Modified-live Vaccine Research Articles

Isolation of a naturally occurring vaccine/wild-type recombinant bovine herpesvirus type 1 (BoHV-1) from an aborted bovine fetus

Bovine herpesvirus type 1 (BoHV-1) causes various disease syndromes in cattle including respiratory disease and abortions. During an investigation into the potential role of BoHV-1 modified-live vaccines (MLV) causing diseases in cattle, we performed whole genome sequencing on six BoHV-1 field strains isolated at Cornell Animal Health Diagnostic Center in the late 1970s. Three isolates (two respiratory and a fetal) were identified as vaccine-derived isolates, having SNP patterns identical to that of a previously sequenced MLV virus that exhibited a deleted US2 and truncated US1.67 genes. Two other isolates (a respiratory and a fetal) were categorized as wild-type (WT) viruses based on their unique SNP pattern that is distinct from MLV viruses. The sixth isolate from an aborted fetus was a recombinant virus with 62% of its genome exhibiting SNPs identical to one of the above-mentioned WT viruses also recovered from an aborted fetus. The remaining 38% consisted of two blocks of sequences derived from the MLV virus. The first block replaced the UL9-UL19 region, and the second vaccine-derived sequence block encompassed all the genes within the unique short region and the internal/terminal repeats containing the regulatory genes BICP4 and BICP22. This is confirmatory evidence that recombination between BoHV-1 MLV and WT viruses can occur under natural conditions and cause disease. It is important in that it underscores the potential for the glycoprotein E negative (gE−) marker vaccine used to eradicate BoHV-1 in some countries, to recombine with virulent field strains allowing them to capture the gE− marker, thereby endangering the control and eradication programs.

A DNA Prime Immuno-Potentiates a Modified Live Vaccine against the Porcine Reproductive and Respiratory Syndrome Virus but Does Not Improve Heterologous Protection.

The porcine reproductive and respiratory syndrome virus (PRRSV), an RNA virus inducing abortion in sows and respiratory disease in young pigs, is a leading infectious cause of economic losses in the swine industry. Modified live vaccines (MLVs) help in controlling the disease, but their efficacy is often compromised by the high genetic diversity of circulating viruses, leading to vaccine escape variants in the field. In this study, we hypothesized that a DNA prime with naked plasmids encoding PRRSV antigens containing conserved T-cell epitopes may improve the protection of MLV against a heterologous challenge. Plasmids were delivered with surface electroporation or needle-free jet injection and European strain-derived PRRSV antigens were targeted or not to the dendritic cell receptor XCR1. Compared to MLV-alone, the DNA-MLV prime- boost regimen slightly improved the IFNγ T-cell response, and substantially increased the antibody response against envelope motives and the nucleoprotein N. The XCR1-targeting of N significantly improved the anti-N specific antibody response. Despite this immuno-potentiation, the DNA-MLV regimen did not further decrease the serum viral load or the nasal viral shedding of the challenge strain over MLV-alone. Finally, the heterologous protection, achieved in absence of detectable effective neutralizing antibodies, was not correlated to the measured antibody or to the IFNγ T-cell response. Therefore, immune correlates of protection remain to be identified and represent an important gap of knowledge in PRRSV vaccinology. This study importantly shows that a naked DNA prime immuno-potentiates an MLV, more on the B than on the IFNγ T-cell response side, and has to be further improved to reach cross-protection.

Oral hydration therapy with water and bovine respiratory disease incidence affects rumination behavior, rumen pH, and rumen temperature in high-risk, newly received beef calves.

The study objectives were to determine the effect of oral hydration therapy and bovine respiratory disease (BRD) on rumination behavior, rumen pH, and rumen temperature. A random subset of high-risk, auction-sourced bulls from 3 truckload blocks (initial BW = 188.9 ± 19.1 kg) were fitted with a collar containing a 3-axis accelerometer to quantify rumination time and activity (n = 58) and administered a rumen pH and temperature data logging bolus (n = 33). At arrival, subset calves (n = 2 per pen) were balanced across treatment pens (n = 15 per treatment; n = 10 animals per pen) and randomized to receive 0.57 L water/45.4 kg BW from a modified oral drenching apparatus (H2O) or no water administration (CON). Standard arrival processing procedures were implemented including surgical castration. Modified-live virus respiratory vaccination was delayed until day 28. Technicians assigned a clinical illness score (CIS) daily; calves with CIS ≥ 2 and rectal temperature ≥ 40 °C were considered a BRD case (RCASE) and treated with an antimicrobial. The fixed effect of BRD cases vs. nontreated cohorts (RCON) was determined retrospectively using data from the accelerometer collar (n = 19 and 29) and rumen bolus (n = 12 and 21, for RCASE and RCON, respectively). Daily means and hourly means across days throughout the 56-d observation period were generated. Fixed effects were analyzed using the mixed model procedure with repeated measures. Daily rumen temperature was altered (P = 0.04) such that peak rumen temperature occurred earlier for H2O, whereas CON had increased (P ≤ 0.01) rumen temperature following delayed vaccination on day 28. Calves diagnosed with BRD had a transiently decreased (P = 0.04) active minutes between days 9 and 32, decreased (P < 0.01) active minutes between 0800 and 2000 h, decreased (P < 0.01) rumination time between 2000 and 0400 h, greater (P < 0.01) rumen temperature until delayed vaccination on day 28, and greater (P < 0.01) hourly rumen temperature between 0900 and 0300, and altered (P < 0.01) rumen pH. Earlier peak rumen temperature observed in H2O may indicate physiological modification enabling a more pronounced inflammatory response. Differences in rumination behavior and activity may be useful for early BRD detection.

A Field Recombinant Strain Derived from Two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-1) Modified Live Vaccines Shows Increased Viremia and Transmission in SPF Pigs.

In Europe, modified live vaccines (MLV) are commonly used to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, they have been associated with safety issues such as reversion to virulence induced by mutation and/or recombination. On a French pig farm, we identified a field recombinant strain derived from two PRRSV-1 MLV (MLV1). As a result, we aimed to evaluate its clinical, virological, and transmission parameters in comparison with both parental strains. Three groups with six pigs in each were inoculated with either one of the two MLV1s or with the recombinant strain; six contact pigs were then added into each inoculated group. The animals were monitored daily for 35 days post-inoculation (dpi) for clinical symptoms; blood samples and nasal swabs were collected twice a week. PRRS viral load in inoculated pigs of recombinant group was higher in serum, nasal swabs, and tonsils in comparison with both vaccine groups. The first viremic contact pig was detected as soon as 2 dpi in the recombinant group compared to 10 and 17 dpi for vaccine groups. Estimation of transmission parameters revealed fastest transmission and longest duration of infectiousness for recombinant group. Our in vivo study showed that the field recombinant strain derived from two MLV1s demonstrated high viremia, shedding and transmission capacities.

Systematic review and meta-analysis comparing arrival versus delayed vaccination of high-risk beef cattle with 5-way modified-live viral vaccines against BHV-1, BRSV, PI3, and BVD types 1 and 2

Bovine respiratory disease (BRD) is a major economic and welfare concern of the beef cattle industry. Various approaches to vaccination against the major viral pathogens have been investigated, including timing. It is thought that beef cattle entering feedlots may not be able to adequately respond to vaccination due to the stresses of shipping and processing. A meta-analysis was performed to compare the effectiveness of on-arrival vaccination to vaccination delayed 7 or more days following arrival, using a modified-live viral (MLV) vaccine against bovine herpesvirus-1 (BHV-1 ), bovine respiratory syncytial virus (BRSV), parainfluenza-3 (PI3), and bovine viral diarrhea types 1 and 2 (BVD 1 and 2), on outcomes of BRO morbidity, retreatment risk, and mortality. Eight studies were identified comparing vaccination timing in feedlot cattle via literature search. Mantel-Haenzsel risk ratios were calculated for each outcome, and Forest plots were constructed. In the studies identified, there was no difference in BRD morbidity risk, retreatment risk, or BRD mortality between calves vaccinated at arrival or delayed. Based on the data from the studies analyzed, it would not appear that there is an advantage or disadvantage in terms or morbidity, retreatment risk, or mortality in delaying vaccination.

Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates.

We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 ( HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012-2017 to determine their clade affiliation. All 14 EIV prototype strains were identified correctly as either Florida sublineage clade 1 or clade 2 using the 2 SNP target positions. Of 341 EIV rtPCR-positive samples, 337 (98.8%) and 4 (1.2%) isolates were classified as belonging to clade 1 and 2 Florida sublineage EIV, respectively. All clade 1 Florida sublineage EIV strains were detected in domestic horses, three clade 2 Florida sublineage EIV strains originated from horses recently imported into the United States, and one clade 2 Florida sublineage EIV strain originated from a healthy horse recently vaccinated with a modified-live intranasal EIV vaccine containing the American lineage strain A/eq/Kentucky/1991. EIV Florida sublineage clade differentiation using a fast and reliable multiplex rtPCR platform will help monitor the introduction of clade 2 Florida sublineage EIV strains into North America via international transportation.

Examination of viraemia and clinical signs after challenge with a heterologous PRRSV strain in PRRS Type 2 MLV vaccinated pigs: A challenge-dose study.

Vaccination with porcine reproductive and respiratory syndrome (PRRS) Type 2 modified-live vaccines (MLVs) has been shown to improve clinical signs and survival rates in PRRS virus (PRRSV)-challenged pigs. This study evaluated the dose of PRRSV challenge needed to cause and maintain viraemia in PRRS Type 2 MLV-vaccinated pigs and assessed clinical responses to various doses of virulent challenge. This controlled, randomised, blinded vaccination-challenge study involved 95 pigs who were either vaccinated with 2 mL of a PRRS Type 2 MLV on Day 0 or left unvaccinated. On Day 28, pigs were challenged intranasally with virulent PRRSV isolate (dose range <1.5 to 4 log10 50% tissue culture infectious dose/mL). Five pigs were left unchallenged and served as environmental controls. Viraemia levels, pyrexia, average daily weight gain and clinical signs were assessed. At all challenge doses, vaccinated groups had reduced viraemia levels and clinical signs, and higher average daily weight gain compared with non-vaccinated groups. Vaccinated groups challenged with ≤2 log had similar viraemia levels and clinical performance (days pyrexic and average daily weight gain) as the non-challenged group. Vaccinated groups had significantly reduced pyrexic days compared with non-vaccinated groups across all challenge doses (P <.05). Vaccinated pigs challenged with <3 log had significantly improved average daily weight gain (P <.05). In vaccinated groups, challenge dose correlated positively with viraemia levels and number of days pyrexic, and negatively with average daily weight gain. This is the first study to use a challenge-dose model to evaluate the efficacy of vaccination against PRRSV. PRRS Type 2 MLV was shown to mitigate the consequences of PRRSV infection at all evaluated PRRSV challenge doses. Lower levels of challenge had minimal impact on health and performance of vaccinated pigs, supporting the benefit of vaccinating swine with PRRS Type 2 MLV.

Efficacy evaluation of three modified-live PRRS vaccines against a local strain of highly pathogenic porcine reproductive and respiratory syndrome virus

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is characterized by high fever and high mortality in pigs of all ages and has severely affected the pork industry of China in the last decades. This study evaluated the differences in protection conferred by of three MLV PRRS vaccines derived from classical PRRSV (C-PRRSV, VR2332) and HP-PRRSV (TJM-F92 and JXA1-R) against the field challenge of HP-PRRSV TP strain (JXA1-R like). Compared to the experiment pigs in control group which were vaccinated with normal saline, the MLV PRRS vaccinated pigs had milder clinical symptoms, fewer pathological changes in the lung, and higher body weight gain at the end of the study. However, piglets vaccinated with VR2332 had higher body temperature, higher viral loads and lower body weight gain when compared with piglets vaccinated with TJM-F92 or JXA1-R vaccines at the end of the study. The results demonstrated that VR2332 vaccine provided a limited cross-protection against the HP-PRRSV TP strain infection, while in contrast the TJM-F92 and JXA1-R vaccines provided more efficacious protection. The findings of this study could serve as a valuable reference guide for the pig producers and veterinarians when considering the choice of which type of MLV PRRS vaccines to protect their pig herds against field challenge by HP-PRRSV TP strain.

PSVIII-38 Oral hydration therapy and bovine respiratory disease affects rumination behavior, rumen pH, and rumen temperature in high risk, newly received beef bulls.

The study objectives were to determine the effect of oral hydration therapy and bovine respiratory disease (BRD) on rumination behavior, rumen pH, and rumen temperature. A random subset of high risk, auction-sourced bulls from 3 truckload blocks (initial BW=188.9 ± 19.1 kg) were fitted with a collar containing a 3-axis accelerometer to quantify rumination time and activity index (n=58), and administered a rumen pH and temperature data logging bolus (n=33). At arrival, subset calves (n = 2/pen) were balanced across treatment pens (n=15/treatment; n=10 animals/pen) and randomized to receive 0.57 L water/45.4 kg BW from a modified oral drenching apparatus (H2O) or no water administration (CON). Standard arrival processing procedures were implemented and bulls were surgically castrated. Modified-live virus respiratory vaccination was delayed until day 28. Daily rumen temperature was altered (P=0.04) such that peak rumen temperature occurred earlier for H2O; whereas, CON had increased (P≤0.01) rumen temperature following delayed vaccination on day 28. Technicians assigned a clinical illness score (CIS) daily; calves with CIS ≥2 and rectal temperature ≥40°C were considered a bovine respiratory disease case (BRD) and treated with an antimicrobial. The fixed effect of BRD cases vs. non-treated cohorts (RCON) was determined retrospectively using data from the accelerometer collar (n= 19 vs. 29) and rumen bolus (n= 12 vs. 21). Calves diagnosed with BRD had decreased (P<0.01) rumination time between 2000 and 0400 hours, greater (P<0.01) rumen temperature until delayed vaccination on day 28, greater (P<0.01) hourly rumen temperature between 0900 and 0300, transiently decreased (P=0.04) activity index between day 9 and 32, decreased (P<0.01) activity index between 0800 and 2000, and altered (P<0.01) rumen pH. Earlier peak rumen temperature observed in H2O may indicate physiological modification enabling a more pronounced inflammatory response. Differences in rumination behavior and activity index are potential tools for early BRD detection.

Differential evolution of antigenic regions of porcine reproductive and respiratory syndrome virus 1 before and after vaccine introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread viral pathogen that has caused tremendous economic losses throughout most pig-producing countries. Nowadays, both PRRSV-1 and PRRSV-2 co-circulate in Korean pig populations, and commercial modified live vaccine (MLV) is predominantly used to control PRRS. Specifically, control strategy using only PRRSV-2 MLV that was used since 1995 cannot prevent the spread of PRRSV-1 and damage from its infection, which led to the first introduction of two additional PRRSV-1 vaccines in 2014. Despite the wide implementation with PRRSV-1 vaccines, there is a lack of knowledge about the currently circulating Korean PRRSV-1 strains. Whole structural genes of PRRSV-1 before (11) and after (17) the introduction of vaccine were compared to determine the genetic evolutionary features of PRRSV. Genetic and phylogenetic analysis indicated that Korean PRRSV-1 shared 91.5 ± 1.7% nucleotide identity but formed a unique clade based on ORF2–7 phylogeny. Bioinformatics showed increased genetic heterogeneity, enhanced diversifying selection, and the emergence of novel glycosylation sites within neutralizing epitopes of minor structural proteins after vaccine introduction. Taken together, our data provide novel insight into the evolution of minor structural proteins of PRRSV-1 in the vaccination era.

High reversion potential of a cell-adapted vaccine candidate against highly pathogenic porcine reproductive and respiratory syndrome

Modified live vaccine (MLV) based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is prone to quick reversion of virulence upon circulating in host animals. The objective of this study was to evaluate the virulence reversion potential of HP-PRRSV MLV and to identify elements within the HP-PRRSV genome contributing to this phenomenon.A blind passage, cell-adaptation strategy was attempted to attenuate a HP-PRRSV strain JX143, which was isolated during the atypical PRRS outbreak in 2006. Two attenuated candidates passage 87 (JXM87) and passage 105 (JXM105) used as MLVs showed the best balance of safety and efficacy in 4 week-old piglets (unpublished data). Two studies were performed during which the candidates were assessed for reversion to virulence through five back passages in susceptible piglets (21 ± 3 days of age). Both study results showed increase in clinical signs, pyrexia and lung lesions as well as decreased average daily weight gain as of passage 3 in susceptible pigs clearly, and it indicated that both candidates regained virulence, irrespective of the passage level. Increase in respective parameters was accompanied by increase in viremia in piglets: JXM87 virus titer increased from Passage 1 (P1) 4.40 Lg TCID50/mL to P4 5.75 Lg TCID50/mL, and JXM105 virus titer increased from P1 3.78 Lg TCID50/mL to P4 6.42 Lg TCID50/mL. Next generation sequencing (NGS) was performed on clinical samples (serum, lung tissue) from P4 animals. Sequence analysis comparing P4 materials with their parental strains revealed 10 amino acid mutations in 4 proteins for JXM87 and 14 amino acid mutations in 9 proteins for JXM105, respectively. Interestingly, five amino acid mutations were identical for the two candidates, which were located in nsp1β, GP5a and nsp10 coding regions, suggesting nsp1β, GP5a and nsp10 could contribute to virulence in HP-PRRSV.

Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR.

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate-phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

Whole-genome sequence analysis reveals unique SNP profiles to distinguish vaccine and wild-type strains of bovine herpesvirus-1 (BoHV-1)

Bovine herpesvirus-1 (BoHV-1) is a major pathogen affecting cattle worldwide causing primarily respiratory illness referred to as infectious bovine rhinotracheitis (IBR), along with reproductive disorders including abortion and infertility in cattle. While modified live vaccines (MLVs) effectively induce immune response against BoHV-1, they are implicated in disease outbreaks in cattle. Current diagnostic methods cannot distinguish between MLVs and field strains of BoHV-1. We performed whole genome sequencing of 18 BoHV-1 isolates from Pennsylvania and Minnesota along with five BoHV-1 vaccine strains using the Illumina Miseq platform. Based on nucleotide polymorphisms (SNPs) the sequences were clustered into three groups with two different vaccine groups and one distinct cluster of field isolates. Using this information, we developed a novel SNP-based PCR assay that can allow differentiation of vaccine and clinical strains and help accurately determine the incidence of BoHV-1 and the association of MLVs with clinical disease in cattle.

Acute porcine reproductive and respiratory syndrome outbreaks in immunized sow herds: from occurrence to stabilization under whole herd vaccination strategy

Outbreaks of porcine reproductive and respiratory syndrome virus (PRRSV) in vaccinated sow herds from occurrence to stabilization were monitored and analyzed in terms of serology and reproductive performance. Three different conventional pig farms experienced severe reproductive failures with the introduction of a type 1 PRRSV. These farms had adopted mass vaccination of sows using a type 2 PRRSV modified live vaccine (MLV). Therefore, to control the type 1 PRRSV, an alternative vaccination program utilizing both type 1 and type 2 MLV was undertaken. Following whole herd vaccinations with both types of MLV, successful stabilization of PRRS outbreaks was identified based on serological data (no viremia and downward trends in ELISA antibody titers in both sows and suckling piglets) and recovery of reproductive performance. Additionally, through comparison of the reproductive parameters between outbreak and non-outbreak periods, it was identified that PRRSV significantly affected the farrowing rate and the number of suckling piglets per litter at all three pig farms. Comparison of reproductive parameters between periods when the different vaccination strategies were applied revealed that the number of piglets born in total and born dead per litter were significantly increased after the introduction of the type 1 PRRS MLV.

Comparison of three vaccine programs on the health, growth performance, and carcass characteristics of high-risk feedlot heifers procured from auction-markets

A total of 2,575 beef heifers (BW = 568 ± 28.1 lb; 258 ± 12.7 kg) at high-risk of developing bovine respiratory disease (BRD) were enrolled in a randomized complete-block design study at a commercial feedlot to evaluate the effect of 3 vaccine programs on health, growth performance, and carcass characteristics. Dates of arrival to the feedyard served as the blocking factor and 10, 3-pen blocks were enrolled in the study (n = 10 replications per vaccine program). Chute- order randomization was used during arrival processing to assign heifers to 1 of 3 vaccine programs that differed by vaccine products or timing of the pentavalent modified-live viral vaccination: 1) Pyramid® 5 and Presponse® SQ during arrival processing (PRE), 2) Titanium® 5 and Nuplura® PH during arrival processing (TNA), and 3) Nuplura® PH during arrival processing with Titanium® 5 delayed until 28 days- on-feed (TND). No booster vaccinations were administered. Overall mortality, BRD morbidity, and BRD treatment success risks did not differ among the vaccine programs (P&gt;0.13). There were numerically fewer mortalities attributable to acute interstitial pneumonia in TNA heifers than the TND and PRE heifers (probability of difference = 0.99 and 1.00, respectively). Mortality attributable to BRD did not differ between vaccine programs (probability of difference &lt; 0.48). Endotoxin concentrations were measured in the Mannheimia haemolytica vaccines, and were lower in Nuplura® PH than Presponse® SQ. An arrival vaccine program implementing Titanium® 5 and Nuplura® PH had similar efficacy on BRD- related health outcomes as vaccinating with Pyramid® 5 and Presponse® SQ. Delaying a pentavalent viral vaccine until 28 days-on-feed did not affect health or growth-performance outcomes in this study.

Genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine

Modified live vaccines (MLVs) based on genotype 1 strains, particularly C-strain, have been used to prevent and control classical swine fever virus (CSFV) worldwide. Nevertheless, a shift in the predominant CSFV strains circulating in the field from genotype 1 or 3 to genotype 2 is seen. Genotype 2 is genetically distant from the vaccine strains and was recently reported during outbreaks after vaccine failure; this has raised concerns that vaccination has influenced viral evolution. In Korea in 2016, there was an unexpected CSF outbreak in a MLV-vaccinated commercial pig herd. The causative CSFV strain was genetically distinct from previously isolated Korean strains but similar to recent Chinese strains exhibiting enhanced capacity to escape neutralization; this suggests the need for global cooperative research on the evolution of CSFV. We analysed global E2 sequences, using bioinformatics tools, revealing the evolutionary pathways of CSFV. Classical swine fever virus genotypes 1 and 2 experienced different degrees and patterns of evolutionary growth. Whereas genotype 1 stayed relatively conserved over time, the genetic diversity of genotype 2 has progressively expanded, with few fluctuations. It was determined that genotype 2 evolved under lower immune pressures and at a higher evolutionary rate than genotype 1. Further, several selected codons, under diversifying selection in genotype 1 but under purifying selection in genotype 2, correspond to antigenic determinants, which could lead to evasion of vaccine-induced immunity. Our findings provide evidence that evolutionary changes in CSFV are the result of the disproportionate usage of the CSF MLVs in endemic areas; this underscores the need to develop mitigation strategies to minimize the substantial risk associated with the emergence of vaccine-escaping mutants.

Expected Net Benefit of Vaccinating Rangeland Sheep against Bluetongue Virus Using a Modified-Live versus Killed Virus Vaccine.

Recurring outbreaks of bluetongue virus in domestic sheep of the US Intermountain West have prompted questions about the economic benefits and costs of vaccinating individual flocks against bluetongue (BT) disease. We estimate the cost of a BT outbreak on a representative rangeland sheep operation in the Big Horn Basin of the state of Wyoming using enterprise budgets and stochastic simulation. The latter accounts for variability in disease severity and lamb price, as well as uncertainty about when an outbreak will occur. We then estimate the cost of purchasing and administering a BT vaccine. Finally, we calculate expected annual net benefit of vaccinating under various outbreak intervals. Expected annual net benefit is calculated for both a killed virus (KV) vaccine and modified-live virus vaccine, using an observed price of $0.32 per dose for modified-live and an estimated price of $1.20 per dose for KV. The modified-live vaccine’s expected annual net benefit has a 100% chance of being positive for an outbreak interval of 5, 10, or 20 years, and a 77% chance of being positive for a 50-year interval. The KV vaccine’s expected annual net benefit has a 97% chance of being positive for a 5-year outbreak interval, and a 42% chance of being positive for a 10-year interval. A KV vaccine is, therefore, unlikely to be economically attractive to producers in areas exposed less frequently to BT disease. A modified-live vaccine, however, requires rigorous authorization before legal use can occur in Wyoming. To date, no company has requested to manufacture a modified-live vaccine for commercial use in Wyoming. The KV vaccine poses less risk to sheep reproduction and less risk of unintentional spread, both of which facilitate approval for commercial production. Yet, our results show an economically consequential tradeoff between a KV vaccine’s relative safety and higher cost. Unless the purchase price is reduced below our assumed $1.20 per dose, producer adoption of a KV vaccine for BT is likely to be low in the study area. This tradeoff between cost and safety should be considered when policymakers regulate commercial use of the two vaccine types.

Does modified-live viral vaccine administration to heifers or cows lack substantial risk?

Modified-live viral (MLV) vaccines are an important tool to limit reproductive loss subsequent to infection from bovine viral diarrhea virus and bovine herpesvirus-1, but are not without risk. Therefore, their utilization must be undertaken with an understanding of the inherent risks of the vaccines and their administration. These risks include the potential causation of undue harm and lack of effective immunization. Consequently, vaccine programs should be designed to minimize the risks while maintaining or maximizing potential benefits of vaccination. The risk of viral transmission from vaccinated calves to naive cows is low but not absent. Therefore, cows and heifers should be effectively immunized prior to gestation, ideally at least 30 days before breeding. Additionally, revaccination of pregnant cows previously vaccinated with the same MLV vaccine carries a low but detectable risk of adverse reproductive consequences. Understanding the level of risk associated with the vaccination of cattle against bovine viral diarrhea virus and bovine herpesvirus-1 will aid in the optimization of vaccination protocols. Proper timing of MLV vaccine administration can maximize protection against reproductive viral pathogens while minimizing the potential for the development of adverse consequences subsequent to vaccination.

Development of an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against wild-type porcine reproductive and respiratory syndrome from the vaccine strain TJM-F92 based on a recombinant Nsp2 protein

An accurate ELISA method to differentiate pigs infected with wild-type porcine reproductive and respiratory syndrome (PRRSV) strains from vaccinated ones would help to monitor PRRSV vaccination compliance. The recombinant protein GST-d120aa derived from the continuous deletion of 120 amino acids in the non-structural protein 2 region of the modified-live vaccine strain TJM-F92 was used to develop an indirect enzyme-linked immunosorbent assay (d120-ELISA) for differentiating serum antibodies against TJM-F92 from other PRRSV strains. At the optimized cut-off value which was calculated at an S/P of 0.25, it yielded a sensitivity of 90.7% and a specificity of 95.1%. Cross-reactivity tests suggested that the d120-ELISA was PRRSV-specific. Coefficient of variations of the repeatability tests ranged between 1.41–17.02%. The results suggest that the d120-ELISA is suitable for differentiating animals infected with wild-type strains from those immunized with MLV TJM-F92.

Genetic diversity in envelope genes of contemporary U.S. porcine reproductive and respiratory syndrome virus strains influences viral antigenicity

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases in swine caused by porcine reproductive and respiratory syndrome virus (PRRSV). Genome sequences of sixty-six PRRSV strains were obtained using metagenomic sequencing of serum samples collected in the U.S. in 2014 to explore contemporary genetic diversity. Phylogenetic analysis of the genes encoding the envelope proteins identified four to eight distinct lineages with >87% intraclade identity. To explore the effect of the observed genetic diversity on antigenicity, the genome regions encoding either GP2a-GP3-GP4 or GP5-M in strain SD95-21 were replaced with alleles from each of eight distinct PRRSV strains using reverse genetics. The GP2a-GP3-GP4 region from only four of the eight strains yielded viable recombinant virus. When viable, both GP2a-GP3-GP4 and GP5-M variably affected antigenicity. A strain-dependent significant loss in cross reactivity was variably observed by indirect immunofluorescence assays using antisera from pigs vaccinated with commercial modified-live vaccines following replacement of GP2a-GP3-GP4 or GP5-M. Significantly reduced neutralization titers were similarly measured using antisera from naturally PRRSV-exposed pigs. These results illustrate the need to consider genomic regions besides GP5 for PRRSV epidemiology and vaccination.