.01). Presence of HEV genotype 3c was demonstrated during a secondary study in the Lower region (Conakry) in 2022. Conclusion: The presence of HEV-3c in pigs calls for an evaluation of seroprevalence in human populations and for a HEV genotype human circulation check. Contribution Heading: This study is the first report, to our knowledge, of seroprevalence and characterization of HEV infection in pigs in Guinea.

Halogenated Rocaglate Derivatives: Pan-antiviral Agents against Hepatitis E Virus and Emerging Viruses.

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4', 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

Correction: Rivas et al. Tendon-Derived Mesenchymal Stem Cells (TDSCs) as an In Vitro Model for Virological Studies in Wild Birds. Viruses 2023, 15, 1455.

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