Aujeszky’s disease in hunting dogs after the ingestion of wild boar raw meat in Sicily (Italy): clinical, diagnostic and phylogenetic features

Background Aujeszky's disease is caused by Suid Herpes Virus-1 and species belonging to the genus Sus scrofa are the main reservoir hosts. This virus, however, is capable of infecting and causing severe disease, with an almost constant fatal outcome in other species, both domestic and wild (carnivores, monogastric herbivores and ruminants). Moreover, the possibility of transmission to humans has been demonstrated. This study reports and describes the clinical, diagnostic, pathological and phylogenetic aspects of two cases of Aujeszky's disease in two hunting dogs following the ingestion of infected wild boar raw meat. These cases are contextualized in the province of Messina (Sicily), where a high prevalence of Aujeszky's disease has been recorded (average of 12,20% in the period 2010–2019) in farmed pig, and with evidence of spread to other species. A severe outbreak in cattle has recently been reported in these areas. Nevertheless, cases of Aujeszky's disease in dogs are rarely reported and this study represents the first well-documented report in this species in Sicily. Case presentation After a wild boar hunt, two dogs showed neurological symptoms and intense itching unresponsive to therapy. Diagnosis of Aujeszky's disease was made based on clinical suspicion, anamnestic information and confirmed by the isolation of the virus from the brain of both dogs. In addition, molecular typing, sequencing and phylogenetic analysis of the Real-Time PCR products were performed. The sequences studied were placed in the Italian Clade 1 along with the sequences obtained from wild boars and hunting dogs from Italy and France. Conclusions The finding of this disease in non-natural hosts in Sicilian multi-host epidemiological contexts suggests that the risk of inter-species transmission is concrete and that attention should be paid to developing disease control programs in these territories. The data obtained from genome sequencing of the two SuHV-1 isolates contribute to the enrichment of the GenBank with unknown sequences and the phylogenetic analysis implementation.

Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including SARS-CoV and SARS-CoV-2 enter cells via engagement of ACE2, while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clade 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from R. alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

Phylogeny of the ingoid clade (Caesalpinioideae, Fabaceae), based on nuclear and plastid data

We investigated generic relationships in the ingoid clade (Fabaceae) (sensu Koenen & al. 2020a), with main focus on genera with a taxonomic history in Calliandra s.l. of the tribe Ingeae (i.e. Afrocalliandra, Calliandra s.s., Sanjappa, Thailentadopsis, Viguieranthus, Zapoteca), and three genera of the tribe Acacieae (i.e., Acacia, Acaciella, Senegalia). The nuclear ribosomal ETS and ITS, and the plastid matK, trnL-trnF and ycf1 DNA-regions were analysed for 246 representatives from 36 genera using maximum likelihood as implemented in IQ-tree. The results show an Ingeae-Acacia clade within the ingoid clade, resolved in three major clades. Clade 1 (Calliandra s.s. and Afrocalliandra) is sister to clades 2 and 3. Clade 2 comprises Faidherbia, Sanjappa, Thailentadopsis, Viguieranthus and Zapoteca. Clade 3 comprises the remaining genera of the Ingeae, plus Acacia. The ingoid genus Senegalia is excluded from the Ingeae-Acacia clade. Acaciella is sister to the remaining ingoid clade when nuclear ribosomal data is included in the analyses, but included in the Ingeae-Acacia clade based on plastid data. Acacia and perhaps also Acaciella are thus nested within Ingeae. Species traditionally referred to Calliandra (Calliandra s.l.) are resolved in two clades, and the Calliandra-pod has apparently evolved independently several times.

Alcohol Acyltransferase Is Involved in the Biosynthesis of C6 Esters in Apricot (Prunus armeniaca L.) Fruit

Short-chain esters derived from fatty acid contribute to the characteristic flavor of apricot fruit, and the biosynthesis of these compounds in fruit is catalyzed by alcohol acyltransferase (AAT). In this work, we investigated the AAT gene family via genome-wide scanning, and three AAT loci were identified in different linkage groups (LGs), with PaAAT1 (PARG22907m01) in LG7, PaAAT2 (PARG15279m01) in LG4, and PaAAT3 (PARG22697m01) in LG6. Phylogenetic analysis showed that PaAAT1 belongs to clade 3, while PaAAT2 and PaAAT3 belong to clade 1 and clade 2, respectively. In contrast, the three AAT genes present different expression patterns. Only PaAAT1 exhibited distinct patterns of fruit-specific expression, and the expression of PaAAT1 sharply increased during fruit ripening, which is consistent with the abundance of C4–C6 esters such as (E)-2-hexenyl acetate and (Z)-3-hexenyl acetate. The transient overexpression of PaAAT1 in Katy (KT) apricot fruit resulted in a remarkable decrease in hexenol, (E)-2-hexenol, and (Z)-3-hexenol levels while significantly increasing the corresponding acetate production (p < 0.01). A substrate assay revealed that the PaAAT1 protein enzyme can produce hexenyl acetate, (E)-2-hexenyl acetate, and (Z)-3-hexenyl acetate when C6 alcohols are used as substrates for the reaction. Taken together, these results indicate that PaAAT1 plays a crucial role in the production of C6 esters in apricot fruit during ripening.

Phylogeny of Amathusiini butterflies based on adult morphology (Lepidoptera, Nymphalidae, Satyrinae)

Based on comparative morphology of adults, a phylogeny is proposed for the butterfly tribe Amathusiini (Nymphalidae, Satyrinae). The dataset includes 92 characters scored for 45 species in 12 genera, representing the most comprehensive phylogenetic analysis for this group. Parsimony analyses produced a well-resolved strict consensus tree where genera were divided in three main groups: (clade 1) Stichophthalma; (clade 2) Aemona, Faunis, Melanocyma and Taenaris; (clade 3) Enispe, Discophora, Thaumantis, Thauria, Amathusia, Amathuxidia, and Zeuxidia. While genera in clades 1 and 2 were found to be morphologically homogeneous, clade 3 showed remarkable morphological divergence between and within genera. The monophyly of most genera was recovered with variable levels of support, but Melanocyma and Taenaris nested within Faunis. Therefore, here Melanocyma NEW SYN. is subsumed within Faunis, and Taenaris STAT. REV. is regarded as a subgenus of Faunis. Mimicry likely evolved a single time within the Faunis-Taenaris assemblage, as species of Taenaris formed a monophyletic group. Results are compared to early classifications and recent DNA-based analyses, and points of agreement and conflicts are discussed.

A clade of telosma mosaic virus from Thailand is undergoing geographical expansion and genetic differentiation in passionfruit of Vietnam and China

Passionfruit (Passiflora edulis) is widely cultivated in tropical and subtropical regions around the world. Several viruses of the genus Potyvirus pose serious threat to passion fruit production. The origin, dispersal and evolution of these potyviruses, however, are poorly understood. Here, we investigated the genetic structure of telosma mosaic virus (TelMV), a potyvirus that infects passionfruit in East and Southeast Asia, after a survey of its incidence in passionfruit plants of China. The phylogeny inferred from 140 nucleotide sequences of the coat protein (CP) gene of TelMV, including 96 determined in this study, separated this virus into 4 clades. TelMV isolates from passionfruit were placed into Clade 1–3, while those from other plant species into Clade 4. Interestingly, TelMV isolates of passionfruit from Thailand were found in all the three clades of Clade 1–3, but those from China and Vietnam were found exclusively in Clade 1. Nevertheless, TelMV isolates within Clade 1 tended to cluster according to their geographical origin. Geographical populations from Thailand, Taiwan and Hainan islands of China showed significant genetic differences with one another and with those from Guangxi, Fujian, Guangdong, Yunnan and Jiangsu provinces of China. Altogether, these data suggest that several distinct TelMV clades had arisen from the passionfruit of Thailand, but only one of which was dispersed. In expanding its distribution, this clade of TelMV has undergone geography-associated evolution. Further studies on this hypothesis may shed new insights into mechanisms underlying the emergence of potyviral diseases in passionfruit plants.

Comparative Genome Analysis of Bacillus amyloliquefaciens Focusing on Phylogenomics, Functional Traits, and Prevalence of Antimicrobial and Virulence Genes

Bacillus amyloliquefaciens is a gram-positive, nonpathogenic, endospore-forming, member of a group of free-living soil bacteria with a variety of traits including plant growth promotion, production of antifungal and antibacterial metabolites, and production of industrially important enzymes. We have attempted to reconstruct the biogeographical structure according to functional traits and the evolutionary lineage of B. amyloliquefaciens using comparative genomics analysis. All the available 96 genomes of B. amyloliquefaciens strains were curated from the NCBI genome database, having a variety of important functionalities in all sectors keeping a high focus on agricultural aspects. In-depth analysis was carried out to deduce the orthologous gene groups and whole-genome similarity. Pan genome analysis revealed that shell genes, soft core genes, core genes, and cloud genes comprise 17.09, 5.48, 8.96, and 68.47%, respectively, which demonstrates that genomes are very different in the gene content. It also indicates that the strains may have flexible environmental adaptability or versatile functions. Phylogenetic analysis showed that B. amyloliquefaciens is divided into two clades, and clade 2 is further dived into two different clusters. This reflects the difference in the sequence similarity and diversification that happened in the B. amyloliquefaciens genome. The majority of plant-associated strains of B. amyloliquefaciens were grouped in clade 2 (73 strains), while food-associated strains were in clade 1 (23 strains). Genome mining has been adopted to deduce antimicrobial resistance and virulence genes and their prevalence among all strains. The genes tmrB and yuaB codes for tunicamycin resistance protein and hydrophobic coat forming protein only exist in clade 2, while clpP, which codes for serine proteases, is only in clade 1. Genome plasticity of all strains of B. amyloliquefaciens reflects their adaption to different niches.

Uncovering Symbionts Across the Psyllid Tree of Life and the Discovery of a New Liberibacter Species, “Candidatus” Liberibacter capsica

Sap-feeding insects in the order Hemiptera associate with obligate endosymbionts that are required for survival and facultative endosymbionts that can potentially modify resistance to stress, enemies, development, and reproduction. In the superfamily Psylloidea, the jumping plant lice (psyllids), less is known about the diversity and prevalence of their endosymbionts compared to other sap-feeding pests such as aphids (Aphididae). To address this knowledge gap, using 16S rRNA sequencing we identify symbionts across divergent psyllid host lineages from around the world. Taking advantage of a new comprehensive phylogenomic analyses of Psylloidea, we included psyllid samples from 44 species of 35 genera of five families, collected from 11 international locations for this study. Across psyllid lineages, a total of 91 OTUs were recovered, predominantly of the Enterobacteriaceae (68%). The diversity of endosymbionts harbored by each psyllid species was low with an average of approximately 3 OTUs. Two clades of endosymbionts (clade 1 and 2), belonging to Enterobacteriaceae, were identified that appear to be long term endosymbionts of the psyllid families Triozidae and Psyllidae, respectively. We also conducted high throughput metagenomic sequencing on three Ca. Liberibacter infected psyllid species (Russelliana capsici, Trichochermes walkeri, and Macrohomotoma gladiata), initially identified from 16S rRNA sequencing, to obtain more genomic information on these putative Liberibacter plant pathogens. The phylogenomic analyses from these data identified a new Ca. Liberibacter species, Candidatus Liberibacter capsica, that is a potential pathogen of solanaceous crops. This new species shares a distant ancestor with Ca. L. americanus, which occurs in the same range as R. capsici in South America. We also detected the first association between a psyllid specializing on woody hosts and the Liberibacter species Ca. L. psyllaurous, which is a globally distributed pathogen of herbaceous crop hosts in the Solanaceae. Finally, we detected a potential association between a psyllid pest of figs (M. gladiata) and a Ca. Liberibacter related to Ca. L. asiaticus, which causes severe disease in citrus. Our findings reveal a wider diversity of associations between facultative symbionts and psyllids than previously reported and suggest numerous avenues for future work to clarify novel associations of ecological, evolutionary, and pathogenic interest.

Clinical Molecular and Genomic Epidemiology of Morganella morganii in China

Objectives: Ongoing acquisition of antimicrobial resistance genes has made Morganella morganii a new clinical treatment challenge. Understanding the molecular epidemiology of M. morganii will contribute to clinical treatment and prevention.Methods: We undertook a 6-year clinical molecular epidemiological investigation of M. morganii from three tertiary hospitals in China since 2014. Antimicrobial susceptibility testing was performed using a VITEK-2 system. All isolates were screened for β-lactam and plasmid-mediated quinolone resistance genes by PCR. Isolates carrying carbapenem-resistant genes were subjected to whole-genome sequencing (WGS). The variation and evolution of these mobile genetic elements (MGEs) were then systematically analyzed.Results: Among all M. morganii isolates (n = 335), forty (11.9%) were recognized as multidrug resistant strains. qnrD1, aac(6′)-Ib-cr, blaTEM–104, and blaCTX–M–162 were the top four most prevalent resistance genes. Notably, phylogenomic and population structure analysis suggested clade 1 (rhierBAPS SC3 and SC5) associated with multiple resistance genes seemed to be widely spread. WGS showed a blaOXA–181-carrying IncX3 plasmid and a Proteus genomic island 2 variant carrying blaCTX–M–3, aac(6′)-Ib-cr coexisted in the same multidrug resistant strain zy_m28. Additionally, a blaIMP–1-carrying IncP-1β type plasmid was found in the strain nx_m63.Conclusion: This study indicates a clade of M. morganii is prone to acquire resistance genes, and multidrug resistant M. morganii are increasing by harboring a variety of MGEs including two newly discovered ones in the species. We should be vigilant that M. morganii may bring more extensive and challenging antimicrobial resistance issue.

Genomic and phenotypic analysis of heat and sanitizer resistance in Escherichia coli from beef in relation to the locus of heat resistance

The locus of heat resistance (LHR) can confer heat resistance to Escherichia coli to various extents. This study investigated the phylogenetic relationships, and genomic and phenotypic characteristics of E. coli with or without LHR recovered from beef by direct plating or from enrichment broth at 42°C. LHR-positive E. coli isolates (n=24) were whole genome-sequenced by short- and long-reads. LHR-negative isolates (n=18) from equivalent sources as LHR-positive isolates were short-read sequenced. All isolates were assessed for decimal reduction time at 60°C ( D 60°C ) and susceptibility to E-SAN and Perox-E. Selected isolates were evaluated for growth at 42°C. The LHR-positive and negative isolates were well separated on the core genome tree, with 22/24 of the positive isolates clustering into three clades. Isolates within clade 1 and 2, despite their different D 60°C values, were clonal, as determined by subtyping (MLST, core genome MLST, and serotyping). Isolates within each clade are of one serotype. The LHR-negative isolates were genetically diverse. The LHR-positive isolates had a larger (p<0.001) median genome size by 0.3 Mbp (5.0 vs 4.7 Mbp), and overrepresentation of genes in plasmid maintenance, stress response and cryptic prophages, but underrepresentation of genes involved in epithelial attachment and virulence. All LHR-positive isolates harbored a chromosomal copy of LHR, and all clade 2 isolates had an additional partial copy of LHR on conjugative plasmids. The growth rates at 42°C were 0.71±0.02 and 0.65±0.02 logOD h −1 for LHR-positive and negative isolates. No meaningful difference in sanitizer susceptibility was noted between LHR-positive and negative isolates. Importance Resistant bacteria are serious food safety and public health concerns. Heat resistance conferred by the LHR varies largely among different strains. The findings in this study show that genomic background and composition of LHR, in addition to the presence of LHR, play an important role in the degree of heat resistance in E. coli , and that strains with certain genetic background are more likely to acquire and maintain the LHR. Also, caution should be exercised when recovering E. coli at elevated temperatures as the presence of LHR may confer growth advantages to some strains. Interestingly, the LHR harboring strains seem to have evolved further from their primary animal host to adapt to their secondary habitat, as reflected by fewer genes in virulence and epithelial attachment. The phylogenetic relationships among the isolates point towards multiple mechanisms for acquiring LHR, likely prior to their deposition on meat.