Discriminatory Weight of SNPs in Spike SARS-CoV-2 Variants: A Technically Rapid, Unambiguous, and Bioinformatically Validated Laboratory Approach

Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

Non-conspecificity ofCylindrocladium quinqueseptatumandCalonectria quinqueseptatabased on a β-tubulin gene phylogeny and morphology

Cylindrocladium quinqueseptatum Boedijn & Reitsma was originally described from leaf spots of Hibiscus sabdariffa L. from Indonesia. This fungus infects many host plants in Southeast Asia and causes severe leaf blight disease of eucalypts. Calonectria quinqueseptata Figueiredo & Namek., which was described from leaf spots on Annona squamosa L. from Brazil, has been regarded as the teleomorph of Cy. quinqueseptatum. Based on morphology and on the phylogeny derived from the DNA sequence of a β-tubulin gene portion spanning several phylogenetically informative introns, the two respective ex-type cultures are shown to be distinct species. Furthermore, Calonectria reteaudii (Bugn.) C. Booth (anamorph Cy. reteaudii (Bugn.) Boesew.), which was described on Smithia bequaertii De Wild. from Vietnam, is shown to be morphologically identical to a comprehensive selection of isolates of Cy. quinqueseptatum from Southeast Asia, Australia, and Madagascar. As Cy. reteaudii represents an older name for Cy. quinqueseptatum, we suggest that the fungus causing widespread damage on eucalypts and other hosts in the above regions be referred to as Cy. reteaudii. Calonectria quinqueseptata should be retained for the fungus that thus far has been found only in Brazil.Key words: Cylindrocladium reteaudii, Eucalyptus, Hypocreales, phylogeny, systematics.

Detection of Mycobacterium avium subsp.paratuberculosis in Infected Tissues by New Species-Specific Immunohistological Procedures

ABSTRACT We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein ofMycobacterium avium subsp. paratuberculosis, the etiological agent of Johne’s disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930–4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947–954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne’s disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp.paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity.