Amplification and sequencing of entire tick mitochondrial genomes for a phylogenomic analysis

The mitochondrial genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be costprohibitive at scale. To address this issue, we developed a cost-effective approach to amplify and sequence the whole mitogenome of individual tick specimens. Using two different primer sites, this approach generated two full-length mitogenome amplicons that were sequenced using the Oxford Nanopore Technologies’ Mk1B sequencer. We used this approach to generate 85 individual tick mitogenomes from samples comprised of the three tick families, 11 genera, and 57 species. Twentysix of these species did not have a complete mitogenome available on GenBank prior to this work. We benchmarked the accuracy of this approach using a subset of samples that had been previously sequenced by low-coverage Illumina genome skimming. We found our assemblies were comparable or exceeded the Illumina method, achieving a median sequence concordance of 99.98%. We further analyzed our mitogenome dataset in a mitophylogenomic analysis in the context of all three tick families. We were able to sequence 72 samples in one run and achieved a cost/sample of ~ $10 USD. This cost-effective strategy is applicable for sample identification, taxonomy, systematics, and population genetics for not only ticks but likely other metazoans; thus, making mitogenome sequencing equitable for the wider scientific community.NIH Grants and the Norman E. Borlaug International Agricultural Science and Technology Fellow.http://www.nature.com/scientificreportsam2023Veterinary Tropical Disease

Monitoring Temporal Changes in SARS-CoV-2 Spike Antibody Levels and Variant-Specific Risk for Infection, Dominican Republic, March 2021-August 2022

To assess changes in SARS-CoV-2 spike binding antibody prevalence in the Dominican Republic and implications for immunologic protection against variants of concern, we prospectively enrolled 2,300 patients with undifferentiated febrile illnesses in a study during March 2021-August 2022. We tested serum samples for spike antibodies and tested nasopharyngeal samples for acute SARS-CoV-2 infection using a reverse transcription PCR nucleic acid amplification test. Geometric mean spike antibody titers increased from 6.6 (95% CI 5.1-8.7) binding antibody units (BAU)/mL during March-June 2021 to 1,332 (95% CI 1,055-1,682) BAU/mL during May-August 2022. Multivariable binomial odds ratios for acute infection were 0.55 (95% CI 0.40-0.74), 0.38 (95% CI 0.27-0.55), and 0.27 (95% CI 0.18-0.40) for the second, third, and fourth versus the first anti-spike quartile; findings were similar by viral strain. Combining serologic and virologic screening might enable monitoring of discrete population immunologic markers and their implications for emergent variant transmission

Characterization of a Zika Virus Isolate from Colombia.

Zika virus (Flavivirus genus) is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments.We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015). Here, we describe the patient's clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3' un-translated region), to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains.We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas

Correction: Mosquito-bite infection of humanized mice with chikungunya virus produces systemic disease with long-term effects.

[This corrects the article DOI: 10.1371/journal.pntd.0009427.]

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations

Aislamiento de Borrelia turicatae de Ornithodoros turicata colectada en México

Las espiroquetas causantes de la fiebre recurrente (FR) son patógenos del género Borrelia transmitidos por garrapatas blandas y duras. Esta enfermedad se considera como desatendida y emergente. Aunque la enfermedad se ha descrito y diagnosticado en México, al momento no se ha logrado el aislamiento de la bacteria causante de la misma. De tal forma, el objetivo de este trabajo fue obtener el primer aislado mexicano de Borrelia spp. causante de FR transmitida por garrapatas de vectores colectados en México. En marzo del 2022 se realizó una colecta de garrapatas en los estados de Sonora y Sinaloa donde previamente se tenían reportes serológicos de exposición de personas a esta enfermedad. Las garrapatas vivas agrupadas en lotes se pusieron en contacto con ratones DBA/2J para su alimentación y posterior vigilancia, se realizaron frotis de sangre periférica y tinción Giemsa del día 02 al día 18 posterior a la alimentación. A las 4 semanas se obtuvo suero y se realizó un Western blot para detectar anticuerpos IgG contra la proteína recombinante glicerofosfodiesterasa (rGlpQ). La prueba de Western blot mostró que un suero fue positivo contra la rGlpQ, por lo que las garrapatas O. turicata positivas se alimentaron en un nuevo ratón DBA/2J y a los 7 días se tomó sangre periférica y se inoculó en medio Barbour-Stoenner-Kelley IIb (mBSK IIb) en donde se observaron espiroquetas a los 9 días de la inoculación. Se realizó la extracción de DNA y se realizó la confirmación por PCR y secuenciación del gen que codifica para el rRNA 16S, que indicó que se trataba de Borrelia turicatae. Posteriormente, el DNA genómico de esta cepa se secuenció con las plataformas Ilumina y Nanopore, las secuencias resultantes se ensamblaron, se anotaron y se analizaron filogenéticamente con otros genomas depositados en el NCBI. El resultado indica que esta cepa de B. turicatae está emparentada con otras aisladas en Norteamérica y que representa una cepa nueva; tiene un genoma de 1.57 Mpb con las siguientes características: 29.4% GC, 1 cromosoma lineal, 13 plásmidos lineales y 3 plásmidos circulares. En conclusión, se obtuvo una cepa de Borrelia turicatae de garrapatas Ornithodoros turicata del estado de Sinaloa, México. Estos resultados sugieren que se debe ampliar el estudio a otras áreas para conocer las zonas de distribución de las garrapatas blandas y de la bacteria causante de FR en México

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure.

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations

Mosquito-bite infection of humanized mice with chikungunya virus produces systemic disease with long-term effects.

Chikungunya virus (CHIKV) is an emerging, mosquito-borne alphavirus responsible for acute to chronic arthralgias and neuropathies. Although it originated in central Africa, recent reports of disease have come from many parts of the world, including the Americas. While limiting human CHIKV cases through mosquito control has been used, it has not been entirely successful. There are currently no licensed vaccines or treatments specific for CHIKV disease, thus more work is needed to develop effective countermeasures. Current animal research on CHIKV is often not representative of human disease. Most models use CHIKV needle inoculation via unnatural routes to create immediate viremia and localized clinical signs; these methods neglect the natural route of transmission (the mosquito vector bite) and the associated human immune response. Since mosquito saliva has been shown to have a profound effect on viral pathogenesis, we evaluated a novel model of infection that included the natural vector, Aedes species mosquitoes, transmitting CHIKV to mice containing components of the human immune system. Humanized mice infected by 3-6 mosquito bites showed signs of systemic infection, with demonstrable viremia (by qRT-PCR and immunofluorescent antibody assay), mild to moderate clinical signs (by observation, histology, and immunohistochemistry), and immune responses consistent with human infection (by flow cytometry and IgM ELISA). This model should give a better understanding of human CHIKV disease and allow for more realistic evaluations of mechanisms of pathogenesis, prophylaxis, and treatments

Aislamiento de Borrelia turicatae de Ornithodoros turicata colectada en México

RESUMEN Las espiroquetas causantes de la fiebre recurrente (FR) son patógenos del género Borrelia transmitidos por garrapatas blandas y duras. Esta enfermedad se considera como desatendida y emergente. Aunque la enfermedad se ha descrito y diagnosticado en México, al momento no se ha logrado el aislamiento de la bacteria causante de la misma. De tal forma, el objetivo de este trabajo fue obtener el primer aislado mexicano de Borrelia spp. causante de FR transmitida por garrapatas de vectores colectados en México. En marzo del 2022 se realizó una colecta de garrapatas en los estados de Sonora y Sinaloa donde previamente se tenían reportes serológicos de exposición de personas a esta enfermedad. Las garrapatas vivas agrupadas en lotes se pusieron en contacto con ratones DBA/2J para su alimentación y posterior vigilancia, se realizaron frotis de sangre periférica y tinción Giemsa del día 02 al día 18 posterior a la alimentación. A las 4 semanas se obtuvo suero y se realizó un Western blot para detectar anticuerpos IgG contra la proteína recombinante glicerofosfodiesterasa (rGlpQ). La prueba de Western blot mostró que un suero fue positivo contra la rGlpQ, por lo que las garrapatas O. turicata positivas se alimentaron en un nuevo ratón DBA/2J y a los 7 días se tomó sangre periférica y se inoculó en medio Barbour-Stoenner-Kelley IIb (mBSK IIb) en donde se observaron espiroquetas a los 9 días de la inoculación. Se realizó la extracción de DNA y se realizó la confirmación por PCR y secuenciación del gen que codifica para el rRNA 16S, que indicó que se trataba de Borrelia turicatae. Posteriormente, el DNA genómico de esta cepa se secuenció con las plataformas Ilumina y Nanopore, las secuencias resultantes se ensamblaron, se anotaron y se analizaron filogenéticamente con otros genomas depositados en el NCBI. El resultado indica que esta cepa de B. turicatae está emparentada con otras aisladas en Norteamérica y que representa una cepa nueva; tiene un genoma de 1.57 Mpb con las siguientes características: 29.4% GC, 1 cromosoma lineal, 13 plásmidos lineales y 3 plásmidos circulares. En conclusión, se obtuvo una cepa de Borrelia turicatae de garrapatas Ornithodoros turicata del estado de Sinaloa, México. Estos resultados sugieren que se debe ampliar el estudio a otras áreas para conocer las zonas de distribución de las garrapatas blandas y de la bacteria causante de FR en México

Phylogenetic tree of full genome nucleotide sequences of 11 ZIKV strains, using only those that were derived from viruses (i.e. not amplicons), available in GenBank.

<p>Alignments were done via Clustal W and analysis was done via Bayesian MCMC. Methods and accession numbers are provided in text. The scale represents substitutions per nucleotide site and the numbers on nodes are the posterior probabilities of branching (max = 1).</p