Host-virus interactions in the Pacific white shrimp, Litopenaeus vannamei

ABSTRACT Aquatic food products are the fastest growing of the food-producing sectors, and an important food source for human consumption. The research described in this dissertation demonstrates the interactions of the Pacific white shrimp, Litopenaeus vannamei (L. vannamei) with two devastating viruses, Infectious myonecrosis virus (IMNV) and White spot syndrome virus (WSSV). Viral diseases cause tremendous negative impacts on the shrimp farming industry and these two virus have caused more that $16 billion in losses. Thus, development of strategies to prevent and control viral diseases is of tremendous interest and importance for the sustainability of this industry by utilizing RNA interference as a tool. To understand the underlying mechanisms of host-virus interactions, the transcriptome of lymphoid organ from shrimp infected with IMNV have been analyzed as described in the first manuscript that characterizes newly revealed sequences in the IMNV genome. This paper provides evidence that additional sequences exist in the IMNV genome resulting in an overall genome length that is 8226 base pair (bp) as compared to the original description of 7561 bp. The second manuscript describes variation in virulence among geographically distinct isolates of IMNV from Brazil and Indonesia and the results show that there is a higher virulence in IMNV (Indonesia). The complete genome sequences of both IMNV isolates were compared and shown to differ in 67 bp and 30 amino acid positions. The third manuscript describes the utilization of RNAi to silence WSSV by targeting early gene (wssv477) expression and that is more effective at suppressing virus replication than suppressing late genes (VP15). This result provides a new target antiviral for WSSV. The fourth manuscript presents the development of a direct delivery methodology for double-stranded RNA (dsRNA) into the foregut lumen of L. vannamei. This manuscript indicates that delivery of dsRNA by reverse gavage elicits protection that is comparable to intramuscular injection methods. This method can be used as a proxy for possible per os vaccination trials. The research presented in this dissertation significantly advances the knowledge in host-virus interactions in the Pacific white shrimp and provides promising results toward shrimp disease control strategies

Direct Delivery of VP19 Double-Stranded RNA into Litopenaeus vannamei by Reverse Gavage Induces Protection against White Spot Syndrome Virus Disease

Double stranded RNA was synthesized in vitro and was delivered by reverse gavage (RG) compared to traditional intramuscular injection (IM) 3 days prior to challenge with a lethal dose of WSSV in both groups

Injection of Double Stranded RNA Enhances Survival of Litopenaeus vannamei Challenged with White Spot Syndrome Virus

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

In Vivo Titration and Development of a Challenge Model for White Spot Syndrome Virus (WSSV) in Pacific White Shrimp (Litopeneus vannamei)

A challenge model was developed using a controlled bioassay system for estimation of lethal infective doses (LD 50) of White Spot Syndrome Virus (WSSV), as a model system to be used in further WSSV studies

Characterization of Newly Revealed Sequences in the Infectious Myonecrosis Virus Genome in \u3ci\u3eLitopenaeus vannamei\u3c/i\u3e

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 5′ terminus and 23 nt at the 3′ 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 5′ RACE, an additional 4 nt were discovered; 3′ 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 5′ end of the IMNV genome in IMNV-infected L. vannamei. An RNAi trigger targeting a 376 bp length of the 5′ 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

Characterization of Newly Revealed Sequences in the Infectious Myonecrosis Virus Genome in \u3ci\u3eLitopenaeus vannamei\u3c/i\u3e

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 5′ terminus and 23 nt at the 3′ 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 5′ RACE, an additional 4 nt were discovered; 3′ 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 5′ end of the IMNV genome in IMNV-infected L. vannamei. An RNAi trigger targeting a 376 bp length of the 5′ 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

Naturally occurring highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b infection in three domestic cats in North America during 2023

The Eurasian strain of highly pathogenic avian influenza (HPAI) H5N1 is a devastating pathogen for birds that also has the capacity to infect mammals. This report describes the presentation, clinical case findings (including haemogram and serum biochemistry), gross and microscopic lesions and virus detection in three HPAI H5N1-infected domestic cats from the USA in 2023. All three cats presented with neurological abnormalities and were euthanized due to a poor prognosis within 2 days (two cats) or 10 days (one cat) of known clinical disease onset. Necropsy consistently revealed pulmonary congestion and oedema, and cerebrocortical malacia with haemorrhage was also seen in the cat that survived for 10 days. On histology, all cats had necrotizing encephalitis and interstitial pneumonia with pulmonary congestion, oedema, vasculitis and vascular thrombosis. One cat also had microscopic multifocal necrosis in the liver, pancreas and an adrenal gland. To our knowledge, this report is the first to detail pathological findings in HPAI H5N1 naturally-infected cats during the widespread outbreak in North America beginning in 2021, and that describes a cat surviving for 10 days after onset of HPAI H5N1 encephalitis

Sequence-optimized and targeted double-stranded RNA as a therapeutic antiviral treatment against infectious myonecrosis virus in \u3ci\u3eLitopenaeus vannamei\u3c/i\u3e

Infectious myonecrosis virus (IMNV) is a significant and emerging pathogen that has a tremendous impact on the culture of the Pacific white shrimp Litopenaeus vannamei. IMNV first emerged in Brazil in 2002 and subsequently spread to Indonesia, causing large economic losses in both countries. No existing therapeutic treatments or effective interventions currently exist for IMNV. RNA interference (RNAi) is an effective technique for preventing viral disease in shrimp. Here, we describe the efficacy of a double-stranded RNA (dsRNA) applied as an antiviral therapeutic following virus challenge. The antiviral molecule is an optimized dsRNA construct that targets an IMNV sequence at the 5’ end of the genome and that showed outstanding antiviral protection previously when administered prior to infection. At least 50% survival is observed with a low dose of dsRNA administered 48 h post-infection with a lethal dose of IMNV; this degree of protection was not observed when dsRNA was administered 72 h post-infection. Additionally, administration of the dsRNA antiviral resulted in a significant reduction of the viral load in the muscle of shrimp that died from disease or survived until termination of the present study, as assessed by quantitative RT-PCR. These data indicate that this optimized RNAi antiviral molecule holds promise for use as an antiviral therapeutic against IMNV

Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis

Tritrichomonas foetus (TF) is a significant reproductive pathogen of cattle, and sample collection, handling, transport, and testing are significant hurdles to surveillance programs. Recent methods have been developed that allow for the direct detection of TF using a reverse transcription real-time PCR (direct RT-qPCR) approach. To evaluate these methods, a comparative analysis was conducted to assess the technical performance of this assay with a commercially available real-time PCR (qPCR) assay. In addition, the evaluation of two types of collection media (PBS and TF transport tube) was conducted that evaluated sample stability from 0 to 3 days when stored at 4°C or 25°C. Extended incubation times for PBS media were also evaluated (5, 7, and 14 days) at both refrigeration and frozen temperatures to evaluate the effect of extended transport time on samples. Limits of detection (LODs), dynamic range, and RNA stability were assessed using lab-cultured TF spiked into samples of normal bovine smegma collected in PBS or TF transport media, and performance was assessed on field samples collected in parallel. 100% agreement was found between direct RT-qPCR and qPCR at 10 parasites/extraction and a LOD of 1 parasite/extraction. Differences in detection were not observed in either collection media when incubated at either temperatures for up to 3 days of incubation. In addition, the extended incubation experiments indicate that samples containing 10 parasites/extraction can be detected at 4°C for 5 days with a mean Cq 26.34 (95% CI: 23.11–29.58) and detected at −20°C for 7 or 14 days, with a mean Cq 29.55 (95% CI: 27.73–31.37). A significant decrease in detectable RNA was observed in samples containing <10 parasites/extraction at −20°C for 14 days, which should be considered for long-term storage. In summary, direct RT-qPCR was found to be equivalent or superior to qPCR and PBS was not significantly different from TF transport media. The findings of the current study allows for more flexibility during sample collection and transport and ultimately enhancement of TF surveillance programs

RNA Nanovaccine Protects against White Spot Syndrome Virus in Shrimp

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(pcarboxyphenoxy) 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