Tracing viral transmission and evolution of Bovine leukemia virus through long read Oxford nanopore sequencing of the proviral genome

Bovine leukemia virus (BLV) causes Enzootic Bovine Leukosis (EBL), a persistent life-long disease resulting in immune dysfunction and shortened lifespan in infected cattle, severely impacting the profitability of the US dairy industry. Our group has found that 94% of dairy farms in the United States are infected with BLV with an average in-herd prevalence of 46%. This is partly due to the lack of clinical presentation during the early stages of primary infection and the elusive nature of BLV transmission. This study sought to validate a near-complete genomic sequencing approach for reliability and accuracy before determining its efficacy in characterizing the sequence identity of BLV proviral genomes collected from a pilot study made up of 14 animals from one commercial dairy herd. These BLV-infected animals were comprised of seven adult dam/daughter pairs that tested positive by ELISA and qPCR. The results demonstrate sequence identity or divergence of the BLV genome from the same samples tested in two independent laboratories, suggesting both vertical and horizontal transmission in this dairy herd. This study supports the use of Oxford Nanopore sequencing for the identification of viral SNPs that can be used for retrospective genetic contact tracing of BLV transmission

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Efficacy and Immunogenicity of Recombinant Pichinde Virus-Vectored Turkey Arthritis Reovirus Subunit Vaccine

We created a recombinant live pichinde virus-vectored bivalent codon optimized subunit vaccine that expresses immunogenic Sigma C and Sigma B proteins of turkey arthritis reovirus. The vaccine virus could be transmitted horizontally immunizing the non-vaccinated pen mates. The vaccine was tested for efficacy against homologous (TARV SKM121) and heterologous (TARV O’Neil) virus challenge. Immunized poults produced serum neutralizing antibodies capable of neutralizing both viruses. The vaccinated and control birds showed similar body weights indicating no adverse effect on feed efficiency. Comparison of virus gene copy numbers in intestine and histologic lesion scores in tendons of vaccinated and non-vaccinated birds showed a decrease in the replication of challenge viruses in the intestine and tendons of vaccinated birds. These results indicate the potential usefulness of this vaccine

Altered Biomechanical Properties of Gastrocnemius Tendons of Turkeys Infected with Turkey Arthritis Reovirus

Turkey arthritis reovirus (TARV) causes lameness and tenosynovitis in commercial turkeys and is often associated with gastrocnemius tendon rupture by the marketing age. This study was undertaken to characterize the biomechanical properties of tendons from reovirus-infected turkeys. One-week-old turkey poults were orally inoculated with O’Neil strain of TARV and observed for up to 16 weeks of age. Lameness was first observed at 8 weeks of age, which continued at 12 and 16 weeks. At 4, 8, 12, and 16 weeks of age, samples were collected from legs. Left intertarsal joint with adjacent gastrocnemius tendon was collected and processed for histological examination. The right gastrocnemius tendon’s tensile strength and elasticity modulus were analyzed by stressing each tendon to the point of rupture. At 16 weeks of age, gastrocnemius tendons of TARV-infected turkeys showed significantly reduced (P<0.05) tensile strength and modulus of elasticity as compared to those of noninfected control turkeys. Gastrocnemius tendons revealed lymphocytic tendinitis/tenosynovitis beginning at 4 weeks of age, continuing through 8 and 12 weeks, and progressing to fibrosis from 12 to 16 weeks of age. We propose that tendon fibrosis is one of the key features contributing to reduction in tensile strength and elasticity of gastrocnemius tendons in TARV-infected turkeys

Investigation of Cyprinid Herpesvirus 3 (CyHV-3) Disease Periods and Factors Influencing CyHV-3 Transmission in A Low Stocking Density Infection Trial

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of koi herpesvirus disease (KHVD) and important pathogen of aquaculture and wild populations of common carp worldwide. Understanding the relative contributions of direct and indirect transmission of CyHV-3 as well as the factors that drive CyHV-3 transmission can clarify the importance of environmental disease vectors and is valuable for informing disease modeling efforts. To study the mechanisms and factors driving CyHV-3 transmission we conducted infection trials that determined the kinetics of KHVD and the contributions of direct and indirect forms of CyHV-3 transmission, as well as the contributions of contact rate, viral load, pathogenicity and contact type. The incubation period of KHVD was 5.88 + 1.75 days and the symptomatic period was 5.31 + 0.87 days. Direct transmission was determined to be the primary mechanism of CyHV-3 transmission (OR = 25.08, 95%CI = 10.73&ndash;99.99, p = 4.29 &times; 10&minus;18) and transmission primarily occurred during the incubation period of KHVD. Direct transmission decreased in the symptomatic period of disease. Transmissibility of CyHV-3 and indirect transmission increased during the symptomatic period of disease, correlating with increased viral loads. Additionally, potential virulence-transmission tradeoffs and disease avoidance behaviors relevant to CyHV-3 transmission were identified

Detection of Astrovirus, Coronavirus and Haemorrhagic Enteritis Virus in Turkeys with Poult Enteritis Mortality Syndrome in Turkey

This study was carried out to investigate the presence of turkey astrovirus 2 (TAstV-2), turkey coronavirus (TCoV) and haemorrhagic enteritis virus (HEV) by molecular methods in cloacal swabs collected from both clinically healthy turkey flocks and those associated with poult enteritis mortality syndrome (PEMS) in Turkey. In the reverse transcriptase polymerase chain reaction (RT-PCR) examination of 230 cloacal swabs collected from 23 turkey flocks associated with PEMS, TAstV-2 was detected in 13.4% (31/230) of the animals and in 43.4% (10/23) of the flocks. In addition, this virus was found in two turkeys originating from one of the four clinically healthy flocks. On the other hand, neither TCoV nor HEV were detected in any of the turkey samples examined in this study. In the partial sequence analysis of four randomly selected DNA samples, 96% nucleotide identity was observed between our strains and reference Turkey astrovirus isolated from turkeys in Italy between 2000 and 2004 (sequence accession number DQ381378.1)

Comparative molecular characterization of novel and known piscine toti-like viruses

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5′ or 3′ ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3′ end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3′ end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters

Host Range of Carp Edema Virus (CEV) during a Natural Mortality Event in a Minnesota Lake and Update of CEV Associated Mortality Events in the USA

Mass mortality events of common carp (Cyprinus carpio, carp) associated with carp edema virus (CEV) alone or in coinfections with koi herpesvirus (KHV), is an emerging issue. Despite recent outbreaks of CEV in wild carp populations, the host range of North American species has not been well studied. To that end, we intensively sampled carp (n = 106) and co-habiting native fish species (n = 5 species; n = 156 total fish) from a CEV-suspect mass-mortality event of carp in a small Minnesota lake (Lake Swartout). Additionally, fecal and regurgitant samples (n = 73 each) from double-crested cormorants (Phalacrocorax auritus, DCCO) were sampled to test the potential of DCCO to act as a vector for virus transmission. CEV was confirmed to be widespread in the Lake Swartout carp population during the outbreak with high viral loads and histological confirmation, suggesting that CEV was the cause of the mortality event. There were no detections of CEV in any native fish species; however, DCCO regurgitant and fecal samples were positive for CEV DNA. In addition, three CEV-positive and one CEV + KHV-positive mortality events were confirmed with no observed mortality or morbidity of non-carp species in other lakes. This study provides evidence that CEV infection and disease may be specific to carp during mortality events with mixed-species populations, identifies DCCO as a potential vector for CEV, and further expands the known range of CEV, as well as coinfections with KHV, in North America

Host Range of Carp Edema Virus (CEV) during a Natural Mortality Event in a Minnesota Lake and Update of CEV Associated Mortality Events in the USA

Mass mortality events of common carp (Cyprinus carpio, carp) associated with carp edema virus (CEV) alone or in coinfections with koi herpesvirus (KHV), is an emerging issue. Despite recent outbreaks of CEV in wild carp populations, the host range of North American species has not been well studied. To that end, we intensively sampled carp (n = 106) and co-habiting native fish species (n = 5 species; n = 156 total fish) from a CEV-suspect mass-mortality event of carp in a small Minnesota lake (Lake Swartout). Additionally, fecal and regurgitant samples (n = 73 each) from double-crested cormorants (Phalacrocorax auritus, DCCO) were sampled to test the potential of DCCO to act as a vector for virus transmission. CEV was confirmed to be widespread in the Lake Swartout carp population during the outbreak with high viral loads and histological confirmation, suggesting that CEV was the cause of the mortality event. There were no detections of CEV in any native fish species; however, DCCO regurgitant and fecal samples were positive for CEV DNA. In addition, three CEV-positive and one CEV + KHV-positive mortality events were confirmed with no observed mortality or morbidity of non-carp species in other lakes. This study provides evidence that CEV infection and disease may be specific to carp during mortality events with mixed-species populations, identifies DCCO as a potential vector for CEV, and further expands the known range of CEV, as well as coinfections with KHV, in North America

Detection of multiple lineages of PRRSV in breeding and growing swine farms

The detection and co-circulation of multiple variants of porcine reproductive and respiratory syndrome virus (PRRSV) have been observed and reported in swine. However, the potential long-term impact of multiple prevailing PRRSV variants on pig-performance is not yet fully understood. The primary objective of this study was to describe the genetic variation of PRRSV in processing fluid (PF), oral fluid (OF), and tonsil scraping (TS) specimens from five swine farms with different production types and PRRS status over a period of time (~1 year). Furthermore, the association between PRRSV prevalence and production parameters was investigated. Results showed that PRRSV was detected by RT-qPCR in 21–25% of all types of specimens. In breeding farms, PRRSV detection in PF and/or TS samples was correlated with stillborn and mummified fetuses, and pre-weaning mortality throughout the study period. Although ORF5 sequences were obtained in <16% of all sample types, simultaneous detection of PRRSV variants including field and vaccine strains within a single sampling event was identified in both breeding and growing pig farms. Phylogenetic analyses based on the ORF5 sequence classified the detected field PRRSV into L1A and L1H, two sub-lineages of lineage 1 (L1). Our study demonstrated the presence of multiple PRRSV lineages, sub-lineages, and variants in swine herds and its potential association with swine reproductive performance under field conditions