Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

Piscichuvirus-Associated Severe Meningoencephalomyelitis in Aquatic Turtles, United States, 2009-2021.

Viruses from a new species of piscichuvirus were strongly associated with severe lymphocytic meningoencephalomyelitis in several free-ranging aquatic turtles from 3 coastal US states during 2009-2021. Sequencing identified 2 variants (freshwater turtle neural virus 1 [FTuNV1] and sea turtle neural virus 1 [STuNV1]) of the new piscichuvirus species in 3 turtles of 3 species. In situ hybridization localized viral mRNA to the inflamed region of the central nervous system in all 3 sequenced isolates and in 2 of 3 additional nonsequenced isolates. All 3 sequenced isolates phylogenetically clustered with other vertebrate chuvirids within the genus Piscichuvirus. FTuNV1 and STuNV1 shared ≈92% pairwise amino acid identity of the large protein, which narrowly places them within the same novel species. The in situ association of the piscichuviruses in 5 of 6 turtles (representing 3 genera) with lymphocytic meningoencephalomyelitis suggests that piscichuviruses are a likely cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles.

Putative Novel Avian Paramyxovirus (AMPV) and Reidentification of APMV-2 and APMV-6 to the Species Level Based on Wild Bird Surveillance (United States, 2016-2018).

Avian paramyxoviruses (APMVs) (subfamily Avulavirinae) have been isolated from over 200 species of wild and domestic birds around the world. The International Committee on Taxonomy of Viruses (ICTV) currently defines 22 different APMV species, with Avian orthoavulavirus 1 (whose viruses are designated APMV-1) being the most frequently studied due to its economic burden to the poultry industry. Less is known about other APMV species, including limited knowledge on the genetic diversity in wild birds, and there is a paucity of public whole-genome sequences for APMV-2 to -22. The goal of this study was to use MinION sequencing to genetically characterize APMVs isolated from wild bird swab samples collected during 2016 to 2018 in the United States. Multiplexed MinION libraries were prepared using a random strand-switching approach using 37 egg-cultured, influenza-negative, hemagglutination-positive samples. Forty-one APMVs were detected, with 37 APMVs having complete polymerase coding sequences allowing for species identification using ICTV's current Paramyxoviridae phylogenetic methodology. APMV-1, -4, -6, and -8 viruses were classified, one putative novel species (Avian orthoavulavirus 23) was identified from viruses isolated in this study, two putative new APMV species (Avian metaavulavirus 24 and 27) were identified from viruses isolated in this study and from retrospective GenBank sequences, and two putative new APMV species (Avian metaavulavirus 25 and 26) were identified solely from retrospective GenBank sequences. Furthermore, coinfections of APMVs were identified in four samples. The potential limitations of the branch length being the only species identification criterion and the potential benefit of a group pairwise distance analysis are discussed. IMPORTANCE Most species of APMVs are understudied and/or underreported, and many species were incidentally identified from asymptomatic wild birds; however, the disease significance of APMVs in wild birds is not fully determined. The rapid rise in high-throughput sequencing coupled with avian influenza surveillance programs have identified 12 different APMV species in the last decade and have challenged the resolution of classical serological methods to identify new viral species. Currently, ICTV's only criterion for Paramyxoviridae species classification is the requirement of a branch length of >0.03 using a phylogenetic tree constructed from polymerase (L) amino acid sequences. The results from this study identify one new APMV species, propose four additional new APMV species, and highlight that the criterion may have insufficient resolution for APMV species demarcation and that refinement or expansion of this criterion may need to be established for Paramyxoviridae species identification.

Nanopore sequencing as a rapid tool for identification and pathotyping of avian influenza A viruses.

We report whole-genome sequencing of influenza A virus (IAV) with 100% diagnostic sensitivity and results available in <24-48 h using amplicon-based nanopore sequencing technology (MinION) on clinical material from wild waterfowl (n = 19), commercial poultry (n = 4), and swine (n = 3). All 8 gene segments of IAV including those from 14 of the 18 recognized hemagglutinin subtypes and 9 of the 11 neuraminidase subtypes were amplified in their entirety at >500× coverage from each of 16 reference virus isolates evaluated. Subgenomic viral sequences obtained in 3 cases using Sanger sequencing as the reference standard were identical to those obtained when sequenced using the MinION approach. An inter-laboratory comparison demonstrated reproducibility when comparing 2 independent laboratories at ≥99.8% across the entirety of the IAV genomes sequenced.

Randomly primed, strand-switching, MinION-based sequencing for the detection and characterization of cultured RNA viruses.

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.

Hyperpigmented melanistic skin lesions of smallmouth bass Micropterus dolomieu from the Chesapeake Bay watershed.

Hyperpigmented melanistic skin lesions (HPMLs) of smallmouth bass Micropterus dolomieu are observed in the Potomac and Susquehanna rivers, Chesapeake Bay watershed, USA. Routine, nonlethal population surveys were conducted at 8 sites on the mainstem Susquehanna River and 9 on the Juniata River, a tributary of the Susquehanna River, between 2012 and 2018, and the prevalence of HPMLs was documented. A total of 4078 smallmouth bass were collected from the mainstem Susquehanna River and 6478 from the Juniata River. Lesions were primarily seen in bass greater than 200 mm, and prevalence in the Susquehanna River (8%) was higher (p < 0.001) than in the Juniata River (2%). As part of ongoing fish health monitoring projects, smallmouth bass were collected at additional sites, primarily tributaries of the Susquehanna (n = 758) and Potomac (n = 545) rivers between 2013 and 2018. Prevalence in the Susquehanna River (13%) was higher (p < 0.001) than the Potomac (3%). Microscopically, HPMLs were characterized by an increased number of melanocytes in the epidermis or within the dermis and epidermis. RNAseq analyses of normal and melanistic skin identified 3 unique sequences in HPMLs. Two were unidentified and the third was a viral helicase (E1). Transcript abundance in 16 normal skin samples and 16 HPMLs showed upregulation of genes associated with melanogenesis and cell proliferation in HPMLs. The E1 transcript was detected in 12 of the 16 melanistic areas but in no samples from normal skin. Further research will be necessary to identify the putative new virus and determine its role in melanocyte proliferation.

Draft Genome Sequence of an Adomavirus Associated with Raised Mucoid Skin Lesions on Smallmouth Bass (Micropterus dolomieu).

Raised mucoid skin lesions have been observed on smallmouth bass (Micropterus dolomieu) for years. Here, we report the draft genome of a novel adomavirus (Micropterus dolomieu adomavirus 2) associated with this disease. The circular genome is 17,561 bp and most similar to that of alpha-adomaviruses.