Viral diseases are significant impediments to the sustainability of shrimp aquaculture. In addition to endemic disease, new viral diseases continue to emerge and cause significant impact on the shrimp industry. Disease caused by infectious myonecrosis virus (IMNV) has caused tremendous losses in farmed Pacific white shrimp (Litopenaeus vannamei) since it emerged in Brazil and translocated to Indonesia. There are no existing antiviral interventions, outside of pathogen exclusion, to mitigate disease in commercial shrimp operations. Here, we describe an iterative process of panning the genome of IMNV to discover RNA interference trigger sequences that initiate a robust and long-lasting protective response against IMNV in L. vannamei. Using this process, a single, low dose (0.02 mg) of an 81 or 153 bp fragment, with sequence corresponding to putative cleavage protein 1 in ORF1, protected 100 % of animals from disease and mortality caused by IMNV. Furthermore, animals that were treated with highly efficacious dsRNA survived an initial infection and were resistant to subsequent infections over 50 days later with a 100-fold greater dose of virus. This protection is probably sequence dependent, because targeting the coding regions for the polymerase or structural genes of IMNV conferred lesser or no protection. Interestingly, non-sequence specific dsRNA did not provide any degree of protection to animals as had been described for other shrimp viruses. Our data indicate that the targeted region for dsRNA is a crucial factor in maximizing the degree of protection and lowering the dose required to induce a protective effect against IMNV infection in shrimp.

PRNP genotypes, number of octarepeats (PHGGGWGQ) and indels in the PRNP promoter can influence the progression of prion disease in mammals. We found no relationship between presence of promoter indels in white-tailed deer and mule deer from Nebraska and CWD presence. White-tailed deer with the 95 H allele and G20D mule deer were more likely to be CWDfree, but unlike other studies white-tailed deer with the 96S allele(s) were equally likely to be CWD-free. We provide the first information on PRNP genotypes and indels in the promoter for Key deer (all homozygous 96SS) and Coues deer (lacked 95 H and 96S alleles, but possessed a uniquely high frequency of 103 T). All deer surveyed were homozygous for three tandem octarepeats.

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In the last 15 years, crustacean fisheries have experienced billions of dollars in economic losses, primarily due to viral diseases caused by such pathogens as white spot syndrome virus (WSSV) in the Pacific white shrimp Litopenaeus vannamei and Asian tiger shrimp Penaeus monodon. To date, no effective measures are available to prevent or control disease outbreaks in these animals, despite their economic importance. Recently, double-stranded RNA-based vaccines have been shown to provide specific and robust protection against WSSV infection in cultured shrimp. However, the limited stability of double-stranded RNA is the most significant hurdle for the field application of these vaccines with respect to delivery within an aquatic system. Polyanhydride nanoparticles have been successfully used for the encapsulation and release of vaccine antigens. We have developed a double-stranded RNA-based nanovaccine for use in shrimp disease control with emphasis on the Pacific white shrimp L. vannamei. Nanoparticles based on copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane exhibited excellent safety profiles, as measured by shrimp survival and histological evaluation. Furthermore, the nanoparticles localized to tissue target replication sites for WSSV and persisted through 28 days postadministration. Finally, the nanovaccine provided ~80% protection in a lethal WSSV challenge model. This study demonstrates the exciting potential of a safe, effective, and field-applicable RNA nanovaccine that can be rationally designed against infectious diseases affecting aquaculture.

Infectious myonecrosis virus (IMNV) causes significant economic losses in farmed shrimp, where associated mortality in ponds can reach 70 %. To explore host/pathogen interactions, a next-generation sequencing approach using lymphoid organ tissue from IMNV-infected Litopenaeus vannamei shrimp was conducted. Preliminary sequence assembly of just the virus showed that there were at least an additional 639 bp at the 59 terminus and 23 nt at the 39 terminus as compared with the original description of the IMNV genome (7561 nt). Northern blot and reverse transcription-PCR analysis confirmed the presence of novel sequence at both ends of the genome. Using 59 RACE, an additional 4 nt were discovered; 39 RACE confirmed the presence of 22 bp rather than 23 bp of sequence. Based on these data, the IMNV genome is 8226 bp in length. dsRNA was used to trigger RNA interference (RNAi) and suppress expression of the newly revealed genome sections at the 59 end of the IMNV genome in IMNVinfected L. vannamei. An RNAi trigger targeting a 376 bp length of the 59 UTR did not improve survival of infected shrimp. In contrast, an RNAi trigger targeting a 381 bp sequence in ORF1 improved survival to 82.2 % as compared with 2.2 % survival in positive control animals. These studies revealed the importance of the new genome sections to produce high-titre infection, and associated disease and mortality, in infected shrimp.

and Implications Double stranded RNA was synthesized in vitro with sequences corresponding to portions of the WSSV genome and were injected into shrimp 3 days prior to challenge with a lethal dose of WSSV.

The temporal trend of novel pathogen identification has increased over the last decade, with viruses being the main contributor to the increase. Currently, the NAHLN provides veterinary surveillance and testing procedures for high consequence animal diseases, where in many cases, the detection of these diseases is based on real-time PCR assay (ASF, CSF, FMD, and IAV-S) or pathogen specific antibodies using ELISA methods (PRV). However, genetic shift and drift in virus genomes can lead to failure in detection of novel or emerging pathogens. Therefore, with the current trend of increased viral disease emergence, novel methods are needed to accurately and rapidly identify and characterize viral pathogens independent of knowing genomic information. In this study, we developed a novel diagnostic approach to identifying swine pathogens, using surrogate viruses. We implemented the use of Oxford Nanopore MinION Technology to detect long read sequences in real time. Surrogate viruses were used in place of common pathogens such as African Swine Fever, Classical Swine Fever, Pseudorabies, Foot and Mouth Disease, and Influenza A to investigate rapid detection limits. To increase the complexity of a sample, both DNA and RNA viruses were spiked into tissues were analyzed using Oxford Nanopore MinION Technology. This method can detect both DNA and RNA viruses simultaneously without prior knowledge. Additionally, as sequences can be detected real-time, we were able to confidently detect viral pathogens within a sample within 30 min. Additionally, we have established a library preparation and sequencing protocol as well as an informatic pipeline that could allow clinics and diagnostics centers to identify pathogens in less than 10 hours.