Rotavirus Research Articles

Effect of HDAC9-induced deacetylation of glycolysis-related GAPDH lysine 219 on rotavirus replication in rotavirus-infected Caco-2 cells.

Post-translational modifications (PTMs), as epigenetic modifications, are significant in the interaction between virus and its host. However, it is unclear whether rotavirus (RV) causes changes in both the host cell epigenetic protein modification and the regulatory mechanism of viral replication. Here, we analyzed the proteome of Caco-2 cells to determine if acetylation modification occurred within the cells after RV infection. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein involved in glycolysis, was deacetylated at lysine 219 via histone deacetylase 9 (HDAC9) in 50h after the RV infection. Remarkably, the deacetylation of GAPDH promoted RV replication. Finally, we found that glycolysis was alterable in Caco-2 cells by RV or the deacetylation of GAPDH lysine 219, using the Seahorse XF Glycolysis Stress Test. In conclusion, our results demonstrate for the first time that RV infection promoted deacetylation of GAPDH at lysine 219 in order to increase its own viral replication in Caco-2 cells.

Foodborne Pathogens Across Different Food Matrices in Sicily (Southern Italy)

Foodborne diseases result from the consumption of foods contaminated with pathogens or their toxins and represent a serious public health problem worldwide. This study aimed to assess the presence of Rotavirus (RoV), Adenovirus (AdV), Norovirus (NoV), Hepatitis A and Hepatitis E viruses (HAV and HEV, respectively), Toxoplasma gondii, Coxiella burnetii and Leptospira spp. across various food matrices in Sicily. The analysis concerned 504 samples, including mussels, farmed meat, game meat, vegetables and bulk milk. Following appropriate pre-treatment, acid nucleic extraction was carried out and amplification of pathogen nucleic acids was carried out by molecular methods. The mussels tested positive for NoVs (3/51, 5.9%) and farm meat resulted positive for T. gondii (1/34, 2.9%). The game offal samples tested positive for HEV, which was detected in 17 out of 222 samples (7.7%), and T. gondii (18/318, 5.7%) and Leptospira spp. (2/318, 0.6%). The milk samples tested positive for C. burnetii (15/ 85, 17.6%), T. gondii (2/85, 2.4%) and Leptospira spp. (1/85, 1.2%). This study highlights the variability in the risk of contamination of different food matrices, confirming the importance of vigilance in the consumption of potentially contaminated food products.

Rapid production of recombinant rotaviruses by overexpression of NSP2 and NSP5 genes with modified nucleotide sequences.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution.

Isoleucine Enhanced the Function of the Small Intestinal Mucosal Barrier in Weaned Piglets to Alleviate Rotavirus Infection.

Rotavirus (RV) is a major cause of diarrhea in young children and animals, especially piglets, leading to substantial economic losses in the global pig industry. Isoleucine (Ile), a branched-chain amino acid, plays an important role in regulating nutrient metabolism and has been shown to improve diarrhea. This study aimed to evaluate the effects of Ile supplementation on the mucosal immune barrier of the small intestine in RV-infected weaned piglets. Forty-eight 21-day-old weaned piglets were randomly divided into three dietary treatments (each treatment was subdivided into two groups, eight replicates per group), with 0%, 0.5%, or 1% Ile added for 15 days, and then, one group from each treatment was challenged with RV. The results showed that 1% Ile added to the diet promoted the healthy development of the intestinal mucosa. Ile could restore the reduced villus height in the ileum and the goblet cell number in the duodenum and ileum to normal levels, improving the intestinal epithelial tight junctions in RV-infected piglets. Additionally, Ile increased the activity of lipase, amylase, and sucrase, as well as superoxide dismutase (SOD) and glutathione (GSH), along with the expression of SIgA, DEFβ1, and DEFβ2 in parts of the small intestine. The addition of Ile to the diet mitigated the effects of RV infection on intestinal morphology and mucosal barrier function, as well as the physiological functions of weaned piglets, and improved the antioxidant and immune functions of the piglets to some extent. These findings offer valuable insights, contributing to a deeper understanding of the role of Ile in supporting intestinal health.

Evaluation of Immunochromatographic Test for Rapid Detection of Norovirus, Rotavirus, and Adenovirus in Stool Samples.

Viral enteric infections are illnesses caused by several types of viruses that primarily affect the gastrointestinal system. Globally, three major viruses associated with gastroenteritis, including norovirus (NoV), rotavirus A (RVA), and human adenovirus (HAdV), have been widely recognized. Accordingly, a rapid and sensitive diagnostic tool, such as immunochromatographic (IC) test, for diarrheal virus detection is required and helpful for rapid diagnosis. The IP-Triple I IC test kit for simultaneous triple virus detections of NoV, RVA, and HAdV was evaluated for its efficacy by using stool specimens that were known positive for these viruses by real-time RT-PCR or PCR, which was used as the gold standard method. The results revealed that the IP-Triple I IC test kit exhibited a very high level of specificity (100%) for NoV, RVA, and HAdV detections, while the sensitivity was slightly different for these three viruses. The sensitivity of detection for RVA was 86.7%, whereas those for NoV and HAdV were 70.6% and 76.2%, respectively. This IP-Triple I IC kit could detect common antigens of a wide range of NoV, RVA, and HAdV genotypes (NoV GII.2, GII.4, GII.6, GII.7, GII.10, GII.17, RVA G1P[8], G2P[4], G3P[8], G8P[8], G9P[8], HAdV-C1, -C2, -C5, -A12, -F40, and -F41). Our results revealed that the IP-Triple I IC test kit is highly effective for simultaneous, direct detection of common antigens of several genotypes of NoV, RVA, and HAdV in stool specimens and useful for screening and rapid diagnosis of diarrheal viruses during the seasonal outbreak.

An investigation of group and subtype diversity and distribution of porcine rotaviruses in Canadian suckling piglets with diarrhea, 2019-2023

Objective: To determine the frequency of detection and group diversity of rotavirus (RV) A, B, and C, and G (glycoprotein antigen) serotype (based on viral protein 7 [VP7] gene analysis) infecting suckling piglets with diarrhea in Canadian farms. Materials and methods: Canadian swine veterinarians submitted 1117 enteric samples from suckling piglets between July 2019 and December 2023 to the University of Guelph Animal Health Laboratory for RV group identification and VP7 sequencing for subtyping. Analysis of the VP7 sequence from 837 samples was performed using the Animal Health Sequivity Dashboard (Merck & Co, Inc) and descriptive statistics. Results: Rotavirus A, B, and C were present in 40.7%, 12.5%, and 46.8% of samples, respectively. The most common RV identified was RVC G6, present in 296 samples, followed by RVA G9 in 205 samples. A single RV group was involved in 444 cases (72.3%), while in 170 cases (27.7%), more than one RV group/subtype was detected. Eighteen subtypes were identified by sequencing the VP7 protein (5 RVA, 9 RVB, and 4 RVC). Implications: Rotavirus protection for suckling piglets comes from colostrum and milk. Knowing which RV group is causing diarrhea is important since vaccination does not generate cross-protection among groups. Using molecular diagnostic testing, it is possible to identify the specific group and subtype of RV circulating on the premises and decide the best treatment strategy for the disease.

1α,25-hydroxyvitamin D3 alleviated rotavirus infection induced ferroptosis in IPEC-J2 cells by regulating the ATF3-SLC7A11-GPX4 axis

Rotavirus (RV) mainly infects mature intestinal epithelial cells and impairs intestinal absorption function, which leads to the death of infected cells and eventually fatal diarrhea. Ferroptosis is a novel regulatory cell death pattern, which can be caused by virus infection. 1α,25-hydroxyvitamin D3 (1,25D3) has an anti-RV infection effect and can regulate ferroptosis. However, whether RV infection can induce ferroptosis, and whether 1,25D3 can inhibit RV infection by regulating ferroptosis has not yet been studied. Present study shows that RV infection or erastin treatment induces IPEC-J2 cell death, which results in mitochondrial shrinkage, decreased mitochondrial membrane potential (MMP) and glutathione (GSH) content, increased MMP, intracellular Fe2+, reactive oxygen species (ROS), and malondialdehyde (MDA) contents. Meanwhile, ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and deferoxamine (DFO) treatment can effectively reverse the increase of intracellular Fe2+, ROS and MDA levels induced by RV infection. Moreover, RV infection increases activating transcription factor 3 (ATF3) mRNA and protein expressions, and inhibited SLC7A11 and glutathione peroxidase 4 (GPX4) expressions, which was partially alleviated by siATF3. 1,25D3 treatment significantly eliminates RV induced ferroptosis via ATF3-SLC7A11-GPX4 axis. Therefore, our results reveals that RV infection induces ferroptosis in IPEC-J2 cell and 1,25D3 alleviates RV induced ferroptosis by regulating the ATF3-SLC7A11-GPX4 axis.

Bifidobacterium breve M-16V and scGOS/lcFOS Supplementation to Dams Ameliorates Infant Rotavirus Infection in Early Life.

The immune system of newborns is underdeveloped, leaving them susceptible to infections like rotavirus (RV). Despite vaccines, RV remains a leading cause of child mortality, especially in developing countries. Maternal immunity is transferred during pregnancy and breastfeeding to the offspring providing protection against RV infection. This study aims to explore how the maternal diet can enhance the newborn's ability to fight early infections. Pregnant rats received orally Bifidobacterium breve M-16V and short chain galacto-oligosaccharides (scGOS)/long chain fructo-oligosaccharides (lcFOS). At day 5 of life pups are infected with RV and at day 8, samples are collected for the infection analysis. Pups whose mothers received the synbiotic have lower RV infection severity. The levels of immunoglobulins (Ig) IgG2c and IgA are raised in pups' plasma and digested milk, respectively. Synbiotic supplementation improves intestinal maturation and increases gene expression of immune-related genes. In conclusion, the administration of this synbiotic to gestating and lactating mothers ameliorates the incidence and severity of the pup's diarrhea caused by the RV infection by improving their immunity.

Spray-dried plasma protects against rotavirus-induced gastroenteritis via regulating macrophage and T cells divergence in weanling pigs.

Infectious gastroenteritis is the major cause for diarrhea in piglets. The protection of spray-dried plasma (SDP) on viral gastroenteritis during the progression of rotavirus (RV) infection remain unclear. In this study, 64 weanling piglets were randomly assigned to control diets (n = 40) and SDP diets (n = 24) for 14 days, and then pigs were challenged with RV on day 15. Pigs were sacrificed on day 14 (normal condition), day 18 (manifestation stage), and day 21 (convalescent stage) of the trial. Prior to RV infection, SDP increased ADG, M1 macrophages and CD4+ T cells in different organs without increasing proinflammatory cytokines, indicating a more robust immunity with less inflammation. During the manifestation of infection, SDP enhanced mucosal immunity by increasing M1 macrophages, M1/M2 ratio and cytokines in mucosa and increasing intraepithelial CD8+ T cells for RV clearance. During the convalescence, SDP promoted M2 macrophage polarization and reduced pro-inflammatory cytokines to facilitate intestinal repair and prevent prolonged inflammation. Collectively, SDP enhanced mucosal immunity to promote viral clearance and maintained immune homeostasis to prevent long-lasting inflammation as a therapeutically approach for infectious gastroenteritis.

Genomic analysis of an acute gastroenteritis outbreak caused by rotavirus C in Hebei, China

Rotavirus group C is an important cause of sporadic cases and outbreaks of gastroenteritis worldwide. Whole-Genome sequences of human rotavirus C (RVC) in public databases are limited. We performed genome sequencing to analyze a RVC outbreak of acute gastroenteritis in China. Samples from 22 patients were screened for pathogens using RT-PCR, and six samples were positive for rotavirus. Whole-Genome sequencing analysis showed that the outbreak strain SJZ217 belongs to the G4-P[2]-I2-R2-C2-M3-A2-N2-T2-E2-H2 genotype and shares almost identical genomic sequences with Chungnam isolated in Korea. Phylogenetic analysis revealed strain SJZ217 also fell into a cluster with rotavirus C strains from Japan and Europe. Reassortment in the VP4 fragment was observed. These results helped to understand the genetic diversity and possible spread of RVC strains.

Unusual G3P[10] bat-like rotavirus strains detected in children with acute gastroenteritis in Thailand.

Rotavirus A (RVA) is the main cause of acute gastroenteritis among children under the age of five globally. The unusual bat-like human RVA strains G3P[10] (RVA/Human-wt/THA/CMH079/05/2005/G3P[10] and RVA/Human-wt/THA/CMH-S015-19/2019/G3P[10]) were detected in children with acute gastroenteritis in 2005 and 2019, respectively, in the same geographical area of Northern Thailand. To elucidate the genetic backgrounds of these unusual or bat-like human RVA strains, the complete genome of these RVA strains was sequenced and phylogenetically analyzed. All eleven genome segments of these G3P[10] strains were genotyped as G3-P[10]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is closely related to bat G3P[10] RVA strain (RVA/Bat-tc/CHN/MYAS33/2013/G3P[10]) and bat-like human RVA strain (RVA/Human-wt/THA/MS2015-1-0001/2015/G3P[10]). The findings indicate that human G3P[10] RVA strains detected in this study (RVA/Human-wt/THA/CMH079/05/2005/G3P[10] and RVA/Human-wt/THA/CMH-S015-19/2019/G3P[10]) contained all eleven genome segments similar to those of bat RVA strains and appeared to be human RVA strains of bat origin. Phylogenetic analysis revealed that several genome segments of these two RVA strains were also closely related with those of other species in addition to bats and had a zoonotic transmission history. The results of this study supported the roles of interspecies transmission of RVA strains among bats and humans in the natural environment and provided convincing evidence that the evolution of human RVAs was closely interrelated with those of animal RVAs. Continuing surveillance of RVAs in humans and animals is imperative to gain a better understanding of the origin and the evolution of these viruses.

TLR3/TRIF and MAVS Signaling Is Essential in Regulating Mucosal T Cell Responses during Rotavirus Infection.

The functions of the natural dsRNA sensors TLR3 (TRIF) and RIG-I (MAVS) are crucial during viral challenge and have not been accurately clarified in adaptive immune responses to rotavirus (RV) infection. In this study, we found that RV infection caused severe pathological damage to the small intestine of TLR3-/- and TRIF-/- mice. Our data found that dendritic cells from TLR3-/- and TRIF-/- mice had impaired Ag presentation to the RV and attenuated initiation of T cells upon viral infection. These attenuated functions resulted in impaired CD4+ T and CD8+ T function in mice lacking TLR3-TRIF signaling postinfection. Additionally, attenuated proliferative capacity of T cells from TLR3-/- and TRIF-/- mice was observed. Subsequently, we observed a significant reduction in the absolute number of memory T cells in the spleen and mesenteric lymph node (MLN) of TRIF-/- recipient mice following RV infection in a bone marrow chimeric model. Furthermore, there was reduced migration of type 2 classical dendritic cells from the intestine to MLNs after RV infection in TLR3-/- and TRIF-/- mice. Notably, RV infection resulted in attenuated killing of spleen and MLN tissues in TRIF-/- and MAVS-/- mice. Finally, we demonstrated that RV infection promoted apoptosis of CD8+ T cells in TRIF-/- and TLR3-/-MAVS-/- mice. Taken together, our findings highlight an important mechanism of TLR3 signaling through TRIF in mucosal T cell responses to RV and lay the foundation for the development of a novel vaccine.

Emergence of non-classical genotype constellations of G9P[8] rotavirus strains in diarrheic children in Sabah, Malaysia

G9P[8] has been the predominant rotavirus A (RVA) genotype in Malaysia since the 2000s. However, the overall genetic makeup and evolution of Malaysian G9P[8] strains are still unknown. Therefore, this study aimed to evaluate and characterize the complete genomes of three G9P[8] RVA strains isolated from diarrheic children under five years old in Sabah. Contrary to the classical Wa-like constellation, these strains contained a DS-1-like genotype. Two strains, namely L202 and L234, were genotype G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1, while one (KN102) was genotype G9-P[8]-I1-R1-C1-M1-A2-N1-T1-E1-H1. Phylogenetic analysis revealed that the NSP4 genes of L202 and L234 strains were closer to that of G9P[8]-E2 strains from Japan, suggesting they might share a common ancestor. The findings from this study provide new insights into the genetic characteristics of circulating G9P[8] strains in Sabah, which are important for rotavirus surveillance and potential vaccine development in the region.

Molecular Detection and Phylogenetic Tree of Rotavirus from Human and Sheep, Iraq

Group A rotaviruses (RVA) have been linked to acute gastroenteritis in children, as well as young domestic and wild animals. In Iraq few molecular studies involved the genotype profile of rota virus and this is the first study target VP1 gene. Data were collected from January to February 2018 and subsequently examined utilizing a PCR and because of low viral concentration we utilized Nested PCR. Sequenced PCR result at 327 bp fragment in 4 A rota virus-positive samples deposited in GenBank under accession number (MH11809.1, MH118097.1) for human and (MH118095.1, MH118094.1) to sheep. Children samples were identical to the other human A rota virus at (99-100%), with Japan feline-like human G6P and G6P[14] isolated from cattle as well as, animal rota virus (99-100%) similarity with cross relation with human Wa-Like and porcine, moreover Morocco characterized caprine, bovine rotavirus and Slovenian Roe deer ARV.

Rhesus rotavirus NSP1 mediates extra-intestinal infection and is a contributing factor for biliary obstruction.

We previously demonstrated that in Ifnar1-/-Ifngr1-/- or Stat1-/- suckling mice lacking intact type I and type II interferon (IFN) signaling, rhesus rotavirus (RRV) infection causes a lethal disease with clinical manifestations similar to biliary atresia, including acholic stools, oily fur, growth retardation, and excess mortality. Elevated levels of viral RNA are detected in the bile ducts and liver of diseased pups together with severe inflammatory responses in these tissues. However, the viral determinants and the molecular mechanisms driving this process remain incompletely understood. Using an optimized rotavirus (RV) reverse genetics system, we generated a panel of recombinant RVs that encode non-structural protein 1 (NSP1) derived from different RV strains. We found that compared to the parental simian SA11 strain that is less biliary pathogenic, SA11 containing an RRV-derived NSP1 resulted in severe biliary obstructive disease comparable to that associated with RRV infection, reflected by high levels of viral RNA and inflammation in the biliary tract, liver, and pancreas. In contrast, RRV containing an SA11-originated NSP1 showed only mild biliary obstruction comparable to what was observed during SA11 infection. Infection with a monoreassortant RRV virus carrying NSP1 from the bovine RV UK strain also showed substantially reduced viral replication in extra-intestinal organs and did not develop clinical biliary diseases. Mechanistically, RRV NSP1 seemed to promote active viral replication in hepatocytes and this expanded tropism led to enhanced infiltration of CD4 and CD8 T cells, causing immunopathology and damage in the hepatobiliary system. These results highlight an unexpectedly important role of RV NSP1 in viral replication and disease progression in extra-intestinal tissues.

Bioprospection for antiviral compounds from selected medicinal plants against RNA polymerase of rotavirus A using molecular modelling and density functional theory

Rotavirus A (RVA) infection remains a significant global health challenge, especially in developing countries, causing severe dehydrating diarrhoea in children under five years of age. Despite the availability of four World Health Organization (WHO) pre-qualified vaccines, their availability, particularly in low-income countries, pose significant challenges. Currently, there are no specific anti-rotaviral medications hence, the urgency to develop novel therapeutics against rotavirus infection. Thus, this study explored the potential of secondary metabolites of Spondias mombin, Macaranga barteri and Dicerocaryum eriocarpum as novel inhibitors of the RNA-dependent RNA polymerase (VP1) of rotavirus A using computational techniques. Pharmacokinetics parameters were adopted to screen the top 20 metabolites with high affinity for the target, initially identified through a docking study. Furthermore, the ability of the resulting compounds to modulate the investigated target was assessed using molecular dynamics (MD) simulation, while density functional theory (DFT) calculations were conducted to predict the molecular properties of the top-ranked compounds. Except for ellagic acid hexoside (-33.14 kcal/mol), all the leads had higher binding free energy values relative to sofosbuvir (-36.58 kcal/mol) following a 120 ns MD simulation. Overall, the resulting complexes with the lead compounds demonstrated acceptable stability, reduced flexibility and compactness, with spiraeoside (-51.02 kcal/mol) displaying more favourable thermodynamics metrics, albeit with a lesser binding free energy relative to chrysoeriol 7-glucuronide (-58.36 kcal/mol). The binding free energy and thermodynamic parameters of the top-hit compounds could be attributed to their respective bond interactions and molecular orbital properties except chrysoeriol 7-glucuronide, with a need for additional structural adjustment to enhance its thermodynamic properties. Thus, these findings indicate the potential modulatory ability of the lead compounds against the VP1 protein of RVA, underscoring the importance of further in vitro and in vivo studies to validate the predicted activity, and ongoing efforts are being made to pursue this line of investigation.

Characterization of viroplasm-like structures by co-expression of NSP5 and NSP2 across rotavirus species A to J.

Rotaviruses (RVs) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLSs) by the co-expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with their cognates NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is required for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species.

Short Communication: Rotavirus Group A Occurrence in Rural Water Source Samples in a Midwest Region State of Brazil, Comparing Wet and Dry Seasons.

Identified as a potential reference pathogen by the WHO Guidelines for Drinking-Water Quality, Rotavirus (RV) is among the main enteric viruses that cause waterborne diseases. The aim of this study was to identify and correlate the presence of RV in collective and individual water sources of rural communities in the state of Goiás, within the seasons in which the collections were made (rainy and dry seasons). For this, 86 water samples in the dry period and 160 samples in the rainy period were collected. Concentration of water samples, extraction of viral genetic material and molecular tests were performed. When analyzing the presence of RV in the samples, taking into consideration the period studied, RV was found to be more prevalent in the dry season (54.7%) than in the rainy season (20%), showing a strong statistical association with the dry season (p-value < 0.001). The presence of pathogenic microorganisms in water is a public risk issue, enabling the emergence of outbreaks, endemics and epidemics. In the present research, there was an association between the presence of Rotavirus and the dry period of the year when compared to the rainy period.

Detection and molecular characterization of major enteric pathogens in calves in central Ethiopia

BackgroundCalf diarrhea is a major cause of morbidity and mortality in the livestock sector worldwide and it can be caused by multiple infectious agents. In Ethiopia, cattle are the most economically important species within the livestock sector, but at the same time the young animals suffer from high rates of morbidity and mortality due to calf diarrhea. However, studies including both screening and molecular characterization of bovine enteric pathogens are lacking. Therefore, we aimed to both detect and molecularly characterize four of the major enteric pathogens in calf diarrhea, Enterotoxigenic Escherichia coli (E. coli K99 +), Cryptosporidium spp., rotavirus A (RVA), and bovine coronavirus (BCoV) in calves from central Ethiopia. Diarrheic and non-diarrheic calves were included in the study and fecal samples were analyzed with antigen-ELISA and quantitative real-time PCR (qPCR). Positive samples were further characterized by genotyping PCRs.ResultsAll four pathogens were detected in both diarrheic and non-diarrheic calves using qPCR and further characterization showed the presence of three Cryptosporidium species, C. andersoni, C. bovis and C. ryanae. Furthermore, genotyping of RVA-positive samples found a common bovine genotype G10P[11], as well as a more unusual G-type, G24. To our knowledge this is the first detection of the G24 RVA genotype in Ethiopia as well as in Africa. Lastly, investigation of the spike gene revealed two distinct BCoV strains, one classical BCoV strain and one bovine-like CoV strain.ConclusionsOur results show that Cryptosporidium spp., E. coli K99 + , RVA and BCoV circulate in calves from central Ethiopia. Furthermore, our findings of the rare RVA G-type G24 and a bovine-like CoV demonstrates the importance of genetic characterization.

Evaluation of a commercial Immunochromatographic Strip Assay (ICT) for rapid detection of bovine, porcine and human Rotavirus A

The most frequent diarrhoeal pathogen “Rotavirus” is commonly detected by Ribo Nucleic Acid- Polyacrylamide Gel Electrophoresis (RNA-PAGE), Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Immunoassay (Enzyme Linked Immunosorbent Assay) in humans. However, use of these tests is limited in animals therefore, penside test like Immunochromatographic test/Lateral Flow Assay (ICT/LFA) is necessary. The present study was carried out to compare the diagnostic efficacy of rapid commercial ICT strip assay, RNA-PAGE and RT-PCR for specific detection of Rotavirus A (RVA) from stool samples of calves, piglets and children. A total of 313 faecal samples were collected between November 2022 to April 2023 from children below 5years of age (n=100), calves (n=100) and piglets (n=113) ≤ 3 months of age and were screened by RNA-PAGE, RT-PCR and commercial ICT kit for rotavirus A.The overall positivity of RVA from human, calves and piglets using RNA-PAGE, RT-PCR and LFA was found to be 35% (35/100), 8% (8/100) and 14.16% (16/113), respectively. Higher positivity of rotavirus was recorded in male children (44.26%, 27/61) than in female children (20.51%, 8/39). The kappa agreement between LFA and RT-PCR was 0.79 (substantial agreement); between LFA and RNA-PAGE was 0.61 (substantially low agreement) and between RNA-PAGE and RT-PCR was found to be 0.75 (substantial agreement). Relative diagnostic sensitivity and specificity of LFA in comparison with RT-PCR was 74.54% and 98.44%, respectively and in comparison with RNA-PAGE was 72.97% and 93.47%, respectively. RNA-PAGE in comparison with RT-PCR is having diagnostic sensitivity and specificity of 65.45% and 99.6%, respectively. In conclusion, the ICT strip assay is a rapid, convenient and effective method with satisfactory efficacy for detection of RVA from children, calves and piglets. ICT fulfilled the WHO ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Delivered) criteria for point-of-care testing. It can be useful in determining rotavirus outbreaks in resource-limited settings.