Global emergence and re-emergence of Porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus which causes a highly contagious enteric disease, have led to several studies addressing its variability. The aim of this study was to characterize the infection of weaned pigs with Swine enteric coronavirus (SeCoV) -a chimeric virus most likely originated from a recombination event between PEDV and Transmissible gastroenteritis virus, or its mutant Porcine respiratory coronavirus-, and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals; piglets in groups B, C and D were orally inoculated with a PEDV variant (B and D) or SeCoV (C), while piglets in group A were mock inoculated and maintained as controls. At day 20 post-infection all groups were exposed to rPEDV-SeCoV; thus, group D was subjected to a homologous re-challenge, groups B and C to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively) and group A was primary challenged (-/rPEDV-SeCoV). Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-γ-secreting cells) were monitored. After primo-infection all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposition to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in feces.

Rotaviruses (RVs) are a major cause of viral gastroenteritis in both animals and humans worldwide. According to the molecular and serological properties of Viral Protein 6 (VP6), RVs are classified into nine species or groups (RVA-RVD and RVF-RVJ). RVA, RVB and RVC are well-recognized as etiological agents of enteric disease on swine farms and have been identified in all countries with a relevant pork production. Contrarily, RVH has only been identified on swine farms from Japan and more recently from Brazil, USA, South Africa and Vietnam but not yet in Europe. The occurrence of RVH was investigated in 103 Spanish pig herds. Nine farms were positive and the complete nucleotide sequences were achieved for four RVH isolates. Mean nucleotide identities with the RVH sequences available in GenBak ranged between 69.4 and 93.7 %. Phylogenetically, all genomic segments of Spanish RVH isolates clustered closely with other porcine RVH strains but were distantly related to human RVH as well as bat RVH strain. Moreover, based on the available tentative genotyping system for RVH, a new genotype for VP7 was proposed. To the best of our knowledge this is the first report of RVH on swine farms in Europe including its characterization by means of complete genome sequencing.

An adult male Kemp’s ridley was found dead on the coast of Kenedy County, Texas in August 2019 with bilateral severe, diffuse granulomatous nephritis. Pan-bacterial 16S rRNA gene PCR and amplicon sequencing of affected tissue indicated the presence of a Neorickettsia. Neorickettsia is a genus of obligate intracellular Alphaproteobacteria that are transmitted by digenean trematodes. For further characterization, primers were designed to amplify and sequence the groEL gene. Phylogenetic analysis found that the organism was distinct from other known species to a degree consistent with a novel species. Immunohistochemistry using an antibody directed against a Neorickettsia surface protein showed bacterial clusters within the renal granulomas. A species-specific quantitative PCR was designed, and detected the organism within the liver and colon of the index case. A qPCR survey of grossly normal kidneys opportunistically collected from additional stranded sea turtle kidneys detected this organism in five of 15 Kemp’s ridley turtles, two of nine green turtles, and neither of two loggerhead turtles. Recognition of this novel organism in an endangered species is concerning; additional work is underway to further characterize the potential of this organism as a pathogen of sea turtles.

Bovine viral diarrhea virus (BVDV) is a major cause of economic loss in the cattle industry, worldwide. Infection results in reduce productive performance, growth retardation, reduced milk production, and increased susceptibility to other diseases leading to early culling of animals. There are two main measures used to control the spread of BVDV: the elimination of persistently infected (PI) animals and vaccination. Currently, modified live or inactivated vaccines are used in BVDV vaccination programs, but there are safety risks or insufficient protection, respectively, with these vaccines. Here we report the development and efficacy of the first targeted subunit vaccine against BVDV. The core of the vaccine is a fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present on antigen-presenting cells. Industrial production of the vaccine is carried out using the baculovirus expression vector system (BEVS) using single-use manufacturing technologies. This new subunit vaccine induces strong BVDV-specific neutralizing antibodies in guinea pigs and cattle. Importantly, in cattle with low levels of natural BVDV-specific neutralizing antibodies, the vaccine induced strong neutralizing antibody levels to above the protective threshold, as determined by a competition ELISA. The APCH-E2 vaccine induced a rapid and sustained neutralizing antibody response compared to a conventional vaccine in cattle. The development of this subunit targeted vaccine provides cattle and dairy producers with an inexpensive, easily administered, safe, and efficacious BVDV vaccine.

Coronavirus (CoV) pandemics have become a huge threat to the public health worldwide in the recent decades. Typically, severe acute respiratory syndrome CoV (SARS-CoV) caused SARS pandemic in 2003 and SARS-CoV-2 caused the ongoing COVID-19 pandemic. Both viruses have been reported to originate from bats. Thus, direct or indirect interspecies transmission from bats to humans is required for the viruses to cause pandemics. Receptor utilization is a key factor determining the host range of viruses which is critical to the interspecies transmission. Angiotensin converting enzyme 2 (ACE2) is the receptor of both SARS-CoV and SARS-CoV-2, but only ACE2s of certain animals can be utilized by the viruses. Here, we employed pseudovirus cell-entry assay to evaluate the receptor-utilizing capability of ACE2s of 20 animals by the two viruses and found that SARS-CoV-2 utilized less ACE2s than SARS-CoV, indicating a narrower host range of SARS-CoV-2. Especially, SARS-CoV-2 tended not to use murine or non-mammal ACE2s. Meanwhile, pangolin CoV, another SARS-related coronavirus highly homologous to SARS-CoV-2 in its genome, yet showed similar ACE2 utilization profile with SARS-CoV rather than SARS-CoV-2. To clarify the mechanism underlying the receptor utilization, we compared the amino acid sequences of the 20 ACE2s and found 5 amino acid residues potentially critical for ACE2 utilization, including the N-terminal 20th and 42nd amino acids that may determine the different receptor utilization of SARS-CoV, SARS-CoV-2 and pangolin CoV. Our studies promote the understanding of receptor utilization of pandemic coronaviruses, potentially contributing to the virus tracing, intermediate host screening and epidemic prevention for pathogenic coronaviruses.

Beijing genotype Mycobacterium tuberculosis strains associate with increased virulence, resistance, and/or higher transmission rates. This study describes a specific Beijing strain predominantly identified in the Panamanian province of Colon with one of the highest incidence of tuberculosis in the country. Retrospective Mycobacterial Interspersed Repetitive-Unit/Variable-Number of Tandem Repeats analysis of 42 isolates collected between January-August 2018, allowed to identify a cluster (Beijing A) with 17 (40.5%) Beijing isolates. Subsequent prospective strain-specific PCR based surveillance from September 2019 to March 2020, confirmed the predominance of the Beijing A strain (44.1%) in this province. Whole genome sequencing revealed higher-than-expected diversity within the cluster, suggesting long-term prevalence of this strain and low number of cases caused by recent transmission. The Beijing A strain belongs to the Asian African 3 (Bmyc13, L2.2.5) branch of the modern Beijing sublineage, with their closest isolates corresponding to cases from Vietnam, probably introduced in Panama between 2000 and 2012.

The current pandemic caused by a novel coronavirus named as SARS-CoV2 has underlined the importance of emerging diseases of zoonotic importance. Along with human beings, several species of wild and pet animals have been demonstrated to be infected by SARS-CoV2, both naturally and experimentally. Additionally, with constant emergence of new variants, the species susceptibility might further change, warranting intensification of screening efforts. India is a vast and second most populated country, with a habitat of a very diverse range of animal species. In this study we are reporting infection of SARS-CoV2 in captive Asiatic lions. Detailed characterization revealed involvement of delta mutant (Pango lineage B.1.617.2) of SARS-CoV2 at two different locations. Interestingly, no other feline species enclosed in the zoo/park was found infected. The epidemiological and molecular analysis in this study will contribute to the understanding of SARS-CoV2 emerging mutants in wild and domesticated animals.

Infection with the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induces the coronavirus infectious disease 19 (COVID-19). Its pandemic form in human population and its probable animal origin, along with recent case reports in pets, make drivers of emergence crucial in carnivore domestic pets, especially cats, dogs and ferrets. Few data are available in these species; we first listed forty-six possible drivers of emergence of COVID-19 in pets, regrouped in eight domains (i.e. pathogen/disease characteristics, spatial-temporal distance of outbreaks, ability to monitor, disease treatment and control, characteristics of pets, changes in climate conditions, wildlife interface, human activity, and economic and trade activities). Secondly, we developed a scoring system per driver, then elicited experts (N = 33) to: (i) allocate a score to each driver, (ii) weight the drivers scores within each domain and (iii) weight the different domains between them. Thirdly, an overall weighted score per driver was calculated; drivers were ranked in decreasing order. Fourthly, a regression tree analysis was used to group drivers with comparable likelihood to play a role in the emergence of COVID-19 in pets. Finally, the robustness of the expert elicitation was verified. Five drivers were ranked with the highest probability to play a key role in the emergence of COVID-19 in pets: availability and quality of diagnostic tools, human density close to pets, ability of preventive/control measures to avoid the disease introduction or spread in a country (except treatment, vaccination and reservoir(s) control), current species specificity of the disease causing agent and current knowledge on the pathogen. As scientific knowledge on the topic is scarce and still uncertain, expert elicitation of knowledge, in addition with clustering and sensitivity analyses, is of prime importance to prioritize future studies, starting from the top five drivers. The present methodology is applicable to other emerging pet diseases.

Crimean-Congo haemorrhagic fever (CCHF) is an emerging tick-borne human disease in Spain. Understanding the spatiotemporal dynamics and exposure risk determinants of CCHF virus (CCHFV) in animal models is essential to predict the time and areas of highest transmission risk. With this goal, we designed a longitudinal survey in two wild ungulate species, the red deer ( Cervus elaphus) and the Eurasian wild boar ( Sus scrofa), in Doñana National Park, a protected Mediterranean biodiversity hotspot with high ungulate and CCHFV vector abundance, and which is also one of the main stopover sites for migratory birds between Africa and western Europe. Both ungulates are hosts to the main CCHFV vector in Spain, Hyalomma lusitanicum. We sampled wild ungulates annually from 2005 to 2020 and analysed the frequency of exposure to CCHFV by a double-antigen ELISA. The annual exposure risk was modelled as a function of environmental traits in an approach to understand exposure risk determinants that allow us to predict the most likely places and years for CCHFV transmission. The main findings show that H. lusitanicum abundance is a major driver of the fine-scale spatial CCHFV transmission risk, while inter-annual variations in the risk are conditioned by virus/vector hosts, by host community structure and by weather variations. The most relevant conclusion of the study is that the emergence of CCHF in Spain might have been associated with recent wild ungulate population changes promoting higher vector abundance. Decreasing wild ungulate population densities could reduce vector abundance and thus virus prevalence and the risk of CCHFV transmission to humans.

The world is confronting a dire situation due to the recent pandemic of the novel coronavirus disease (2019-nCoV) with so far mortality cases of 100,000 all over the world. Currently, there are no effective enough treatment options for this previously unknown virus. The current drugs in pipline and some plausible drug are overviewed in this paper. The potential molecular targets of each steps of the 2019-nCoV drug life cycle is discussed and highlights here. Although different types of anti-viral targets are applicable for 2019-nCoV drug screenings, the more promising targets can be considered as protease and RNA polymerase. Based on the results from antiviral agents repurposing and clinical studies, the remdesivir could be an encouraging drug in the frontline to be administrated for 2019-nCoV. Much progress in understanding the 2019-nCoV the molecular details of its life cycle followed by the identification of therapeutic targets seems to be an efficient approach in discovering potential drugs.

Peste des petits ruminants (PPR) is a viral transboundary disease of small ruminants that causes significant damage to agriculture. This disease has not been previously registered in the Republic of Kazakhstan (RK). This paper presents an assessment of the susceptibility of the RK’s territory to the spread of the disease in the event of its importation from infected countries. The Generalized Linear Negative Binomial regression model that was trained on the PPR outbreaks in China was used to rank municipal districts in the RK in terms of PPR spread risk. The outbreaks count per administrative district was used as a risk indicator, while a number of socio-economic, landscape and climatic factors were considered as explanatory variables. Summary road length, altitude, the density of small ruminants, the maximum green vegetation fraction, cattle density and the Engel coefficient were the most significant factors. The model demonstrated a good performance in training data (R 2 = 0.69) and was transferred to the RK, suggesting a significantly lower susceptibility of this country to the spread of PPR. Hot Spot analysis identified three clusters of districts at the highest risk, located in the western, eastern and southern parts of Kazakhstan. As part of the study, a countrywide survey was conducted to collect data on the distribution of livestock populations, which resulted in the compilation of a complete geo-database of small ruminant holdings in the RK. The research results may be used to formulate a national strategy for preventing the importation and spread of PPR in Kazakhstan through targeted monitoring in high-risk areas.

Leptospirosis has been widely reported in insular environments worldwide, characterizing a major public health threat. Although low genetic biodiversity is expected in these regions, the introduction of domestic and synanthropic mammals may contribute to the wider diversity leptospiral strains in insular settings. This study proposes a large-scale investigation of Leptospira infection in animals from Fernando de Noronha archipelago, Brazil. A total of 1,265 blood samples from domestic (n=682), synanthropic (n=133) and wild (n=450) animals were collected between 2007 and 2014, totaling 12 species. The presence of anti-Leptospira spp. antibodies was investigated by the Microscopic Agglutination Test (MAT) and kidney samples from synanthropic rodents were collected for the isolation of Leptospira spp. The leptospires recovered were further characterized by MAT with polyclonal antibodies, whole genome sequencing and Multilocus Sequence Typing (MLST). The MAT results revealed the presence of agglutinins in 90 samples (7.1%), and the most frequently found serogroup was Icterohaemorrhagiae (n=57) in practically all species included. Viable leptospires were recovered from one brown rat, and characterization revealed that the isolate belongs to L. interrogans serogroup Pyrogenes. This study stands as the most comprehensive investigation of Leptospira spp. infection in Fernando de Noronha archipelago, also providing the characterization of the first leptospiral strain ever isolated from an insular setting in Brazil. The results suggest that synanthropic rodents play a major role in the transmission of leptospirosis among wildlife and domestic species in the archipelago.

Canine parvovirus (CPV) is a major enteric pathogen of dogs worldwide that emerged in the late 1970s from a feline parvovirus (FPV)-like ancestral virus. Shortly after its emergence, variant CPVs were generated by acquiring amino-acid (aa) mutations in key capsid residues, associated with biological and/or antigenic changes. This study aimed to identify CPV variants amongst Australian dogs, to gain insights into the evolution of CPV in Australia through phylogenetic analysis of these variants, and to investigate relationships between the disease and vaccination status of dogs from which isolates were collected. CPV VP2 sequences were amplified from 79 faecal samples collected from dogs with parvoviral enteritis at 20 veterinary practices in 5 Australian states. The median age at diagnosis was 4 months (range 1 to 96 months). Only 3.7% of dogs with vaccination histories had completed recommended vaccination schedules, while 49% were incompletely vaccinated and 47.2% were unvaccinated. For the first time, CPV-2b has emerged as the dominant antigenic CPV variant circulating in dogs with parvoviral enteritis in Australia, comprising 54.4% of strains, while CPV-2a and CPV-2 comprised 43.1% and 2.5% of strains. CPV-2c strains were not identified. Analysis of translated VP2 sequences revealed a vast repertoire of aa mutations. Several Australian CPV strains displayed signatures in the VP2 protein typical of Asian CPVs, suggestion introduction of CPV strains from Asia, and/or CPV circulation between Asia and Australia. Strains of CPV were identified containing aa residues typical of FPV at capsid (VP2) key positions, representing reverse mutations or residual mutations retained from CPV-2 during adaptation from an FPV-like ancestor, suggesting that evolutionary intermediates between CPV-2 and FPV are circulating in the field. Similarly, intermediates between CPV-2a-like viruses and CPV-2 were also identified. These findings help inform a better understanding of the evolution of CPV in dogs.

The emergence of drug-resistant tuberculosis (DR-TB) is becoming a challenge to the national TB control programs including Ethiopia. Different risk factors are associated with the emergence of DR-TB. Identifying these risk factors in a local setting is important to strengthen the effort to prevent and control DR-TB. Thus, this study aimed to assess the risk factors associated with drug-resistant TB in Ethiopia. The Preferred Reporting Items for Systematic Reviews and Meta-analysis checklist was followed to conduct this study. We systematically searched the articles from electronic databases and gray literature sources. We used the Joanna Briggs Institute Critical Appraisal tools to assess the quality of studies. Data were analyzed using STATA version 15. We estimated the pooled OR along with 95%CI for each risk factor. The heterogeneity of the studies was assessed using the forest plot and I 2 heterogeneity test. Besides, we explored the presence of publication bias through visual inspection of the funnel plot and Egger’s regression test. After intense searching, we found 2238 articles, and 27 eligible studies were included in the final analysis. Based on the pooled analysis of the odds ratio, unemployment (OR; 2.71, 95% CI; 1.64, 3.78), having a history of the previous TB (OR; 4.83, 95% CI; 3.02, 6.64), having contact with a known TB patient (OR; 1.72, 95% CI; 1.05, 2.40), having contact with a known MDR-TB patient (OR; 2.54, 95% CI; 1.46, 3.63), and having pulmonary TB (OR; 1.80, 95% CI; 1.14, 2.45) were found to be the risk factors of drug-resistant TB. While older age TB patients (OR; 0.77, 95% CI; 0.60, 0.95) including age above 45 years OR; (0.76, 95% CI; 0.55, 0.97), and males (OR; 0.86, 95% CI; 0.76, 0.97) were found to had lower risk of DR-TB compared to their counterparts. A previous history of TB treatment is a major risk factor for acquiring DR-TB in Ethiopia that might be due to poor adherence during the first-line anti TB treatment. Besides, having contact with a known TB patient, having contact with a known MDR-TB patient, having pulmonary TB, and being unemployed were the risk factors of DR-TB in Ethiopia. Thus, active screening of TB contacts for DR-TB might help to detect DR-TB cases as early as possible and could help to mitigate its further transmission across the community.

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporter and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

Atypical porcine pestivirus (APPV), which has been confirmed to be associated with congenital tremor (CT) in pigs, is a newly discovered porcine virus that has been found in the Americas, Europe, and Asia; however, no report of APPV in Japan has been published. We identified an APPV in the central nervous system of Japanese piglets with CT, and firstly determined and analyzed the complete genome sequence. Phylogenetic analysis using the complete genome nucleotide sequence of the Japanese APPV, named Anna/2020, and those of APPVs from the NCBI database showed that APPVs were divided into three genotypes (genotypes 1 to 3), and that Anna/2020 clustered with the genotype 3 APPV strains, but distantly branched from these strains. Pairwise complete coding region nucleotide sequence comparisons revealed that there was 94.0% to 99.7% sequence identity among the genotype 3 strains, while Anna/2020 showed 87.0% to 89.3% identity to those genotype 3 strains, suggesting that Anna/2020 represents a novel APPV lineage within genotype 3. Retrospective examinations using RT-PCR revealed one genotype 1 and two novel genotype 3 APPVs from pigs without CT, and that novel genotype 3 APPVs have been prevalent in Japan since at least 2007.

Porcine circovirus 4 (PCV4), a novel and unclassified member of the genus Circovirus, was first reported in China in 2019. Aimed at providing more evidence about the active circulation of PCV4, this study screened 335 pooled internal organs and detected the virus (i) at the rates of 3.28%, (ii) from both clinical healthy and clinical sick pigs of various age groups, and (iii) in six out of nice provinces of Korea. The complete genomic sequence of a Korean PCV4 strain (E115) was 1,770 nucleotides in length and had 98.5% to 98.9% identity to three PCV4 strains available at GenBank up to date. Utilizing a set of bioinformatic programs, it was revealed that the Korean PCV4 strain contained several genomic features of (i) a palindrome stem-loop structure with conserved nonanucleotide, (ii) packed overlapping ORFs oriented in different directions, and (iii) two intergenic regions in between genes encoding putative replication- associated protein (Rep) and capsid (Cap) proteins. This study also predicted the presence of essential elements known so far for the replication of circoviruses, for example, the origin of DNA replication, endonuclease and helicase domains of Rep, the nuclear localization signal on the putative Cap protein. Finally, based on the phylogeny inferred from sequences of the putative Rep protein, it was suggested that PCV4 belong to genus Circovirus of family Circoviridae and losely related to three previous known porcine circoviruses of PCV1, PCV2 and PCV3.

The emergence of mobile mcr genes mediating resistance to colistin is a critical public health issue that has hindered the treatment of serious infections caused by multidrug-resistant pathogens in humans and other animals. We report the emergence of the mcr-9.1 gene in a polymyxin-resistant extended-spectrum β-lactamase (ESBL)-producing Enterobacter kobei infecting a free-living Franciscana dolphin (Pontoporia blainvillei), threatened with extinction in South America. Genomic analysis confirmed a wide resistome with additional presence of genes conferring resistance to clinically relevant β-lactam [blaCTX-M-15, blaACT-9, blaOXA-1 and blaTEM-1B], aminoglycoside [aac(3)-IIa, aadA1, aph(3’‘)-Ib and aph(6)-Id], trimethoprim [dfrA14], tetracycline [tetA], quinolone [aac(6’)-Ib-cr and qnrB1], fosfomycin [fosA], sulphonamide [sul2], and phenicol [catA1 and catB3] antibiotics. The identification of mcr-9.1 in a CTX-M-15-producing pathogen infecting a critically endangered animal is worryingly, due to the restricted therapeutic options, and should be interpreted as a sign of further spread of critical-priority pathogens and their resistance genes in threatened ecosystems.