Patterns of Hemorrhagic Disease in White-Tailed Deer (Odocoileus Virginianus) in the Great Plains of the USA, 1982-2020.

Hemorrhagic disease (HD) of deer is caused by epizootic hemorrhagic disease virus (EHDV) or bluetongue virus (BTV) and is considered one of the most important viral diseases of white-tailed deer (Odocoileus virginianus). Despite evidence of changing patterns of HD in the northeastern and upper midwestern US, the historical and current patterns of HD in the Great Plains remain poorly described. We used results from an annual survey documenting HD mortality to characterize historic and current patterns of HD in the northern and central Great Plains (North Dakota, South Dakota, Nebraska, Kansas, and Oklahoma), US, between 1982 and 2020. Further, we assessed temporal change using linear regression to determine change in annual reporting intensity (percentage of counties in a state with reported HD) and change in reporting frequency (the number of years a county or state reported HD) during each decade between 1982 and 2020. Across the 38-yr study period, HD reports expanded northeast across latitude and longitude. Intensity of HD reports significantly increased during this period for three (North Dakota, South Dakota, Kansas) of five states examined. Frequency of reports also increased for all five states. Such changes in northern latitudes might lead to increased deer mortality in regions where HD epizootics have been historically less frequent. Understanding how patterns of HD are changing on the landscape is important when considering future deer management in the face of other mortality factors.

Molecular detection and characterization of highly pathogenic H5N1 clade 2.3.4.4b avian influenza viruses among hunter-harvested wild birds provides evidence for three independent introductions into Alaska.

We detected and characterized highly pathogenic avian influenza viruses among hunter-harvested wild waterfowl inhabiting western Alaska during September-October 2022 using a molecular sequencing pipeline applied to RNA extracts derived directly from original swab samples. Genomic characterization of 10 H5 clade 2.3.4.4b avian influenza viruses detected with high confidence provided evidence for three independent viral introductions into Alaska. Our results highlight the utility and some potential limits of applying molecular processing approaches directly to RNA extracts from original swab samples for viral research and monitoring.

H5N1 highly pathogenic avian influenza clade 2.3.4.4b in wild and domestic birds: Introductions into the United States and reassortments, December 2021-April 2022.

Highly pathogenic avian influenza viruses (HPAIVs) of the A/goose/Guangdong/1/1996 lineage H5 clade 2.3.4.4b continue to have a devastating effect on domestic and wild birds. Full genome sequence analyses using 1369 H5N1 HPAIVs detected in the United States (U.S.) in wild birds, commercial poultry, and backyard flocks from December 2021 to April 2022, showed three phylogenetically distinct H5N1 virus introductions in the U.S. by wild birds. Unreassorted Eurasian genotypes A1 and A2 entered the Northeast Atlantic states, whereas a genetically distinct A3 genotype was detected in Alaska. The A1 genotype spread westward via wild bird migration and reassorted with North American wild bird avian influenza viruses. Reassortments of up to five internal genes generated a total of 21 distinct clusters; of these, six genotypes represented 92% of the HPAIVs examined. By phylodynamic analyses, most detections in domestic birds were shown to be point-source transmissions from wild birds, with limited farm-to-farm spread.

Field Research Is Essential to Counter Virological Threats.

The interface between humans and wildlife is changing and, with it, the potential for pathogen introduction into humans has increased. Avian influenza is a prominent example, with an ongoing outbreak showing the unprecedented expansion of both geographic and host ranges. Research in the field is essential to understand this and other zoonotic threats. Only by monitoring dynamic viral populations and defining their biology in situ can we gather the information needed to ensure effective pandemic preparation.

Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America.

Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b underwent an explosive geographic expansion in 2021 among wild birds and domestic poultry across Asia, Europe, and Africa. By the end of 2021, 2.3.4.4b viruses were detected in North America, signifying further intercontinental spread. Here we show that the western movement of clade 2.3.4.4b was quickly followed by reassortment with viruses circulating in wild birds in North America, resulting in the acquisition of different combinations of ribonucleoprotein genes. These reassortant A(H5N1) viruses are genotypically and phenotypically diverse, with many causing severe disease with dramatic neurologic involvement in mammals. The proclivity of the current A(H5N1) 2.3.4.4b virus lineage to reassort and target the central nervous system warrants concerted planning to combat the spread and evolution of the virus within the continent and to mitigate the impact of a potential influenza pandemic that could originate from similar A(H5N1) reassortants.

Bald eagle mortality and nest failure due to clade 2.3.4.4 highly pathogenic H5N1 influenza a virus.

The bald eagle (Haliaeetus leucocephalus) is a culturally and ecologically vital species in North America that embodies conservation success but continues to face threats that include emerging pathogens. The introduction of A/goose/Guangdong/1/1996 lineage highly pathogenic (HP) clade 2.3.4.4b H5N1 influenza A virus (IAV) in North America in late 2021 resulted in high rates of mortality among bald eagles. Here we show an alarming rate of bald eagle nest failure and mortality attributed to HP IAV. We documented fatal, systemic HP IAV infection in breeding adult and nestling bald eagles along the southeastern U.S. coast. Concurrently, annual bald eagle nest surveys in Georgia and Florida revealed a precipitous drop in success in coastal counties compared with previous years, portending negative impacts on population recruitment. As an apex predator and efficient scavenger, it is likely that bald eagles become infected through consumption of infected waterfowl. These results and similar reports of raptor mortality in Europe, Asia, and Africa, indicate a clear threat to raptor health. The possible long-term persistence of HP H5N1 IAV in North America poses an impending threat to bald eagle populations not only related to direct mortality but also decreased recruitment and warrants continued efforts to understand these potential impacts.

North American Arboviruses and White-Tailed Deer (Odocoileus virginianus): Associated Diseases and Role in Transmission.

Background: Arboviral disease is of increasing concern to human and animal health professionals as emerging and re-emerging arboviruses are more frequently recognized. Wildlife species are known to play a role in the transmission and maintenance of arboviruses and infections can result in morbidity and mortality in wildlife hosts. Materials and Methods: In this review, we detail existing evidence of white-tailed deer (Odocoileus virginianus) as an important host to a diverse collection of arboviruses and evaluate the utility of this species as a resource to better understand the epidemiology of related viral diseases. Results: Relevant veterinary and zoonotic viral pathogens endemic to North America include epizootic hemorrhagic disease virus, bluetongue virus, orthobunyaviruses, vesicular stomatitis virus, Eastern equine encephalitis virus, West Nile virus, and Powassan virus. Exotic viral pathogens that may infect white-tailed deer are also identified with an emphasis on zoonotic disease risks. The utility of this species is attributed to the high degree of contact with humans and domestic livestock and evidence of preferential feeding by various insect vectors. Conclusions: There is mounting evidence that white-tailed deer are a useful, widely available source of information regarding arboviral circulation, and that surveillance and monitoring of deer populations would be of value to the understanding of certain viral transmission dynamics, with implications for improving human and domestic animal health.

Spatiotemporal changes in influenza A virus prevalence among wild waterfowl inhabiting the continental United States throughout the annual cycle.

Avian influenza viruses can pose serious risks to agricultural production, human health, and wildlife. An understanding of viruses in wild reservoir species across time and space is important to informing surveillance programs, risk models, and potential population impacts for vulnerable species. Although it is recognized that influenza A virus prevalence peaks in reservoir waterfowl in late summer through autumn, temporal and spatial variation across species has not been fully characterized. We combined two large influenza databases for North America and applied spatiotemporal models to explore patterns in prevalence throughout the annual cycle and across the continental United States for 30 waterfowl species. Peaks in prevalence in late summer through autumn were pronounced for dabbling ducks in the genera Anas and Spatula, but not Mareca. Spatially, areas of high prevalence appeared to be related to regional duck density, with highest predicted prevalence found across the upper Midwest during early fall, though further study is needed. We documented elevated prevalence in late winter and early spring, particularly in the Mississippi Alluvial Valley. Our results suggest that spatiotemporal variation in prevalence outside autumn staging areas may also represent a dynamic parameter to be considered in IAV ecology and associated risks.

Maintenance and dissemination of avian-origin influenza A virus within the northern Atlantic Flyway of North America.

Wild waterbirds, the natural reservoirs for avian influenza viruses, undergo migratory movements each year, connecting breeding and wintering grounds within broad corridors known as flyways. In a continental or global view, the study of virus movements within and across flyways is important to understanding virus diversity, evolution, and movement. From 2015 to 2017, we sampled waterfowl from breeding (Maine) and wintering (Maryland) areas within the Atlantic Flyway (AF) along the east coast of North America to investigate the spatio-temporal trends in persistence and spread of influenza A viruses (IAV). We isolated 109 IAVs from 1,821 cloacal / oropharyngeal samples targeting mallards (Anas platyrhynchos) and American black ducks (Anas rubripes), two species having ecological and conservation importance in the flyway that are also host reservoirs of IAV. Isolates with >99% nucleotide similarity at all gene segments were found between eight pairs of birds in the northern site across years, indicating some degree of stability among genome constellations and the possibility of environmental persistence. No movement of whole genome constellations were identified between the two parts of the flyway, however, virus gene flow between the northern and southern study locations was evident. Examination of banding records indicate direct migratory waterfowl movements between the two locations within an annual season, providing a mechanism for the inferred viral gene flow. Bayesian phylogenetic analyses provided evidence for virus dissemination from other North American wild birds to AF dabbling ducks (Anatinae), shorebirds (Charidriformes), and poultry (Galliformes). Evidence was found for virus dissemination from shorebirds to gulls (Laridae), and dabbling ducks to shorebirds and poultry. The findings from this study contribute to the understanding of IAV ecology in waterfowl within the AF.

A lesser scaup (Aythya affinis) naturally infected with Eurasian 2.3.4.4 highly pathogenic H5N1 avian influenza virus: Movement ecology and host factors.

Despite the recognized role of wild waterfowl in the potential dispersal and transmission of highly pathogenic avian influenza (HPAI) virus, little is known about how infection affects these birds. This lack of information limits our ability to estimate viral spread in the event of an HPAI outbreak, thereby limiting our abilities to estimate and communicate risk. Here, we present telemetry data from a wild Lesser Scaup (Aythya affinis), captured during a separate ecology study in the Chesapeake Bay, Maryland. This bird tested positive for infection with clade 2.3.4.4 HPAI virus of the A/goose/Guangdong/1/1996 (Gs/GD) H5N1 lineage (results received post-release) during the 2021-2022 ongoing outbreaks in North America. While the infected bird was somewhat lighter than other adult males surgically implanted with transmitters (790 g, x̅ = 868 g, n = 11), it showed no clinical signs of infection at capture, during surgery, nor upon release. The bird died 3 days later-pathology undetermined as the specimen was not able to be recovered. Analysis of movement data within the 3-day window showed that the infected individual's maximum and average hourly movements (3894.3 and 428.8 m, respectively) were noticeably lower than noninfected conspecifics tagged and released the same day (x̅ = 21,594.5 and 1097.9 m, respectively; n = 4). We identified four instances where the infected bird had close contact (fixes located within 25 m and 15 min) with another marked bird during this time. Collectively, these data suggest that the HPAI-positive bird observed in this study may have been shedding virus for some period prior to death, with opportunities for direct bird-to-bird or environmental transmission. Although limited by low sample size and proximity to the time of tagging, we hope that these data will provide useful information as managers continue to respond to this ongoing outbreak event.

Naturally Acquired Antibodies to Influenza A Virus in Fall-Migrating North American Mallards.

Although waterfowl are the primary reservoir for multiple subtypes of influenza A virus (IAV), our understanding of population immunity in naturally infected waterfowl is poorly understood. Population immunity may be an important driver of seasonal subtype predominance in waterfowl populations and may affect the potential for establishment of introduced IAV such as the Eurasian-like A/Goose/Guangdong/1/1996 lineage in these populations. Here, we examine the prevalence of naturally acquired antibodies to nucleoprotein (NP), hemagglutinin (H3, H4, H5), and neuraminidase (N1, N2, N6, N8) in early migrating mallards (Anas platyrhynchos) sampled in Northwest Minnesota during staging and early fall migration in September 2014, 2015, 2017, and 2018. Serologic results were compared to historic and contemporary virus isolation results from these same study sites. The prevalence of antibodies to NP ranged from 60.8−76.1% in hatch-year (HY) birds and from 86.0−92.7% in after-hatch-year (AHY, >1-year-old) mallards indicating a high level of previous infection with IAV early in the fall migration season. Neutralizing antibodies were detected against H3, H4, and H5 in all years as were antibodies to N1, N2, N6, and N8. A high proportion of NP seropositive ducks tested positive for antibodies to multiple HA and NA subtypes, and this was more common in the AHY age class. Antibody prevalence to the HA and NA subtypes included in this study were consistent with the predominance of H4N6 in these populations during all years and reflected a broadening of the antibody response with age. Additional work is needed to document the longevity of these immune responses, if and how they correlate with protection against IAV transmission, infection, and disease, and if, as detected in this study, they adequately describe the true extent of exposure to IAV or specific HA or NA subtypes.

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.

Evidence for interannual persistence of infectious influenza A viruses in Alaska wetlands.

Influenza A viruses (IAVs) deposited by wild birds into the environment may lead to sporadic mortality events and economically costly outbreaks among domestic birds. There is a paucity of information, however, regarding the persistence of infectious IAVs within the environment following deposition. In this investigation, we assessed the persistence of 12 IAVs that were present in cloacal and/or oropharyngeal swabs of naturally infected ducks. Infectivity of these IAVs was monitored over approximately one year with each virus tested in five water types: (1) distilled water held in the lab at 4 °C and (2-5) filtered surface water from each of four Alaska sites and maintained in the field at ambient temperature. By evaluating infectivity of IAVs in ovo following sample retrieval at four successive time points, we observed declines in IAV infectivity through time. Many viruses persisted for extended periods, as evidenced by ≥25% of IAVs remaining infectious in replicate samples for each treatment type through three sampling time points (144-155 days post-sample collection) and two viruses remaining viable in a single replicate sample each when tested upon collection at a fourth time point (361-377 days post-sample collection). The estimated probability of persistence of infectious IAVs in all five water types was estimated to be between 0.25 and 0.75 during days 50-200 post-sample collection as inferred through Kaplan-Meier survival analysis. Our results provide evidence that IAVs may remain infectious for extended periods, up to or even exceeding one year, when maintained in surface waters under ambient temperatures. Therefore, wetlands may represent an important medium in which infectious IAVs may reside outside of a biotic reservoir.

Age-Associated Changes in Recombinant H5 Highly Pathogenic and Low Pathogenic Avian Influenza Hemagglutinin Tissue Binding in Domestic Poultry Species.

The 2014 outbreak of clade 2.3.4.4A highly pathogenic avian influenza (HPAI) led to the culling of millions of commercial chickens and turkeys and death of various wild bird species. In this outbreak, older chickens and turkeys were commonly infected, and succumbed to clinical disease compared to younger aged birds such chicken broilers. Some experimental studies using waterfowl species have shown age-related differences in susceptibility to clinical disease with HPAI viruses. Here, we evaluate differences in H5 Hemagglutinin (HA) tissue binding across age groups, using recombinant H5 HA (rHA) proteins generated using gene sequences from low pathogenic (A/mallard/MN/410/2000(H5N2 (LPAIV)) and a HPAIV (A/Northern pintail/Washington/40964/2014(H5N2)) influenza A virus (IAV). Respiratory and intestinal tracts from chickens, ducks (Mallard, Pekin, Muscovy) and turkeys of different age groups were used to detect rHA binding with protein histochemistry, which was quantified as the median area of binding (MAB) used for statistical analysis. There were species and tissue specific differences in the rHA binding among the age groups; however, turkeys had significant differences in the HPAIV rHA binding in the respiratory tract, with younger turkeys having higher levels of binding in the lung compared to the older group. In addition, in the intestinal tract, younger turkeys had higher levels of binding compared to the older birds. Using LPAIV, similar species and tissues, specific differences were seen among the age groups; however, only turkeys had overall significant differences in the respiratory tract MAB, with the older birds having higher levels of binding compared to the younger group. No age-related differences were seen in the overall intestinal tract rHA binding. Age-related differences in rHA binding of the LPAIV and HPAIV demonstrated in this study may partially, but not completely, explain differences in host susceptibility to infection observed during avian influenza outbreaks and in experimental infection studies.

Low-pathogenicity influenza viruses replicate differently in laughing gulls and mallards.

Wild aquatic birds are natural reservoirs of low-pathogenicity avian influenza viruses (LPAIVs). Laughing gulls inoculated with four gull-origin LPAIVs (H7N3, H6N4, H3N8, and H2N3) had a predominate respiratory infection. By contrast, mallards inoculated with two mallard-origin LPAIVs (H5N6 and H4N8) became infected and had similar virus titers in oropharyngeal (OP) and cloacal (CL) swabs. The trend toward predominate OP shedding in gulls suggest a greater role of direct bird transmission in maintenance, whereas mallards shedding suggests importance of fecal-oral transmission through water contamination. Additional infectivity and pathogenesis studies are needed to confirm this replication difference for LPAI viruses in gulls.

Spatial Analysis of the 2017 Outbreak of Hemorrhagic Disease and Physiographic Region in the Eastern United States.

Hemorrhagic disease (HD) is considered one of the most significant infectious diseases of white-tailed deer in North America. Investigations into environmental conditions associated with outbreaks suggest drought conditions are strongly correlated with outbreaks in some regions of the United States. However, during 2017, an HD outbreak occurred in the Eastern United States which appeared to be associated with a specific physiographic region, the Appalachian Plateau, and not drought conditions. The objective of this study was to determine if reported HD in white-tailed deer in 2017 was correlated with physiographic region. There were 456 reports of HD from 1605 counties across 26 states and 12 physiographic regions. Of the 93 HD reports confirmed by virus isolation, 76.3% (71/93) were identified as EHDV-2 and 66.2% (47/71) were from the Appalachian Plateau. A report of HD was 4.4 times more likely to occur in the Appalachian Plateau than not in 2017. Autologistic regression models suggested a statistically significant spatial dependence. The underlying factors explaining this correlation are unknown, but may be related to a variety of host, vector, or environmental factors. This unique outbreak and its implications for HD epidemiology highlight the importance for increased surveillance and reporting efforts in the future.

The Effect of Maternal Antibodies on Clinical Response to Infection with Epizootic Hemorrhagic Disease Virus in White-Tailed Deer (Odocoileus virginianus) Fawns.

We investigated whether naturally acquired maternal antibodies to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) would protect white-tailed deer (Odocoileus virginianus) fawns against infection and clinical disease following an EHDV-2 challenge. We compared viremia and clinical response in 27-47-d-old, experimentally infected fawns with and without maternally derived antibodies to EHDV-2. Mild to moderate clinical signs were observed in four seronegative (maternal antibody-negative) fawns, which were viremic from 3 to 14 d postinoculation. Individual peak blood virus titers for seronegative fawns ranged from 104.3 to 106.3 median tissue culture infective doses (TCID50)/mL. In contrast, clinical signs were not observed in seropositive (maternal antibody-positive) fawns and a transient low-level viremia (≤102.4 TCID50/mL) occurred in two of six fawns. Our results indicated that the presence of maternally derived EHDV-2 antibodies in fawns prevents or greatly reduces clinical disease and the level and duration of EHDV-2 viremia.

Influenza A Viruses in Whistling Ducks (Subfamily Dendrocygninae).

As compared to other Anseriformes, data related to influenza A virus (IAV) detection and isolation, and IAV antibody detection in whistling ducks (Dendrocygna spp. and Thalassornis leuconotus; subfamily Dendrocygninae) are limited. To better evaluate the potential role of whistling ducks in the epidemiology of IAV, we (1) conducted surveillance for IAV from black-bellied whistling ducks (BBWD, Dendrocygnaautumnalis) sampled in coastal Louisiana, USA, during February 2018 and 2019, and (2) reviewed the published literature and Influenza Resource Database (IRD) that reported results of IAV surveillance of whistling ducks. In the prospective study, from 166 BBWD sampled, one H10N7 IAV was isolated (0.6% prevalence), and overall blocking enzyme-linked immunosorbent assay (bELISA) antibody seroprevalence was 10%. The literature review included publications and data in the IRD from 1984 to 2020 that reported results from nearly 5000 collected samples. For any given collection, the IAV isolation rate never exceeded 5.5%, and seroprevalence estimates ranged from 0 to 42%. Results from our prospective study in Louisiana are consistent with this historic literature; however, although all data consistently demonstrated a low prevalence of infection, the potential role of this species in the epidemiology of IAV should not be totally discounted. In sum, whistling ducks can be infected with IAV, they represent important species on many areas where waterfowl winter, and their distribution across the globe appears to be changing.

Coding-Complete Genome Sequence of Avian orthoavulavirus 16, Isolated from Emperor Goose (Anser canagicus) Feces, Alaska, USA.

We sequenced the coding-complete genome of an avian orthoavulavirus serotype 16 (AOAV-16) isolate recovered from emperor goose (Anser canagicus) feces collected in Alaska. The detection of AOAV-16 in North America and genomic sequencing of the resultant isolate confirms that the geographic distribution of this virus extends beyond Asia.

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.