Rapid longitudinal SARS-CoV-2 intra-host emergence of novel haplotypes regardless of immune deficiencies

On February 2020, the municipality of Vo’, a small town near Padua (Italy), was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. The entire population was swab tested in two sequential surveys. Here we report the analysis of the viral genomes, which revealed that the unique ancestor haplotype introduced in Vo’ belongs to lineage B and, more specifically, to the subtype found at the end of January 2020 in two Chinese tourists visiting Rome and other Italian cities, carrying mutations G11083T and G26144T. The sequences, obtained for 87 samples, allowed us to investigate viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo’. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptoms severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype, carried by an index case. The lockdown extinguished both viral spread and the emergence of new variants, confirming the efficiency of this containment strategy. The information gathered from household was used to reconstructs possible transmission events.

Global analysis of Hemileia vastatrix populations shows clonal reproduction for the coffee leaf rust pathogen throughout most of its range

Hemileia vastatrix is the most important fungal pathogen of coffee and the causal agent of recurrent disease epidemics that have invaded nearly every coffee-growing region in the world. The development of coffee varieties resistant to H. vastatrix requires fundamental understanding of the biology of the fungus. However, the complete life cycle of H. vastatrix remains unknown and conflicting studies and interpretations exist as to whether the fungus is undergoing sexual reproduction. Here we used population genetics of H. vastatrix to infer the reproductive mode of the fungus across most of its geographic range including Central Africa, SE Asia, the Caribbean, and South and Central America. The population structure of H. vastatrix was determined using eight simple sequence repeat markers (SSRs) developed for this study. The analyses of the standardized index of association, Hardy Weinberg equilibrium, and clonal richness all strongly support asexual reproduction of H. vastatrix in all sampled areas. Similarly, a minimum spanning network tree reinforces the interpretation of clonal reproduction in the sampled H. vastatrix populations. These findings may have profound implications for resistance breeding and management programs against H. vastatrix.

Clinical Investigation and Molecular Diagnosis of Microsporidian Enterocytozoon hepatopenaei (EHP) in Shrimps from Selangor, Malaysia

In recent years, shrimp aquaculture production in Southeast Asia countries was highly infected by, microsporidian parasite, EHP. Recently, shrimps from farms located in Selangor were encountering growth retardation and it highly concerned us to carry out a clinical investigation in both farms (Location A and B). EHP infected P. monodon samples were collected from both locations. A total of 43 shrimp samples were collected and diagnosed via PCR using the 18S rRNA gene. Environmental parameters were found relatively higher than the advised values. It indicates that the farms are highly infected and toxic which had led to growth retardation and mortality. However, no significant differences were observed (except water temperatures) between the locations. The phylogenetic analysis expressed EHP detected from Malaysia formed in one clade and all the reference sequences clustered based on geographical continents (Asian-Latin America). Minimum-spanning network analysis shows that Malaysia samples Location A are genetically related with other Asian EHP samples even though the total positive rate of EHP infection was lower. However, samples from Location B were not clustered within the EHP clade and shows the highest genetic variance within Malaysia and among Asian samples. This preliminary investigation results confirmed the incidence of EHP outbreak in Malaysia.

Tracking of Anopheles stephensi in Ethiopia using mitochondrial DNA reveals pattern of spread

The recent detection of the South Asian malaria vector Anopheles stephensi in the Horn of Africa (HOA) raises concerns about the impact of this mosquito on malaria transmission in the region. The mode and history of introduction is important for predicting the likelihood of continued introduction and future spread. Analysis of An. stephensi genetic diversity and population structure can provide insight into the history of the mosquito in the HOA. We investigated genetic diversity of An. stephensi in eastern Ethiopia where detection suggests a range expansion to this region to understand the history of this invasive population. We sequenced the cytochrome oxidase subunit I (COI) and cytochrome B gene (CytB) in 187 An. stephensi collected from 10 sites in Ethiopia in 2018. Phylogenetic analyses using a maximum-likelihood approach and minimum spanning network were conducted for Ethiopian sequences. Molecular identification of bloodmeal sources was also performed using universal vertebrate CytB sequencing. Six COI-CytB haplotypes were observed based on five segregating sites, with the highest number of haplotypes in the northeastern sites (Semera, Bati, and Gewana towns) relative to the southeastern sites (Kebridehar, Godey, and Degehabur) in eastern Ethiopia. In the phylogenetic and network analysis, we observed population differentiation based on the distribution of the haplotypes across the northeastern and central sites (Erer Gota, Dire Dawa, and Awash Sebat Kilo) compared to the southeastern sites and evidence of a South Asian origin of the HOA An. stephensi lineages. The presence of the putative South Asian haplotype of origin at sites closest to Ethiopia's northeastern borders support route of introductions into Ethiopia from the northeast. Finally, molecular bloodmeal analysis revealed evidence of feeding on bovines, goats, dogs, and humans, as well as evidence of multiple (mixed) blood meals. In conclusion, we find support for the hypothesis for the recent expansion of An. stephensi into southeastern Ethiopia with multiple introductions. We also find evidence that supports the hypothesis that HOA An. stephensi populations originate from South Asia rather than the Arabian Peninsula. The evidence of both zoophagic and anthropophagic feeding support the potential for livestock movement to play a role in vector spread in this region.

A Microsatellite Analysis Used to Identify Global Pathways of Movement of Phytophthora cinnamomi and the Likely Sources of Wildland Infestations in California and Mexico

The genetic structure of a sample of isolates of the oomycete plant pathogen Phytophthora cinnamomi from natural and agricultural outbreaks and the long-distance movement of individual genotypes were studied using four microsatellite markers to genotype 159 isolates of Californian, Mexican, and worldwide origins. Allelic profiles identified 75 multilocus genotypes. A STRUCTURE analysis placed them in three groups characterized by different geographic and host ranges, different genic and genotypic diversity, and different reproductive modes. When relationships among genotypes were visualized on a minimum spanning network (MSN), genotypes belonging to the same STRUCTURE group were contiguous, with rare exceptions. A putatively ancestral group 1 had high genic diversity, included all A1 mating type isolates and all Papuan isolates in the sample, was rarely isolated from natural settings in California and Mexico, and was positioned at the center of the MSN. Putatively younger groups 2 and 3 had lower genic diversity, were both neighbors to group 1 but formed two distinct peripherical sectors of the MSN, and were equally present in agricultural commodities and natural settings in Mexico and California. A few genotypes, especially in groups 2 and 3, were isolated multiple times in different locations and settings. The presence of identical genotypes from the same hosts in different continents indicated that long-distance human-mediated movement of P. cinnamomi had occurred. The presence of identical genotypes at high frequencies in neighboring wildlands and agricultural settings suggest that specific commodities may have been the source of recent wild infestations caused by novel invasive genotypes.

Microbe-ID: an open source toolbox for microbial genotyping and species identification

Development of tools to identify species, genotypes, or novel strains of invasive organisms is critical for monitoring emergence and implementing rapid response measures. Molecular markers, although critical to identifying species or genotypes, require bioinformatic tools for analysis. However, user-friendly analytical tools for fast identification are not readily available. To address this need, we created a web-based set of applications called Microbe-ID that allow for customizing a toolbox for rapid species identification and strain genotyping using any genetic markers of choice. Two components of Microbe-ID, named Sequence-ID and Genotype-ID, implement species and genotype identification, respectively. Sequence-ID allows identification of species by using BLAST to query sequences for any locus of interest against a custom reference sequence database. Genotype-ID allows placement of an unknown multilocus marker in either a minimum spanning network or dendrogram with bootstrap support from a user-created reference database. Microbe-ID can be used for identification of any organism based on nucleotide sequences or any molecular marker type and several examples are provided. We created a public website for demonstration purposes called Microbe-ID (microbe-id.org) and provided a working implementation for the genusPhytophthora(phytophthora-id.org). InPhytophthora-ID, the Sequence-ID application allows identification based on ITS orcoxspacer sequences. Genotype-ID groups individuals into clonal lineages based on simple sequence repeat (SSR) markers for the two invasive plant pathogen speciesP. infestansandP. ramorum. All code is open source and available on github and CRAN. Instructions for installation and use are provided athttps://github.com/grunwaldlab/Microbe-ID.

Microbe-ID: An open source toolbox for microbial genotyping and species identification

Development of tools to identify species, genotypes, or novel strains of invasive organisms is critical for monitoring emergence and implementing rapid response measures. Molecular markers, although critical to identifying species or genotypes, require bioinformatic tools for analysis. However, user-friendly analytical tools for fast identification are not readily available. To address this need, we created a web-based set of applications called Microbe-ID that allow for customizing a toolbox for rapid species identification and strain genotyping using any genetic markers of choice. Two components of Microbe-ID, named Sequence-ID and Genotype-ID, implement species and genotype identification, respectively. Sequence-ID allows identification of species by using BLAST to query sequences for any locus of interest against a custom reference sequence database. Genotype-ID allows placement of an unknown multilocus marker in either a minimum spanning network or dendrogram with bootstrap support from a user-created reference database. Microbe-ID can be used for identification of any organism based on nucleotide sequences or any molecular marker type and several examples are provided. We created a public website for demonstration purposes called Microbe-ID ( www.microbe-id.org ) and provided a working implementation for the genus Phytophthora ( www.phytophthora-id.org ). In Phytophthora-ID, the Sequence-ID application allows identification based on ITS or cox spacer sequences. Genotype-ID groups individuals into clonal lineages based on simple sequence repeat (SSR) markers for the two invasive plant pathogen species P. infestans and P. ramorum. All code is open source and available on github and CRAN. Instructions for installation and use are provided at https://github.com/grunwaldlab/Microbe-ID.

Microbe-ID: An open source toolbox for microbial genotyping and species identification

Development of tools to identify species, genotypes, or novel strains of invasive organisms is critical for monitoring emergence and implementing rapid response measures. Molecular markers, although critical to identifying species or genotypes, require bioinformatic tools for analysis. However, user-friendly analytical tools for fast identification are not readily available. To address this need, we created a web-based set of applications called Microbe-ID that allow for customizing a toolbox for rapid species identification and strain genotyping using any genetic markers of choice. Two components of Microbe-ID, named Sequence-ID and Genotype-ID, implement species and genotype identification, respectively. Sequence-ID allows identification of species by using BLAST to query sequences for any locus of interest against a custom reference sequence database. Genotype-ID allows placement of an unknown multilocus marker in either a minimum spanning network or dendrogram with bootstrap support from a user-created reference database. Microbe-ID can be used for identification of any organism based on nucleotide sequences or any molecular marker type and several examples are provided. We created a public website for demonstration purposes called Microbe-ID ( www.microbe-id.org ) and provided a working implementation for the genus Phytophthora ( www.phytophthora-id.org ). In Phytophthora-ID, the Sequence-ID application allows identification based on ITS or cox spacer sequences. Genotype-ID groups individuals into clonal lineages based on simple sequence repeat (SSR) markers for the two invasive plant pathogen species P. infestans and P. ramorum. All code is open source and available on github and CRAN. Instructions for installation and use are provided at https://github.com/grunwaldlab/Microbe-ID.

First Report of Puccinia triticina (Leaf Rust) Race FBPT on Wheat in South Africa

Eleven isolates of Puccinia triticina Erikss. collected from bread wheat (Triticum aestivum L.) in the Western Cape during the 2010 annual rust survey were pathotyped to a race not previously recorded in South Africa. Replicated race analysis on seedlings of 16 Thatcher (Tc) near-isogenic differential lines at two rust laboratories confirmed avirulence (infection types [ITs] 0; to 2+) for lines with Lr1, Lr2a, Lr9, Lr11, Lr16, Lr24, and Lr26 and virulence (ITs 3 to 4) for lines with Lr2c, Lr3, Lr3ka, Lr10, Lr14a, Lr17, Lr18, Lr30, and LrB. Thatcher lines with LrB, Lr10, Lr14a, and Lr18 were added as the fourth set to the 12 original differential lines (1,2). This profile codes to race FBPT according to the North American system, and, based on these differentials, resembles a P. triticina isolate from Gwebi, Zimbabwe, in 2012 (Z. A. Pretorius, unpublished data). When additional single gene lines were tested with FBPT (race 3SA147 according to the ARC-Small Grain Institute rust notation procedure), lines with Lr2b, Lr15, Lr19, Lr20, Lr21, Lr25, Lr27+31, Lr28, Lr29, Lr32, Lr36, Lr38, Lr45, Lr47, Lr50, Lr51, and Lr52 were effective, whereas lines with Lr3bg, Lr23, Lr28, and Lr33 were ineffective. In adult plant tests in a greenhouse, Thatcher lines containing Lr12 (IT ;1c), Lr13 (IT ;1c), Lr22a (IT 1), Lr35 (IT ;1+), and Lr37 (IT ;1) were resistant, whereas Thatcher (Lr22b) was susceptible. Of 146 South African cultivars and lines infected as seedlings with 3SA147 (FBPT), 83% were resistant (IT ≤ 2) and 5% showed within-line variation. Entries showing compatible ITs with 3SA147 (FBPT) were also susceptible to either or both of 3SA133 (PDRS) and 3SA146 (MCDS). In addition, the new race was genotyped using 16 simple sequence repeat (SSR) primer-combinations (3). Three single pustule isolates of 3SA147 (FBPT) were identical and showed 82% and 76% similarity with the recently described races 3SA146 (MCDS) and 3SA145 (CCPS), respectively (4). Minimum spanning network analysis confirmed this close genetic relationship among the three races. However, since their virulence phenotypes differ, it is proposed that 3SA147 (FBPT) is not a stepwise mutation from either 3SA145 (CCPS) or 3SA146 (MCDS), but rather a foreign introduction into South Africa. As most current breeding lines and wheat cultivars are resistant, it is unlikely that race FBPT will threaten wheat production in South Africa, but its detection underlines the fact that new P. triticina variants have been occurring at regular intervals in the region. References: (1) J. A. Kolmer et al. Austr. J. Agric. Res. 58:631, 2007. (2) D. L. Long and J. A. Kolmer. Plant Dis. 79:525, 1989. (3) L. J. Szabo and J. A. Kolmer. Mol. Ecol. Notes 7:708, 2007. (4) T. Terefe et al. Plant Dis. 95:611, 2011.

A New Subspecies Identification and Population Study of the Asian Small-Clawed Otter (Aonyx cinereus) in Malay Peninsula and Southern Thailand Based on Fecal DNA Method

Three species of otter can be found throughout Malay Peninsula: Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata. In this study, we focused on the A. cinereus population that ranges from the southern and the east coast to the northern regions of Malay Peninsula up to southern Thailand to review the relationships between the populations based on the mitochondrial D-loop region. Forty-eight samples from six populations were recognized as Johor, Perak, Terengganu, Kelantan, Ranong, and Thale Noi. Among the 48 samples, 33 were identified as A. cinereus, seven as L. sumatrana, and eight as L. perspicillata. Phylogenetically, two subclades formed for A. cinereus. The first subclade grouped all Malay Peninsula samples except for samples from Kelantan, and the second subclade grouped Kelantan samples with Thai sample. Genetic distance analysis supported the close relationships between Thai and Kelantan samples compared to the samples from Terengganu and the other Malaysian states. A minimum-spanning network showed that Kelantan and Thailand formed a haplogroup distinct from the other populations. Our results show that Thai subspecies A. cinereus may have migrated to Kelantan from Thai mainland. We also suggest the classification of a new subspecies from Malay Peninsula, the small-clawed otter named A. cinereus kecilensis.