A Repeat Pattern of Founder Events for SARS-CoV-2 Variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska, the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 27 February 2022, reaching 48.5% of sequenced cases. On the contrary, in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska's unique dynamics influence the emergence of SARS-CoV-2 variants.

Identification of a novel SARS-CoV-2 variant with a truncated protein in ORF8 gene by next generation sequencing.

Using next generation sequencing technology, we identified a novel SARS-CoV-2 variant with a truncated ORF8 protein mutation near the end of the viral genome from nucleotides 27,878 to 27,958. This point mutation from C to T at nucleotide 27,956 changed the amino acid codon CAA (glutamine) to a stop codon, TAA, created a novel stop codon in ORF8 gene, resulting in a much smaller ORF8 protein (26 aa) than the wild type ORF8 protein (121 aa). This variant belongs to Pango lineage B.1.1291, which also contains the D614G mutation in the Spike (S) gene. The B.1.1291 lineage is predominantly circulated in the United States of America (97.18%), although it was also found in other counties (Russia, Canada, Latvia, Chile, India, Japan, Colombia, Germany, Greece, Mexico, and UK). A total of 340 closely related variants to this novel variant were identified in GISAID database with collection dates ranged from 3/6/2020 to 10/21/2020. In addition, a search within NCBI Genbank database found that 108,405 of 873,230 (12.4%) SAR-CoV-2 complete genomes contain this truncated ORF8 protein mutation, indicating this mutation may arise spontaneously in other lineages as well. The wide distribution of this mutation indicates that this truncated ORF8 protein mutation may provide the virus a growth advantage and adaptive evolution.

A repeat pattern of founder events for SARS-CoV-2 variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 2022-02-27, reaching 48.5% of sequenced cases. On the contrary in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska’s unique dynamics influence the emergence of SARS-CoV-2 variants.

Pattern of SARS-CoV-2 variant B.1.1.519 emergence in Alaska.

Alaska has the lowest population density in the United States (US) with a mix of urban centers and isolated rural communities. Alaska's distinct population dynamics compared to the contiguous US may have contributed to unique patterns of SARS-CoV-2 variants observed in early 2021. Here we examined 2323 SARS-CoV-2 genomes from Alaska and 278,635 from the contiguous US collected from December 2020 through June 2021 because of the notable emergence and spread of lineage B.1.1.519 in Alaska. We found that B.1.1.519 was consistently detected from late January through June of 2021 in Alaska with a peak prevalence in April of 77.9% unlike the rest of the US at 4.6%. The earlier emergence of B.1.1.519 coincided with a later peak of Alpha (B.1.1.7) compared to the contiguous US. We also observed differences in variant composition over time between the two most populated regions of Alaska and a modest increase in COVID-19 cases during the peak incidence of B.1.1.519. However, it is difficult to disentangle how social dynamics conflated changes in COVID-19 during this time. We suggest that the viral characteristics, such as amino acid substitutions in the spike protein, likely contributed to the unique spread of B.1.1.519 in Alaska.

Identification of a Novel SARS-CoV-2 Strain with Truncated Protein in ORF8 Gene by Next Generation Sequencing.

Using next generation sequencing technology, we identified a truncated protein mutation located in the ORF8 gene which is near the end of the genome from nucleotides 27,878 to 27,958. The mutation in this novel strain created a stop codon and translates to the novel truncated ORF8 protein, creating a much smaller protein than most other strains of SARS-CoV-2. The novel truncated mutation is most closely related to nine SARS-CoV-2 strains found in Washington state. Our results show a novel strain of SARS-CoV-2 with a truncated ORF8 gene. This shortens the translated ORF8 protein. The effects of ORF8 protein and its functions are still uncertain but a truncated ORF8 could affect antibody response, severity of infection and inflammatory response.

BioRad BioPlex® HIV Ag-Ab assay: Incidence of false positivity in a low-prevalence population and its effects on the current HIV testing algorithm.

BACKGROUND: The BioPlex® HIV Ag-Ab assay, unlike other HIV 1/2 antigen/antibody immunoassays, is capable of differentiating positive HIV-1 antibodies (Groups M and O) from HIV-2 antibodies and/or HIV-1 p24 antigen in a single test. OBJECTIVE: The Alaska State Virology Laboratory (ASVL) adopted the BioPlex® HIV Ag-Ab assay early 2017 and can report on its performance in terms of false positivity in a low-prevalence population and its effects on the current HIV testing algorithm recommended by the Centers for Disease Control and Prevention (CDC). STUDY DESIGN: Specimens received between March 2017 and August 2018 were screened using the BioPlex® HIV Ag-Ab assay. Specimens screening positive for HIV antibodies or antigen were further confirmed using the Geenius™ HIV 1/2 Supplemental Assay and/or HIV RNA testing. RESULTS: Of the 12,338 sera screened by the BioPlex assay for HIV, 35 specimens were positive. Only 22 of the specimens were confirmed by supplemental testing and were considered to be truly positive (PPV, 62.9%). RNA was not detected in these cases suggesting initial false positivity on the BioPlex® HIV Ag-Ab assay. True positive results had index values (IDX) of >180 whereas false positive IDX's were between 1 and 4, with the exception of one specimen. CONCLUSIONS: We suggest that specimens demonstrating positivity with low IDX values <4 on the BioPlex® HIV Ag-Ab assay proceed directly to RNA testing, essentially bypassing supplemental antibody confirmation tests, to reduce turnaround time and cost of HIV confirmation.

Correction: Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

[This corrects the article DOI: 10.1371/journal.pone.0143164.].

Application of next generation sequencing for the detection of human viral pathogens in clinical specimens.

BACKGROUND: Next generation sequencing (NGS) is a new technology that can be used for broad detection of infectious pathogens and is rapidly becoming an essential platform in clinical laboratories. It is not known how NGS will displace or enhance gold standard methodologies in infectious disease diagnosis. OBJECTIVES: To investigate the feasibility and application of NGS technology in public health laboratories and compare NGS technology with conventional methods. STUDY DESIGN: Illumina MiSeq system was used to detect viral pathogens alongside other conventional virology methods using typical clinical specimen matrices. Sixteen clinical specimens and two CDC proficiency panels containing seventeen specimens were analyzed. RESULTS: Known pathogenic viral nucleic acid was positively identified in all clinical specimens, correlating and building upon results obtained by more conventional laboratory methods. Sequencing depths ranged from 0.008X to 319 and genome coverage ranged from 0.6% to 99.9%. To substantiate the described methods used to analyze data derived from clinical specimens, the results of a clinical proficiency panel are also presented. DISCUSSION: Our results reveal true scarcity of known pathogenic viral nucleic acids in clinical specimens. NGS outperforms more conventional detection methods in this study by turnaround time as well as the improved depth of knowledge in regards to serotyping and drug resistance.

Investigation of a Canine Parvovirus Outbreak using Next Generation Sequencing.

Canine parvovirus (CPV) outbreaks can have a devastating effect in communities with dense dog populations. The interior region of Alaska experienced a CPV outbreak in the winter of 2016 leading to the further investigation of the virus due to reports of increased morbidity and mortality occurring at dog mushing kennels in the area. Twelve rectal-swab specimens from dogs displaying clinical signs consistent with parvoviral-associated disease were processed using next-generation sequencing (NGS) methodologies by targeting RNA transcripts, and therefore detecting only replicating virus. All twelve specimens demonstrated the presence of the CPV transcriptome, with read depths ranging from 2.2X - 12,381X, genome coverage ranging from 44.8-96.5%, and representation of CPV sequencing reads to those of the metagenome background ranging from 0.0015-6.7%. Using the data generated by NGS, the presence of newly evolved, yet known, strains of both CPV-2a and CPV-2b were identified and grouped geographically. Deep-sequencing data provided additional diagnostic information in terms of investigating novel CPV in this outbreak. NGS data in addition to limited serological data provided strong diagnostic evidence that this outbreak most likely arose from unvaccinated or under-vaccinated canines, not from a novel CPV strain incapable of being neutralized by current vaccination efforts.

Laboratory-based respiratory virus surveillance pilot project on select cruise ships in Alaska, 2013-15.

BACKGROUND: Influenza outbreaks can occur among passengers and crews during the Alaska summertime cruise season. Ill travellers represent a potential source for introduction of novel or antigenically drifted influenza virus strains to the United States. From May to September 2013-2015, the Alaska Division of Public Health, the Centers for Disease Control and Prevention (CDC), and two cruise lines implemented a laboratory-based public health surveillance project to detect influenza and other respiratory viruses among ill crew members and passengers on select cruise ships in Alaska. METHODS: Cruise ship medical staff collected 2-3 nasopharyngeal swab specimens per week from passengers and crew members presenting to the ship infirmary with acute respiratory illness (ARI). Specimens were tested for respiratory viruses at the Alaska State Virology Laboratory (ASVL); a subset of specimens positive for influenza virus were sent to CDC for further antigenic characterization. RESULTS: Of 410 nasopharyngeal specimens, 83% tested positive for at least one respiratory virus; 71% tested positive for influenza A or B virus. Antigenic characterization of pilot project specimens identified strains matching predominant circulating seasonal influenza virus strains, which were included in the northern or southern hemisphere influenza vaccines during those years. Results were relatively consistent across age groups, recent travel history, and influenza vaccination status. Onset dates of illness relative to date of boarding differed between northbound (occurring later in the voyage) and southbound (occurring within the first days of the voyage) cruises. CONCLUSIONS: The high yield of positive results indicated that influenza was common among passengers and crews sampled with ARI. This finding reinforces the need to bolster influenza prevention and control activities on cruise ships. Laboratory-based influenza surveillance on cruise ships may augment inland influenza surveillance and inform control activities. However, these benefits should be weighed against the costs and operational limitations of instituting laboratory-based surveillance programs on ships.

Draft Genome Sequence of a Taxonomically Unique Neisseria Strain Isolated from a Greater White-Fronted Goose (Anser albifrons) Egg on the North Slope of Alaska.

We report here the draft genome sequence of a unique Neisseria strain that was isolated from a greater white-fronted goose (Anser albifrons) egg. The sequencing was performed with an Illumina MiSeq system, and the sequence consists of 275 contigs. The total genome is 2,397,978 bp long and has a G+C content of 46.4%.

Preferential Amplification of Pathogenic Sequences.

The application of next generation sequencing (NGS) technology in the diagnosis of human pathogens is hindered by the fact that pathogenic sequences, especially viral, are often scarce in human clinical specimens. This known disproportion leads to the requirement of subsequent deep sequencing and extensive bioinformatics analysis. Here we report a method we called "Preferential Amplification of Pathogenic Sequences (PATHseq)" that can be used to greatly enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and 10-mer oligonucleotides called "non-human primers" that do not match the most abundant human transcripts, but instead selectively match transcripts of human pathogens. Instead of using random primers in the construction of cDNA libraries, the PATHseq method recruits these short non-human primers, which in turn, preferentially amplifies non-human, presumably pathogenic sequences. Using this method, we were able to enrich pathogenic sequences up to 200-fold in the final sequencing library. This method does not require prior knowledge of the pathogen or assumption of the infection; therefore, it provides a fast and sequence-independent approach for detection and identification of human viruses and other pathogens. The PATHseq method, coupled with NGS technology, can be broadly used in identification of known human pathogens and discovery of new pathogens.

Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95-100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/µL using qPCR. Analytical sensitivity between the two methods varied from 1.2-1280.8 copies/µL (0.08-3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2-8.4 copies/µL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4-1280.8 copies/µL, 2.50-3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6-94.8 copies/µL, 0.20-1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.

Draft Genome Sequence of an Atypical Strain of Streptococcus pneumoniae Isolated from a Respiratory Infection.

Next-generation sequencing was used to investigate an unknown clinical respiratory infection. This new strain of Streptococcus pneumoniae, ASVL_JC_0001, was isolated from a clinical specimen from a patient with bronchitis and pulmonary inflammation. The draft genome sequence, obtained with an Illumina MiSeq sequencing system, consists of 83 large contigs, a total of 2,092,532 bp long, and has a GC content of 40.3%.