Development and Application of a gp60-Based Subtyping Tool for Cryptosporidium bovis

Cryptosporidium bovis is a common enteric pathogen in bovine animals. The research on transmission characteristics of the pathogen is hampered by the lack of subtyping tools. In this study, we retrieve the nucleotide sequence of the 60 kDa glycoprotein (GP60) from the whole genome sequences of C. bovis we obtained previously and analyze its sequence characteristics. Despite a typical structure of the GP60 protein, the GP60 of C. bovis had only 19.3–45.3% sequence identity to those of other Cryptosporidium species. On the basis of the gene sequence, a subtype typing tool was developed for C. bovis and used in the analysis of 486 C. bovis samples from dairy cattle, yaks, beef cattle, and water buffalos from China. Sixty-eight sequence types were identified from 260 subtyped samples, forming six subtype families, namely XXVIa to XXVIf. The mosaic sequence patterns among subtype families and the 121 potential recombination events identified among the sequences both suggest the occurrence of genetic recombination at the locus. No obvious host adaptation and geographic differences in the distribution of subtype families were observed. Most farms with more extensive sampling had more than one subtype family, and the dominant subtype families on a farm appeared to differ between pre- and post-weaned calves, indicating the likely occurrence of multiple episodes of C. bovis infections. There was an association between XXVId infection and occurrence of moderate diarrhea in dairy cattle. The subtyping tool developed and the data generated in the study might improve our knowledge of the genetic diversity and transmission of C. bovis.

Subtyping Cryptosporidium xiaoi, a Common Pathogen in Sheep and Goats

Cryptosporidiosis is a significant cause of diarrhea in sheep and goats. Among the over 40 established species of Cryptosporidium, Cryptosporidium xiaoi is one of the dominant species infecting ovine and caprine animals. The lack of subtyping tools makes it impossible to examine the transmission of this pathogen. In the present study, we identified and characterized the 60-kDa glycoprotein (gp60) gene by sequencing the genome of C. xiaoi. The GP60 protein of C. xiaoi had a signal peptide, a furin cleavage site of RSRR, a glycosylphosphatidylinositol anchor, and over 100 O-glycosylation sites. Based on the gp60 sequence, a subtyping tool was developed and used in characterizing C. xiaoi in 355 positive samples from sheep and goats in China. A high sequence heterogeneity was observed in the gp60 gene, with 94 sequence types in 12 subtype families, namely XXIIIa to XXIIIl. Co-infections with multiple subtypes were common in these animals, suggesting that genetic recombination might be responsible for the high diversity within C. xiaoi. This was supported by the mosaic sequence patterns among the subtype families. In addition, a potential host adaptation was identified within this species, reflected by the exclusive occurrence of XXIIIa, XXIIIc, XXIIIg, and XXIIIj in goats. This subtyping tool should be useful in studies of the genetic diversity and transmission dynamics of C. xiaoi.

Subtype Characterization and Zoonotic Potential of Cryptosporidium felis in Cats in Guangdong and Shanghai, China

Cryptosporidiumfelis is an important cause of feline and human cryptosporidiosis. However, the transmission of this pathogen between humans and cats remains controversial, partially due to a lack of genetic characterization of isolates from cats. The present study was conducted to examine the genetic diversity of C. felis in cats in China and to assess their potential zoonotic transmission. A newly developed subtyping tool based on a sequence analysis of the 60-kDa glycoprotein (gp60) gene was employed to identify the subtypes of 30 cat-derived C. felis isolates from Guangdong and Shanghai. Altogether, 20 C. felis isolates were successfully subtyped. The results of the sequence alignment showed a high genetic diversity, with 13 novel subtypes and 2 known subtypes of the XIXa subtype family being identified. The known subtypes were previously detected in humans, while some of the subtypes formed well-supported subclusters with human-derived subtypes from other countries in a phylogenetic analysis of the gp60 sequences. The results of this study confirmed the high genetic diversity of the XIXa subtype family of C. felis. The common occurrence of this subtype family in both humans and cats suggests that there could be cross-species transmission of C. felis.

Development of a Subtyping Tool for Zoonotic Pathogen Cryptosporidium Canis

Cryptosporidium canis is an important cause of cryptosporidiosis in canines and humans. Studies of the transmission characteristics of C. canis are currently hampered by lack of suitable subtyping tools. In this study, we conducted a genomic survey of the pathogen and developed a subtyping tool targeting the partial 60-kDa glycoprotein (gp60) gene. Seventy-six isolates previously identified as C. canis were analyzed using the new subtyping tool. Amplicons of expected size were obtained from 49 isolates, and phylogenetic analysis identified 10 subtypes clustered in five distinct groups (XXa–XXe). The largest group XXa contained 43 isolates from four subtypes that differed slightly from each other at the nucleotide level, while groups XXb–XXe contain one to three isolates each. The similar distribution of subtypes in humans and canines suggests that zoonotic transmission might play an important role in the epidemiology of C. canis. In addition, a suspected zoonotic transmission of C. canis between dogs and humans in a household was confirmed using the subtyping tool. The subtyping tool and data generated in this study might improve our understanding of the transmission of this zoonotic pathogen.

Covidex: an ultrafast and accurate tool for virus subtyping

SummaryCovidex is an open-source, alignment-free machine learning subtyping tool for viral species. It is a shiny app that allows a fast and accurate classification in pre-defined clusters for SARS-CoV-2 and FMDV genome sequences. The user can also build its own classification models with the Covidex model generator.AvailabilityCovidex is open-source, cross-platform compatible, and is available under the terms of the GNU General Public License v3 (http://www.gnu.org/licenses/gpl.txt). Covidex is available via SourceForge https://sourceforge.net/projects/covidex or the web application https://cacciabue.shinyapps.io/shiny2/[email protected]; [email protected]

Subtyping Cryptosporidium ryanae: A Common Pathogen in Bovine Animals

Cryptosporidium ryanae is one of the most common species for cryptosporidiosis in cattle. However, little is known of the genetic characteristics of C. ryanae due to the lack of subtyping tools. In the present study, the 60-kDa glycoprotein (gp60) gene of C. ryanae was identified in whole genome sequence data and analyzed for sequence characteristics using bioinformatics tools. The protein it encodes had some of the typical characteristics of GP60 proteins, with a signal peptide, a furin cleavage site, and a glycosylphosphatidylinositol anchor at the C terminus of the protein, and numerous O-glycosylation sites. The gene sequence was used in the development of a subtyping tool, which was used in characterizing C. ryanae from 110 specimens from dairy cattle, 2 from beef cattle, 6 from yaks, and 4 from water buffaloes in China. Altogether, 17 subtypes from 8 subtype families were recognized, namely XXIa to XXIh. Possible host adaption was identified within this species, reflected by the unique occurrence of XXIa, XXIc, and XXIh in dairy cattle, yaks, and water buffaloes, respectively. Some geographical differences were detected in the distribution of subtype families in dairy cattle; specimens from southern China showed higher genetic diversity than from northern China, and the XXIa subtype family was only seen in dairy cattle in southern and eastern China. The gp60-based subtyping tool should be useful in molecular epidemiological studies of the transmission of C. ryanae.

Pervasive and non-random recombination in near full-length HIV genomes from Uganda

Recombination is an important feature of HIV evolution, occurring both within and between the major branches of diversity (subtypes). The Ugandan epidemic is primarily composed of two subtypes, A1 and D, that have been co-circulating for 50 years, frequently recombining in dually infected patients. Here, we investigate the frequency of recombinants in this population and the location of breakpoints along the genome. As part of the PANGEA-HIV consortium, 1,472 consensus genome sequences over 5 kb have been obtained from 1,857 samples collected by the MRC/UVRI & LSHTM Research unit in Uganda, 465 (31.6 per cent) of which were near full-length sequences (>8 kb). Using the subtyping tool SCUEAL, we find that of the near full-length dataset, 233 (50.1 per cent) genomes contained only one subtype, 30.8 per cent A1 (n = 143), 17.6 per cent D (n = 82), and 1.7 per cent C (n = 8), while 49.9 per cent (n = 232) contained more than one subtype (including A1/D (n = 164), A1/C (n = 13), C/D (n = 9); A1/C/D (n = 13), and 33 complex types). K-means clustering of the recombinant A1/D genomes revealed a section of envelope (C2gp120-TMgp41) is often inherited intact, whilst a generalized linear model was used to demonstrate significantly fewer breakpoints in the gag–pol and envelope C2-TM regions compared with accessory gene regions. Despite similar recombination patterns in many recombinants, no clearly supported circulating recombinant form (CRF) was found, there was limited evidence of the transmission of breakpoints, and the vast majority (153/164; 93 per cent) of the A1/D recombinants appear to be unique recombinant forms. Thus, recombination is pervasive with clear biases in breakpoint location, but CRFs are not a significant feature, characteristic of a complex, and diverse epidemic.

REZISTENŢA LA INRT LA PACIENŢII MULTIPLU EXPERIMENTAŢI DIN CONSTANŢA: IMPLICAŢII TERAPEUTICE

Obiective. Identificarea prevalenţei rezistenţei dobândite la INRT (inhibitorii non-nucleozidici de reverstranscriptază) şi al profilelor de rezistenţă la un lot de pacienţi cu multiple scheme de terapie antiretrovirală din Constanţa şi evaluarea opţiunilor terapeutice remanente. Material şi metode. Studiu retrospectiv ce a inclus 144 pacienţi seropozitivi HIV, multiplu experimentaţi terapeutic, aflaţi în eşec virusologic. Tulpinile izolate de la aceşti pacienţi au fost analizate în Laboratorul de Genetică Moleculară al Institutului Naţional de Boli Infecţioase „Matei Balş“ din Bucureşti, secvenţele rezultate fiind salvate în format Fasta. Subtiparea HIV-1 s-a efectuat pe baza algoritmului „REGA HIV-1&2 Automated subtyping tool version 2.0“. Pentru determinarea opţiunile terapeutice s-a utilizat „Stanford HIVdb Program version 8.4“. Datele au fost prelucrate statistic cu programul R-Project. Reprezentările grafice au fost realizate cu programul GNUPLOT. Rezultate. Prevalenţa rezistenţei dobândite a fost de 92,36%. Cea mai frecventă mutaţie a fost la nivelul codonului 184. Calea TAM-2 a fost mai frecvent selectată decât TAM-1, existând şi asociaţii între cele două căi; în schimb, mutaţia K65R a fost rar întâlnită. Concluzii. Prevalenţa rezistenţei dobândite la INRT a fost crescută. Opţiunea terapeutică cea mai valoroasă în clasa INRT a ramas tenofovirul, datorită profilului mutaţional selectat, mai ales din cauza neutilizării lui şi a folosirii extensive anterioare a analogilor timidinici.