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In France during winter 2016-2017, 487 outbreaks of clade 2.3.4.4b H5N8 subtype high pathogenicity (HP) avian influenza A virus (AIV) infections were detected in poultry and captive birds. During this epizootic, HPAIV A/decoy duck/France/161105a/2016 (H5N8) was isolated and characterized in an experimental infection transmission model in conventional mule ducks. To investigate options to possibly protect such ducks against this HPAIV, three vaccines were evaluated in controlled conditions. The first experimental vaccine was derived from the hemagglutinin gene of another clade 2.3.4.4b A(H5N8) HPAIV. It was injected at three weeks of age, either alone (Vac1) or after a primer injection at day-old (Vac1 + boost). The second vaccine (Vac2) was a commercial bivalent adjuvanted vaccine containing an expressed hemagglutinin modified from a clade 2.3.2 A(H5N1) HPAIV. Vac2 was administered as a single injection at two weeks of age. The third experimental vaccine (Vac3) also incorporated a homologous 2.3.4.4b H5 HA gene and was administered as a single injection at three weeks of age. Ducks were challenged with HPAIV A/decoy duck/France/161105a/2016 (H5N8) at six weeks of age. Post-challenge virus excretion was monitored in vaccinated and control birds every 2-3 days for two weeks using real-time reverse-transcription polymerase chain reaction and serological analyses (haemagglutination inhibition test against H5N8, H5 ELISA and AIV ELISA) were performed. Vac1 abolished oropharyngeal and cloacal shedding to almost undetectable levels, whereas Vac3 abolished cloacal shedding only (while partially reducing respiratory shedding) and Vac2 only partly reduced the respiratory and intestinal excretion of the challenge virus. These results provided relevant insights in the immunogenicity of recombinant H5 vaccines in mule ducks, a rarely investigated hybrid between Pekin and Muscovy duck species that has played a critical role in the recent H5 HPAI epizootics in France.

Alphavirus-derived RNA replicon particle (RP) vaccines represent the next generation of swine influenza A virus (IAV) vaccines, as they were shown to be safe, effective, and offer advantages over traditional vaccine platforms. IAV is a significant respiratory pathogen of swine and there is a critical need to improve current commercial swine IAV vaccine platforms. Adjuvanted whole inactivated virus (WIV) IAV swine vaccines provide limited heterologous protection and may lead to vaccine-associated enhanced respiratory disease (VAERD). This study investigated the ability of RP IAV hemagglutinin (HA) vaccines to avoid VAERD and evaluated experimental multivalent HA and neuraminidase (NA) RP vaccines. RP vaccines were formulated with HA or NA heterologous or homologous to the challenge virus in monovalent HA or HA and NA bivalent combinations (HA/NA bivalent). Pigs were vaccinated with an HA RP, HA/NA bivalent RP, or heterologous HA WIV, followed by IAV challenge and necropsy 5 days post infection. RP vaccines provided homologous protection from challenge and induced robust peripheral and local antibody responses. The RP vaccine did not induce VAERD after challenge with a virus containing the heterologous HA, in contrast to the traditional WIV vaccine. The HA monovalent and HA/NA bivalent RP vaccines showed superior protection compared to traditional WIV. Additionally, the RP platform allows greater flexibility to adjust HA and NA content to reflect circulating IAV in swine antigenic diversity.

Background Porcine parainfluenza virus 1 (PPIV-1) is a respiratory virus in the family Paramyxoviridae and genus Respirovirus. It is closely related to bovine parainfluenza virus 3, human parainfluenza virus 1, and Sendai virus. Recent reports suggest PPIV-1 is widespread in swine herds in the United States and abroad. However, seroprevalence studies and the ability to evaluate cross neutralization between heterologous strains is not possible without validated antibody assays. This study describes the development of an indirect fluorescence antibody (IFA) assay, a whole virus enzyme-linked immunosorbent assay (wv-ELISA) and a serum virus neutralization (SVN) assay for the detection of PPIV-1 antibodies using 521 serum samples collected from three longitudinal studies and two different challenge strains in swine. Results The area under the curve (AUC) of the wv-ELISA (95% CI, 0.93–0.98) was significantly higher (p = 0.03) compared to the IFA (95% CI, 0.90–0.96). However, no significant difference was observed between the IFA and wv-ELISA when compared to the SVN (95% CI, 0.92–0.97). All three assays demonstrated relatively uniform results at a 99% true negative rate, with only 11 disagreements observed between the IFA, wv-ELISA and SVN. Conclusions All three serology assays detected PPIV-1 antibody in swine serum of known status that was collected from experimental studies. The SVN detected seroconversion earlier compared to the IFA and the wv-ELISA. Both the wv-ELISA and the SVN had similar diagnostic performance, while the IFA was not as sensitive as the wv-ELISA. All three assays are considered valid for routine diagnostic use. These assays will be important for future studies to screen seronegative swine for research, determine PPIV-1 seroprevalence, and to evaluate vaccine efficacy against PPIV-1 under experimental and field conditions.

Early in the global SARS-CoV-2 pandemic concerns were raised regarding infection of other animal hosts and whether these could play a significant role in the viral epidemiology. Infection of animals could be detrimental by causing clinical disease but also of concern if they become a viral reservoir allowing further mutations, plus having the potential to infect other animals or humans. The first reported animals to be infected both under experimental conditions and from anecdotal field evidence were cats described in China early in 2020. Given the concerns this finding raised and the close contacts between humans and cats, we aimed to determine whether a vaccine candidate could be developed that was suitable for use in multiple susceptible animal species and whether this vaccine could reduce infection of cats in addition to preventing spread to other cats.Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus (VEEV), known to be safe and efficacious for use in a variety of animals, expressing a stabilised Spike antigen, could induce neutralising antibody titers in guinea pigs and cats. After two intramuscular vaccinations, virus neutralising antibodies were detected in the respiratory tract of the guinea pigs and a cell mediated immune response was induced. The design of the SARS-CoV-2 antigen was shown to be critical in developing a strong neutralising antibody response. Vaccination of cats was able to induce a serum neutralising antibody response which lasted for the course of the experiment. Interestingly, in contrast to control animals, infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after challenge. Moreover, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit virus. The results show that the RP vaccine induces sterile immunity preventing SARS-CoV-2 infection and transmission. This data suggests that this RP vaccine could be a multi-species vaccine useful for preventing spread to and between other animals should that approach be required.

Oral administration of maize-expressed H3N2 nucleoprotein induced antibody responses in mice showing the immunogenicity of plant-derived antigen and its potential to be utilized as a universal flu vaccine. Influenza A viruses cause influenza epidemics that are devastating to humans and livestock. The vaccine for influenza needs to be reformulated every year to match the circulating strains due to virus mutation. Influenza virus nucleoprotein (NP) is a multifunctional RNA-binding protein that is highly conserved among strains, making it a potential candidate for a universal vaccine. In this study, the NP gene of H3N2 swine origin influenza virus was expressed in maize endosperm. Twelve transgenic maize lines were generated and analyzed for recombinant NP (rNP) expression. Transcript analysis showed the main accumulation of rNP in seed. Protein level of rNP in T1 transgenic maize seeds ranged from 8.0 to 35 µg of NP/g of corn seed. The level increased up to 70 µg of NP/g in T3 seeds. A mouse study was performed to test the immunogenicity of one line of maize-derived rNP (MNP). Mice were immunized with MNP in a prime-boost design. Oral gavage administration showed that a humoral immune response was elicited in the mice treated with MNP indicating the immunogenicity of MNP. NP-specific antibody responses in the MNP group showed comparable antibody titer with the groups receiving positive controls such as Vero cell-derived NP (VNP) or alphavirus replicon particle-derived NP (ANP). Cytokine analysis showed antigen-specific stimulation of IL-4 cytokine elicited in splenocytes from mice treated with MNP further confirming a TH2 humoral immune response induced by MNP administration.

In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111–130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61–110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.

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 thera- peutic following virus challenge. The antiviral molecule is an optimized dsRNA construct that tar- gets an IMNV sequence at the 5' end of the genome and that showed outstanding antiviral protec- tion 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 protec- tion was not observed when dsRNA was administered 72 h post-infection. Additionally, adminis- tration 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 prom- ise for use as an antiviral therapeutic against IMNV.

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 µg) 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.

Three methods for enrichment of Shiga toxin-producing Escherichia coli (STEC) were compared using waste pit samples from swine production facilities housing 50 to 3,000 animals. The STEC gene stx2 was detected in 5 of 17 pooled samples using a U.S. Department of Agriculture (USDA) enrichment procedure, 6 of 17 samples using a U.S. Food and Drug Administration (FDA) enrichment procedure, and 8 of 17 samples using an experimental acid enrichment. All isolates were non-O157 and 5 of 6 were positive for enterotoxigenic E. coli-associated heat stable toxins a and b. The three enrichment procedures were also tested for their ability to support growth of 31 strains of STEC. The acid enrichment media supported growth of 100% of the strains, the FDA medium supported 77% of the strains, and the USDA medium supported 16% of the strains.

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.

The advent of reverse genetic approaches to manipulate the genomes of both positive (+) and negative (-) sense RNA viruses allowed researchers to harness these genomes for basic research. Manipulation of positive sense RNA virus genomes occurred first largely because infectious RNA could be transcribed directly from cDNA versions of the RNA genomes. Manipulation of negative strand RNA virus genomes rapidly followed as more sophisticated approaches to provide RNA-dependent RNA polymerase complexes coupled with negative-strand RNA templates were developed. These advances have driven an explosion of RNA virus vaccine vector development. That is, development of approaches to exploit the basic replication and expression strategies of RNA viruses to produce vaccine antigens that have been engineered into their genomes. This study has led to significant preclinical testing of many RNA virus vectors against a wide range of pathogens as well as cancer targets. Multiple RNA virus vectors have advanced through preclinical testing to human clinical evaluation. This review will focus on RNA virus vectors designed to express heterologous genes that are packaged into viral particles and have progressed to clinical testing.

During 2012 and 2013, an apparent increase in porcine circovirus associated disease occurred in the USA. A variant PCV2b strain designated mPCV2b was recovered from many of these cases. This raised concerns of a decrease in efficacy of commercially available PCV2 vaccines. The objective of this study was to compare the ability of a commercial PCV2a-based vaccine and an experimental mPCV2b-based vaccine to control mPCV2b-associated disease, lesions, and viremia in a challenge model. Twenty-six caesarian-derived, colostrum-deprived pigs were randomly assigned to one of four groups: (1) vaccinated with a commercial PCV2a-based vaccine and challenged (PCV2a-VAC; n=7), (2) vaccinated with an experimental mPCV2b-based vaccine and challenged (mPCV2b; n=7), (3) sham-vaccinated with saline and challenged (positive controls; n=7), and (4) sham-vaccinated with saline without challenge (negative controls; n=5). Vaccination was done on D0 and D14, challenge was done on D28 using a tissue homogenate containing PRRSV and mPCV2b and the experiment was terminated on D49. Among the challenged pigs, 47.6% (10/21) developed severe clinical disease and either died or had to be humanely euthanized between D39 and D48 (11-20 days after challenge). PCV2 viremia was almost completely absent in the vaccinated groups regardless of vaccine type except for two PCV2a-vaccinated pigs which had detectable PCV2 DNA levels on individual days after challenge. Microscopic lesions typical of PCV2 infection were limited to the positive control group which developed mild-to-severe lesions associated with low-to-abundant PCV2 antigen. Under the conditions of this study, PCV2 vaccines regardless of PCV2 type were effective against mPCV2b challenge.

Vaccination of swine with an alphavirus-derived replicon particle vaccine stimulates a non-specific immune response. This effect was seen when animals were challenged with PRRSV at 24 hours post-vaccination. Animals that received vaccine had reduced viremia as measured by quantitative RT-PCR when compared to placebo. These results highlight the potential of replicon particle vaccines to induce robust immune responses in swine.

Pandemic H1N1 (pH1N1) influenza was first reported in the United States in April 2009. Since then, the virus has spread worldwide in both human and swine populations. Currently, pH1N1 influenza is the most common H1N1 virus infecting pigs in the United States. Vaccination of swine against pH1N1 represents the single best method of protecting against infection.

A single-cycle, propagation-defective replicon particle (RP) vaccine expressing a swine influenza virus hemagglutinin (HA) gene was constructed and evaluated in several different animal studies. Studies done in both the intended host (pigs) and non-host (mice) species demonstrated that the RP vaccine is not shed or spread by vaccinated animals to comingled cohorts, nor does it revert to virulence following vaccination. In addition, vaccinated pigs develop both specific humoral and IFN-γ immune responses, and young pigs are protected against homologous influenza virus challenge.

Recombinant hemagglutinin (HA) from a novel H1N1 influenza strain was produced using an alphavirus replicon expression system. The recombinant HA vaccine was produced more rapidly than traditional vaccines, and was evaluated as a swine vaccine candidate at different doses in a challenge model utilizing the homologous influenza A/California/04/2009 (H1N1) strain. Vaccinated animals showed significantly higher specific antibody response, reduced lung lesions and viral shedding, and higher average daily gain when compared to non-vaccinated control animals. These data demonstrate that the swine vaccine candidate was efficacious at all of the evaluated doses.

Replicon particles (RPs) expressing the GP5 and Matrix structural proteins of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) were created and compared to inactivated PRRSV in a challenge study. Pigs that received the RP vaccine had lower live virus titers and showed lower IDEXX ELISA values following challenge when compared to other groups. These results show that the RP vaccine provided partial protection against challenge with virulent PRRSV. Also, the RP vaccine allows for differentiation between vaccinated and naturally infected animals.

Polyvalent hyperimmune plasma (HP) with high-titers of virus neutralizing (VN) antibody to porcine reproductive and respiratory syndrome virus (PRRSV) strains was produced in gilts and used to passively immunize 3 week old piglets. The piglets were subsequently challenged with live virus. Results showed delay of viremia, decrease in live virus titers, decrease in gross lung lesions, or delay in transmission to naive, non-immunized sentinel pigs.

Horses were inoculated with several strains of virulent PRRS virus. Sera from the horses were tested for the presence of PRRS virus-neutralizing antibodies. Large volumes of equine plasma were collected and used to passively immunize piglets. Sera were collected at various time points after immunization and tested for virus-neutralizing activity. These results show that horses are capable of generating high neutralizing antibody titers to PRRSV when exposed to virulent virus. Piglets develop neutralizing antibody titers to PRRSV when passively immunized with a sufficient volume of ά-PRRSV equine plasma.