Ruminants Virus Research Articles

Metavirome analysis of domestic sheep in Shaanxi, Gansu, and Ningxia, China.

Sheep play an important role in China's agricultural development, but they are also potential hosts for many viruses, some of which have been identified as zoonotic pathogens, which may pose a serious threat to social public health and animal husbandry. Therefore, clarifying the characteristics of viruses in sheep will provide an important basis for the study of pathogenic ecology and viral evolution of viruses carried by sheep. We collected nasal and anal swabs from 688 sheep in 22 counties in Shaanxi, Gansu, and Ningxia, China, between January 2022 and July 2023, and utilized next-generation sequencing technology and bioinformatics approaches to identify the viruses in the samples. A total of 38 virus families carried by sheep were identified, including 12 ssRNA (+) virus families, 2 dsRNA virus families, 8 ssDNA (+) virus families, and 18 dsDNA virus families. Among them, Astroviridae, Coronaviridae, Picornaviridae, and Tobaniviridae in RNA virus families and Herpesviridae, Adenoviridae, and Circoviridae in DNA virus families are all viruses that are frequently detected in most ruminants. Alpha and beta diversity results showed that there was no difference in the overall richness and diversity of RNA and DNA viruses among the three provinces (p > 0.05). The evolutionary analysis demonstrated a tight link between the viral members carried by sheep and other ruminant viruses, implying that these viruses may spread across different species of ruminants. This study established a library of RNA and DNA viruses carried by sheep in the Shaanxi-Gansu-Ningxia region, providing an overview of the viruses present in this population. The findings offer valuable data for further research on virus evolution and monitoring in sheep.

Establishment and validation of a real-time fluorescent PCR freeze-dried type assay for 11 sheep and goats pathogens

Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99–1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for M. ovis, Pathogenic Leptospira, C. burnetii, BTV, PPRV, Brucella, exJSRV, C. abortus, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00 %; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were Mycoplasma ovis (M. ovis), Pathogenic Leptospira, Coxiella burnetii (C. burnetii), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), Brucella, exogenous jaagsiekte sheep retrovirus (exJSRV), Chlamydia abortus (C. abortus), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).

Serological and Molecular Survey of Hepatitis E Virus in Small Ruminants from Central Portugal

Hepatitis E virus (HEV) is currently recognized as an emerging problem and a growing concern for public health in developed countries, with HEV infections mainly attributable to foodborne transmission of HEV-3. The zoonotic HEV genotype 3 infects a wide range of mammalian hosts, with swine considered as the primary host. This study investigates the occurrence of HEV among small ruminants in Portugal. The primary aim of the present research was to evaluate the circulation and the potential for HEV infection among sheep and goats. A total of 400 bile samples and 493 blood samples were collected from sheep and goats at a slaughterhouse in the center region of Portugal, between January 2022 and March 2023. The HEV RNA detection in bile samples was performed using a nested broad-spectrum RT-PCR targeting the ORF1 region. Serological analysis to detect anti-HEV antibodies was conducted using a commercial double-antigen sandwich multi-species ELISA. The HEV RNA was not detected in any bile samples using the nested broad-spectrum RT-PCR. Serological analysis revealed an overall HEV antibody seroprevalence of 2% (10/493, 95% CI: 0.98–3.70) among the small ruminants, namely 2.2% in goats and 2.0% in sheep. Curiously, no statistically significant association among the factors, age, sex and species and HEV seroprevalence was observed. Although HEV RNA was not detected in the bile of sheep and goats, this study the evidence of seroprevalence in these small ruminant species. Further research could provide additional insights into the factors influencing HEV transmission dynamics in small ruminants in Portugal and its potential implications for public health.

Large-Scale Serological Survey of Crimean-Congo Hemorrhagic Fever Virus and Rift Valley Fever Virus in Small Ruminants in Senegal.

Crimean-Congo hemorrhagic fever (CCHF) and Rift Valley fever (RVF) are among the list of emerging zoonotic diseases that require special attention and priority. RVF is one of the six priority diseases selected by the Senegalese government. Repeated epidemic episodes and sporadic cases of CCHF and RVF in Senegal motivated this study, involving a national cross-sectional serological survey to assess the distribution of the two diseases in this country throughout the small ruminant population. A total of 2127 sera from small ruminants (goat and sheep) were collected in all regions of Senegal. The overall seroprevalence of CCHF and RVF was 14.1% (IC 95%: 12.5-15.5) and 4.4% (95% CI: 3.5-5.3), respectively. The regions of Saint-Louis (38.4%; 95% CI: 30.4-46.2), Kolda (28.3%; 95% CI: 20.9-35.7), Tambacounda (22.2%; 95% CI: 15.8-28.6) and Kédougou (20.9%; 95% CI: 14.4-27.4) were the most affected areas. The risk factors identified during this study show that the age, species and sex of the animals are key factors in determining exposure to these two viruses. This study confirms the active circulation of CCHF in Senegal and provides important and consistent data that can be used to improve the surveillance strategy of a two-in-one health approach to zoonoses.

Epidemiological exploration of fleas and molecular identification of flea-borne viruses in Egyptian small ruminants

The study aimed to investigate molecularly the presence of flea-borne viruses in infested small ruminants with fleas. It was carried out in Egypt’s Northern West Coast (NWC) and South Sinai Governorate (SSG). Three specific primers were used targeting genes, ORF103 (for Capripoxvirus and Lumpy skin disease virus), NS3 (for Bluetongue virus), and Rdrp (for Coronavirus), followed by gene sequencing and phylogenetic analyses. The results revealed that 78.94% of sheep and 65.63% of goats were infested in the NWC area, whereas 49.76% of sheep and 77.8% of goats were infested in the SSG region. Sheep were preferable hosts for flea infestations (58.9%) to goats (41.1%) in the two studied areas. Sex and age of the animals had no effects on the infestation rate (p > 0.05). The season and site of infestation on animals were significantly different between the two areas (p < 0.05). Ctenocephalides felis predominated in NWC and Ctenocephalides canis in SSG, and males of both flea species were more prevalent than females. Molecular analysis of flea DNA revealed the presence of Capripoxvirus in all tested samples, while other viral infections were absent. Gene sequencing identified three isolates as sheeppox viruses, and one as goatpox virus. The findings suggest that Capripoxvirus is adapted to fleas and may be transmitted to animals through infestation. This underscores the need for ongoing surveillance of other pathogens in different regions of Egypt.

Establishment of a RAA-CRISPR Cas12a based diagnostic method for peste des petits ruminants virus N gene and M gene

Peste des petis ruminants (PPR) is an acute, highly contagious fatal disease affecting both domestic and wild small ruminants, caused by Morbillivirus caprinae (also known as peste des petis ruminants virus (PPRV)). Herein, a rapid method based on recombinase aided amplification-clustered regularly interspaced short palindromic repeats-Cas12a (RAA-CRISPR Cas12a) to detect PPRV was developed. CRISPR RNAs and RAA primers for PPRV-N (nucleocapsid) and PPRV-M (matrix) fragments were designed. The reaction system was constructed following screening and optimization. Detection could be completed within in 50 minutes at 37°C. Detection of gradient dilutions of plasmids carrying of PPRV N and M gene fragments indicated a minimum limit of detection of 10 copies/μL. There were no cross-reactions with related viruses and all tested lineages of PPRV were detected successfully. The method also showed good repeatability. The detection of clinical samples (previously detected using reverse transcription polymerase chain reaction (RT-PCR)) indicated good consistency between the RAA-CRISPR Cas12a method and RT-PCR. Thus, the RAA-CRISPR Cas12a method for rapid PPRV diagnosis has strong specificity, high sensitivity, and stable repeatability. Moreover, the results can be observed visually under blue or UV light or using lateral flow strips without complex instruments.

Foot-and-mouth disease seroprevalence is higher in young female crossbred sheep and goats compared to their counterparts in the border area between Pakistan and Afghanistan.

Foot-and-mouth disease (FMD) is a highly contagious disease in ruminants that causes significant economic losses worldwide. However, the prevalence of FMD virus (FMDV) in small ruminants has been overlooked in Pakistan. This study aimed to determine the prevalence of FMD in sheep and goats in the border area between Pakistan and Afghanistan. 800 sheep and goats belongs to age groups of 6 month to > 2 years. A total of 800 serum samples were collected from sheep (n = 424) and goats (n = 376) and subjected to structural protein (SP) and 3ABC non-SP (NSP) ELISAs for the detection of antibodies against SP and NSP of the FMDV. For NSP, 340/800 (42.5%) of samples were positive, while SP analysis revealed that serotype O (44.5%) was the most common in sheep and goats, followed by Asia-1 (42%) and A (32%) serotypes. Sheep (39%; 95% CI, 34 to 44) had a higher (P < .05) prevalence of FMD than goats (46%; 95% CI, 41 to 51). Statistically significant (P < .05) differences in the seroprevalence of FMD-SP and FMD-NSPs were observed between various agencies (areas) of the study area. Risk factors such as age, sex, breed, season, flock size, body condition, animal movement, and production system were significantly (P < .05) associated with FMDV prevalence. This study showed that FMD is highly prevalent in sheep and goats in the border areas of Pakistan and Afghanistan. Therefore, outbreak investigation teams should be arranged at the border level to develop FMD risk-based surveillance and control plans for small ruminants in order to mitigate infection risks.

Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3–8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

Seroprevalence of Bluetongue and Schmallenberg viruses in ruminants in Al-Batinah Governorates, Oman

Bluetongue (BTV) and Schmallenberg viruses (SBV) are the causative agents of Bluetongue and Schmallenberg dis-eases, respectively. Both BTV and SBV are vector-borne viruses that are transmitted mainly by Culicoides (Diptera; Ceratopogo-nidae) and infect do-mestic, wild ruminants, and camelid species. Bluetongue virus be-longs to the genus Orbi-virus, family Reoviridae, whereas Schmallenberg virus belongs to the ge-nus Orthobunyavirus, family Bunyaviridae. In this study, we used en-zyme-linked immuno-sorbent assay (ELISA) to determine the sero-prevalence of both vi-ruses in ruminants in Al-Batinah Gover-norates, Sultanate of Oman. A total of 529 serum samples were randomly collected from 207 sheep, 265 goats, and 57 cattle from five wilayat of Al-Batinah North and Al-Batinah South Governorates. The serum samples were screened for the presence of BTV-specific antibodies against the BTV VP7 protein using ID Screen® Bluetongue Competition (cELISA) and screened for the presence of SBV-specific antibodies against the recombinant SBV nucleoprotein anti-gen using ID Screen® Schmallenberg virus Indirect Mult (iELISA). The overall seropreva-lence of BTV and SBV infections was 69.8% and 44.8%. In cattle, the prevalence of BTV and SBV antibodies was comparatively higher (94.7% and 82.5%) than in goats (83% and 43%) and sheep (45.9% and 36.7%). The high-est BTV seroprevalence was recorded in Nakhal (85.5%), followed by Wadi Al-Maawil (81%), and was lowest in Bar-ka (59.6%). However, the highest seropreva-lence of SBV was ob-served in Wadi Al-Maawil (50%), followed by Barka (48.7%), and lowest in Sohar (34.2%). Overall, the seroprevalence of BTV and SBV in domesticat-ed ruminants was high-er in adults than in young animals. Females showed a higher sero-prevalence of BTV and SBV compared to males. The study pro-vides an update on the epidemiological status of BTV, and to our knowledge, this is the first study on the sero-prevalence of SBV in ruminants in Oman. Future studies are re-quired for the isolation and identification of BTV and SBV along with other potential bio-logical vectors in Oman.

Vimentin inhibits peste des petits ruminants virus replication by interaction with nucleocapsid protein

The Peste des petits ruminant virus (PPRV) is a member of the Paramyxoviridae family and is classified into the genus Measles virus. PPRV predominantly infects small ruminants, leading to mortality rates of nearly 100%, which have caused significant economic losses in developing countries. Host proteins are important in virus replication, but the PPRV nucleocapsid (N) protein-host interacting partners for regulating PPRV replication remain unclear. The present study confirmed the interaction between PPRV-N and the host protein vimentin by co-immunoprecipitation and co-localization experiments. Overexpression of vimentin suppressed PPRV replication, whereas vimentin knockdown had the opposite effect. Mechanistically, N was subjected to degradation via the ubiquitin/proteasome pathway, where vimentin recruits the E3 ubiquitin ligase NEDD4L to fulfill N-ubiquitination, resulting in the degradation of the N protein. These findings suggest that the host protein vimentin and E3 ubiquitin ligase NEDD4L have an anti-PPRV effect.

First Report of the Emergence of Peste des Petits Ruminants Lineage IV Virus in Senegal.

Peste des petits ruminants (PPR) is a highly contagious viral disease and one of the deadliest affecting wild goats, sheep, and small ruminants; however, goats are generally more sensitive. The causative agent is the Peste des Petits Ruminants virus (PPRV), which is a single-stranded RNA virus of negative polarity belonging to the Paramyxoviridae family. In February 2020, an active outbreak of PPR was reported in a herd of a transhumant farmer in the village of Gainth Pathé (department of Kounguel, Kaffrine region, Senegal). Of the ten swabs collected from the goats, eight returned a positive result through a quantitative real-time PCR. The sample that yielded the strongest signal from the quantitative real-time PCR was further analyzed with a conventional PCR amplification and direct amplicon sequencing. A phylogenetic analysis showed that the sequence of the PPR virus obtained belonged to lineage IV. These results confirm those found in the countries bordering Senegal and reinforce the hypothesis of the importance of animal mobility between these neighboring countries in the control of PPRV. In perspective, following the discovery of this lineage IV in Senegal, a study on its dispersion is underway throughout the national territory. The results that will emerge from this study, associated with detailed data on animal movements and epidemiological data, will provide appropriate and effective information to improve PPR surveillance and control strategies with a view to its eradication.

Survey of antibodies to Peste des petits ruminants virus in small ruminants in the Mediterranean Region of Turkey

Peste des petits ruminants (PPR) is a viral disease affecting sheep and goats caused by peste des petits ruminants virus (PPRV) has a serious economic impact due to the restrictions on animal trade and animal movements and high mortality rates in small ruminant populations. The common clinical sings of the PPR are fever, muco-purulent nasal discharge, diarrhoea and abortion. Seroepidemiological studies of PPRV infection in sheep and goats in Turkey are scant. Therefore, this study was aimed to evaluate the seroprevalence of PPR in small ruminants in Turkey. Ovine blood samples were collected by random sampling method from sheep (n = 77) and goats (n = 61) from unvaccinated flocks (n = 40) in the Antalya Province in the Mediterranean region of Turkey. A competitive enzyme-linked immunosorbent assay (c-ELISA) kit was used to detect antibodies against PPRV in sera samples. Out of 138 sera samples analysed, eighteen sera samples (13%, 95% CI: 7.4 - 18.7) were PPRV seropositive, of which 18.2% (95% CI: 9.6 - 26.8; 14/77) were from sheep, whereas 6.6% (95% CI: 0.3 - 12.8; 4/61) were from goats. Although PPRV seropositivity rate was higher in sheep than goats, it was not statistically significant (P = 0.07). PPRV seropositivity was higher in small ruminants older than 24 months (19.4%) compared with less than or equal to 24 months (7%) (P = 0.04). Although, there was no statistically significant difference between sexes, PPRV seropositivity rate was higher (14.5%) in females than males (10.9%) (P = 0.61). The flock-level seroprevalence was 30% (12/40). The result of the present study showed that seroprevalence of PPRV infection is high in sheep and goats in the Antalya Province. However, results of the study are not enough to determine the regional and country-based profile of the PPRV infection in Turkey. Further epidemiological studies are required to get more epidemiology data on PPR in Turkey.

Molecular Epidemiology of Foot-and-Mouth Disease Viruses in the Emirate of Abu Dhabi, United Arab Emirates.

Foot-and-mouth disease (FMD) is an endemic disease in the United Arab Emirates (UAE) in both wild and domestic animals. Despite this, no systematic FMD outbreak investigation accompanied by molecular characterisation of FMD viruses (FMDVs) in small ruminants or cattle has been performed, and only a single report that describes sequences for FMDVs in wildlife from the Emirate has been published. In this study, FMD outbreaks that occurred in 2021 in five animal farms and one animal market in the Emirate of Abu Dhabi were investigated. Cases involved sheep, goats, and cattle, as well as Arabian oryx (Oryx leucoryx). Twelve samples were positive for FMDV via RT-qPCR, and four samples (Arabian oryx n = 1, goat n = 2, and sheep n = 1) were successfully genotyped using VP1 nucleotide sequencing. These sequences shared 88~98% identity and were classified within the serotype O, Middle East-South Asia topotype (O/ME-SA). Phylogenetic analysis revealed that the Arabian oryx isolate (UAE/2/2021) belonged to the PanAsia-2 lineage, the ANT-10 sublineage, and was closely related to the FMDVs recently detected in neighbouring countries. The FMDV isolates from goats (UAE/10/2021 and UAE/11/2021) and from sheep (UAE/14/2021) formed a monophyletic cluster within the SA-2018 lineage that contained viruses from Bangladesh, India, and Sri Lanka. This is the first study describing the circulation of the FMDV O/ME-SA/SA-2018 sublineage in the UAE. These data shed light on the epidemiology of FMD in the UAE and motivate further systematic epidemiological studies and genomic sequencing to enhance the ongoing national animal health FMD control plan.

Clinical Manifestation and Phylogenetic Analysis of Peste des Petits Ruminants in Local Iraqi Breed Sheep in Al-Diwaniyah Province.

Peste des petits ruminants (PPR), a contagious virus that infects sheep and goats, damages livestock globally. This study examined the clinical features and phylogenetic analysis of the PPR virus in Iraqi breed sheep from Al-Diwaniyah province. A clinical trial of 610 sheep from different flocks found 150 oral lesions. Special primers for RT-PCR and Mega11 for phylogenetic analysis were used to study the PPR virus nucleoprotein (N) gene. The PPR infection rate was 44.6% in 4-12 month olds (n = 33/131) and 4.8% in 36-48 month olds (n = 3/75). A 608-bp PPR virus partial N gene sequence was found in 49.3% of samples by RT-PCR. In leucine, isoleucine, proline, glycine, alanine, glutamine, asparagine, threonine, serine, arginine, and lysine codons, 25 amino acid alterations were found. The protein codon 56 alanine-valine alteration was most significant. Moving from a smaller hydrophobic amino acid to one with a bigger side chain may reduce protein stability. Steric hindrance or protein shape change from Valine's extended side chain may impact folding, stability, functionality, and interactions with other molecules. Furthermore, phylogenetic analysis showed that the Nigerian strain (MN271586) was most similar to our Iraqi strain, with 100% identity and coverage. This study found the Peste des Petits Ruminants (PPR) virus in sheep flocks in Al-Diwaniyah Governorate, Iraq, which is genetically similar to neighboring countries. PPR virus strains must be monitored and genetically characterized since N gene alterations can affect infection and propagation.

Infectivity and molecular epidemiology of Peste des Petits ruminants virus in slaughtered goats at the local meat market of Mymensingh division, Bangladesh.

The purpose of this study was to characterize the circulating Peste des Petits Ruminants virus (PPRV) from slaughtered goats and conduct a phylogenetic analysis of the N gene of PPRV. A total of 196 slaughtered goats were investigated at the marketplaces of Mymensingh division from January 2019 to March 2021. Lungs, spleen, and mesenteric lymph nodes were collected for histology and molecular study. In-house developed Reverse-Transcriptase-Polymerase Chain Reaction (RT-PCR) protocols were carried out using designed primer sets (PPRV NF-gctctgtgattgcggctgagc and PPRV NR-cctggtcctccagaatcttggcc). The CLC sequence viewer was used for phylogenetic analysis. Grossly pneumonic lungs, shrinkage spleen, and enlarged mesenteric lymph nodes with hemorrhages were recorded. Both intracytoplasmic and intranuclear inclusion bodies were seen in lymphocytes of the mesenteric lymph node, spleen, and lungs. PPRV was detected in 37 goats (18.9%) by RT-PCR test. The 402-bp amplicon was generated in PPRV-positive cases. The phylogenetic analysis showed that the studied PPRV isolates of the Mymensingh division belonged to lineage IV. The prevalence of PPR was 18.9% in slaughtered goats at marketplaces in the Mymensingh division. Slaughterhouses may be a source of PPRV, and it can be horizontally transmitted from the meat market to the farm. Restricting sick animal movement within the country, mass PPR vaccination campaigns, increased awareness, and improved biosecurity in the meat market may lessen the incidence of PPR in goats.

Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

Molecular detection and characterization of Rift Valley fever virus in humans and domestic ruminants in Nigeria

Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne disease of domestic livestock, wildlife and humans majorly in Africa and the Arabian Peninsula. The virus is a Phlebovirus belonging to the Family Phenuiviridae and constitutes a threat to both human and animal health. Although there has been serological evidence of RVFV in humans and animals in Nigeria, reports on its molecular detection and characterization are absent. Blood samples (n = 1101) were collected between January 2019 and March 2021 from humans, cattle and sheep in four States of Nigeria. The harvested plasma samples were tested using a commercial ELISA kit. All the ELISA-positive, ‘-doubtful’ and some ‘-negative’ samples (n = 436) were further analyzed using nested reverse transcriptase-polymerase chain reaction (nRT-PCR). Eight samples (humans, n = 1; cattle, n = 6; sheep, n = 1) that gave the expected band size of approximately 668 base pairs following gel electrophoresis were sequenced by Sanger method. The sequence data were edited using BioEdit software and RVFV was successfully identified in all of them. Nucleotide sequence and phylogenetic analyses revealed that these eight RVFV strains were 98.3–99.9% identical and clustered with the Ugandan Smithburn strain. Genotyping showed that they belonged to Lineage K. This report, which is the first molecular detection and characterization of RVFV in Nigeria, confirms the circulation of the virus in the country. It also highlights the need for large-scale surveillance for RVFV in Nigeria, including among mosquito vectors, to provide better understanding of its spatiotemporal epidemiology, and thus, aid in preventing potential RVF outbreaks in the country.

Direct Dengue Virus Genome Sequencing from Antigen NS1 Rapid Diagnostic Tests: A Proof-of-Concept with the Standard Q Dengue Duo Assay.

With nearly half of the world's population being at risk of infection, dengue virus represents a major global health issue. The use of dengue antigen rapid diagnostic tests (Ag-RDTs) represents an alternative to PCR methods for the diagnosis of acute infections since they display excellent sensitivities and specificities and can be performed outside the laboratory. The high genetic diversity of the dengue virus genome represents a challenge for vaccine development, and the progressive expansion of this virus into previously nonendemic regions justifies the implementation of a genomic surveillance program. In this proof-of-concept study, we show the feasibility of sequencing dengue virus genomes directly from positive Ag-RDT (Standard Q Dengue Duo Test assay, n = 7) cassettes stored up to 31 days at room temperature after testing. For 5 of the 7 samples, a high number of reads were obtained allowing phylogenetic analyses to be carried out to determine not only the serotypes (dengue 1, 2, 3 and 4 were detected) but also the genotypes. Furthermore, in one sample, our unbiased metagenomic next-generation sequencing approach made it possible to detect epizootic hemorrhagic disease virus sequences, an arthropod-transmitted virus in ruminants. To conclude, as such an approach requires no cold storage or freezing of samples, dengue Ag-RDTs represent a very pragmatic and robust alternative for the genomic surveillance of dengue virus.

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells.

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC50 ) value of each peptide was greater than 1000 μg mL-1 . The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 μg mL-1 concentration showed a reduction of more than log10 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log10 4.8 to log10 2.5 and log10 3.1 respectively. The concentration of SLAM homologous peptide (25 μg mL-1 ) to exert its anti-PPRV effect was much less than its CC50 level (>1000 μg mL-1 ). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.

The genomic and epidemiological investigations of enteric viruses of domestic caprine (Capra hircus) revealed the presence of multiple novel viruses related to known strains of humans and ruminant livestock species.

In this study, the enteric virome of diarrheic caprine was investigated using next-generation sequencing, reverse transcription PCR methods, and bioinformatics analyses. The complete or nearly complete genomes of seven novel viruses including (i) a caprine astrovirus (OQ758025) of family Astroviridae, four picornaviruses in genera (ii) Boosepivirus (OQ758026), (iii) Enterovirus (OQ758028), (iv) Kobuvirus (OQ758029), and (v) an unassigned picornavirus provisionally called as capripivirus (from the term caprine picornavirus) of the family Picornaviridae (OQ758027), as well as (vi) a tusavirus of family Parvoviridae (OL692339.2) and an (vii) unassigned CRESS DNA virus (OQ758030) have been identified. Structural analyses of the internal ribosomal entry sites revealed the presence of unique motifs and suggest the modular exchange of certain elements between co-infecting viruses. Epidemiological investigations and genotyping PCR reactions of identified RNA virus groups were also conducted using multiple "in-house" developed SYBRgreen-based screening quantitative PCR (qPCR) assays and generic capsid primers on additional fecal samples from ruminant livestock species (n = 62 caprine, n = 32 ovine, and n = 94 cattle) of three different age groups from n = 12 geographically distant sampling sites in Hungary. The results of qPCR screening assays as well as K-means cluster and phylogenetic analyses of the determined sequences suggest that diverse members of the above RNA virus groups were found in all age groups, but mostly in <1-year-old ruminants. Members of the analyzed virus groups were found frequently (37.2%) in multiple (even in quintuple) co-infections, while strains related closely to the identified index viruses were detectable only in certain caprine and ovine samples indicating a wide range of viral diversity. IMPORTANCE Compared with other domestic animals, the virome and viral diversity of small ruminants especially in caprine are less studied even of its zoonotic potential. In this study, the enteric virome of caprine was investigated in detail using next-generation sequencing and reverse transcription PCR techniques. The complete or nearly complete genomes of seven novel viruses were determined which show a close phylogenetic relationship to known human and ruminant viruses. The high similarity between the identified caprine tusavirus (family Parvoviridae) and an unassigned CRESS DNA virus with closely related human strains could indicate the (reverse) zoonotic potential of these viruses. Others, like astroviruses (family Astroviridae), enteroviruses, or novel caripiviruses (named after the term caprine picornavirus) of family Picornaviridae found mostly in multiple co-infections in caprine and ovine, could indicate the cross-species transmission capabilities of these viruses between small ruminants.