Immunity raised by recent European subtype 1 PRRSV strains allows better replication of East European subtype 3 PRRSV strain Lena than that raised by an older strain

International audienceAbstractStable spatial distribution of porcine reproductive and respiratory syndrome (PRRSV)-1 subtypes in Europe is accompanied by a strong population immunity induced by local PRRSV strains. In the present study, it was examined if the immunity induced by three West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) offers protection against the highly virulent East European subtype 3 PRRSV strain Lena. The number of fever days was greater (p < 0.05) in the control group (7.6 ± 1.7 days) compared to the immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (p < 0.05) of AUC values of nasal shedding (control: 14.6, 07V063-immune: 3.4, 13V091-immune: 8.9, 13V117-immune: 8.0) and viremia (control: 28.1, 07V063-immune: 5.4, 13V091-immune: 9.0, 13V117-immune: 8.3). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (p < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation with subtype 1 PRRSV strains caused priming of the Lena-specific virus neutralization antibody response. Upon challenge with Lena, we observed a very strong serological booster effect for neutralizing antibodies against strains used for the first inoculation. Our results indicate that inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. The lower protection level elicited by recently isolated subtype 1 PRRSV strains may impair the outcome of the spatial expansion of subtype 3 strains from East Europe to West Europe

The immunity raised by recent European subtype 1 PRRSV strains allows a better replication of East European subtype 3 PRRSV strain Lena than the immunity raised by an older strain -increased risk for spatial expansion of PRRSV Lena-like strains

The spatial distribution of PRRSV-1 subtypes in Europe is quite stable, most probably due to a strong population immunity induced by the local PRRSV strains. In this study, we evaluated the potential of the immunity induced by several West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) to provide a protection against the highly virulent East European subtype 3 PRRSV strain Lena. Eleven-week-old pigs were inoculated with subtype 1 PRRSV strains (07V063, 13V091 or 13V117). Seven weeks later, the pigs were challenged with PRRSV strain Lena. Clinical, virological and serological parameters were monitored upon challenge. Number of fever days was higher (P < 0.05) in the non-immune control group (7.6 ± 1.7 days) compared to animals from immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (P < 0.05) of AUC values of nasal shedding (control: 14.6 ± 5.6, 07V063-immune: 3.4 ± 3.4, 13V091-immune: 8.9 ± 6.1, 13V117-immune: 8.0 ± 6.1) and viremia (control: 28.1 ± 11.0, 07V063-immune: 5.4 ± 4.4, 13V091-immune: 9.0 ± 1.5, 13V117-immune: 8.3 ± 4.8). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (P < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation of animals with subtype 1 PRRSV strains caused a priming of Lena-specific VN antibody response. Upon challenge with PRRSV Lena a serological booster effect was observed for neutralizing antibodies against strains used for the first inoculation. Our results indicate that immunity elicited by inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. We conclude that the lower protection level elicited by recently isolated subtype 1 PRRSV strains may facilitate spatial expansion of subtype 3 strains from East Europe to West Europe

CRISPR/Cas9 Editing of Duck Enteritis Virus Genome for the Construction of a Recombinant Vaccine Vector Expressing <i>ompH</i> Gene of <i>Pasteurella multocida</i> in Two Novel Insertion Sites

Duck enteritis virus (DEV) and Pasteurella multocida, the causative agent of duck plague and fowl cholera, are acute contagious diseases and leading causes of morbidity and mortality in duck. The NHEJ-CRISPR/Cas9-mediated gene editing strategy, accompanied with the Cre–Lox system, have been employed in the present study to show that two new sites at UL55-LORF11 and UL44-44.5 loci in the genome of the attenuated Jansen strain of DEV can be used for the stable expression of the outer membrane protein H (ompH) gene of P. multocida that could be used as a bivalent vaccine candidate with the potential of protecting ducks simultaneously against major viral and bacterial pathogens. The two recombinant viruses, DEV-OmpH-V5-UL55-LORF11 and DEV-OmpH-V5-UL44-44.5, with the insertion of ompH-V5 gene at the UL55-LORF11 and UL44-44.5 loci respectively, showed similar growth kinetics and plaque size, compared to the wildtype virus, confirming that the insertion of the foreign gene into these did not have any detrimental effects on DEV. This is the first time the CRISPR/Cas9 system has been applied to insert a highly immunogenic gene from bacteria into the DEV genome rapidly and efficiently. This approach offers an efficient way to introduce other antigens into the DEV genome for multivalent vector

Inhibition of Marek’s Disease Virus Replication and Spread by 25-hydroxycholesterol and 27-hydroxycholesterol In Vitro

Marek’s disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. It has been suggested that MDV suppresses the induction of type I interferons and thus escapes immune control. Cholesterol 25-hydroxylase (CH25H), a gene that encodes an enzyme that catalyses cholesterol to 25-hydroxycholesterol (25-HC), is an interferon-stimulating gene (ISG) known to exert antiviral activities. Other oxysterols, such as 27-hydroxycholesterols (27-HC), have also been shown to exert antiviral activities, and 27-HC is synthesised by the catalysis of cholesterol via the cytochrome P450 enzyme oxidase sterol 27-hydroxylase A1 (CYP27A1). At 24 h post infection (hpi), MDV stimulated a type I interferon (IFN-α) response, which was significantly reduced at 48 and 72 hpi, as detected using the luciferase assay for chicken type I IFNs. Then, using RT-PCR, we demonstrated that chicken type I IFN (IFN-α) upregulates chicken CH25H and CYP27A1 genes in chicken embryo fibroblast (CEF) cells. In parallel, our results demonstrate a moderate and transient upregulation of CH25H at 48 hpi and CYP27A1 at 72hpi in MDV-infected CEF cells. A significant reduction in MDV titer and plaque sizes was observed in CEFs treated with 25-HC or 27-HC in vitro, as demonstrated using a standard plaque assay for MDV. Taken together, our results suggest that 25-HC and 27-HC may be useful antiviral agents to control MDV replication and spread

Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro

Infectious bursal disease virus (IBDV) vaccines do not induce sterilizing immunity, and vaccinated birds can become infected with field strains. Vaccine-induced immune selection pressure drives the evolution of antigenic drift variants that accumulate amino acid changes in the hypervariable region (HVR) of the VP2 capsid, which may lead to vaccine failures. However, there is a lack of information regarding how quickly mutations arise, and the relative contribution different residues make to immune escape. To model IBDV antigenic drift in vitro, we serially passaged a classical field strain belonging to genogroup A1 (F52/70) ten times, in triplicate, in the immortalized chicken B cell line, DT40, in the presence of sub-neutralizing concentrations of sera from birds inoculated with IBDV vaccine strain 2512, to generate escape mutants. This assay simulated a situation where classical strains may infect birds that have suboptimal vaccine-induced antibody responses. We then sequenced the HVR of the VP2 capsid at passage (P) 5 and 10 and compared the sequences to the parental virus (P0), and to the virus passaged in the presence of negative control chicken serum that lacked IBDV antibodies. Two escape mutants at P10 had the same mutations, D279Y and G281R, and a third had mutations S251I and D279N. Furthermore, at P5, the D279Y mutation was detectable, but the G281R mutation was not, indicating the mutations arose with different kinetics

Isolation and seroprevalence of Aeromonas spp. among common food animals slaughtered in Nagpur, Central India

Aeromonads are ubiquitous foodborne pathogens with a global distribution. Animal-origin foods and contaminated animals are the main sources of Aeromonas infection to humans. So far little is known about the occurrence of Aeromonas spp. in food-producing animals in India. The present study was conducted to determine the prevalence and seroprevalence of Aeromonas species from 50 each of meat, blood, and sera samples collected from cattle, buffaloes, goats, and pigs slaughtered in and around Nagpur, Central India. Alkaline peptone water and ampicillin dextrin agar were used to isolate Aeromonas spp. An indirect enzyme-linked immunosorbent assay (ELISA) was standardized by use of whole-cell antigen (WC) and outer membrane protein (OMP) of Aeromonas hydrophila (MTCC 646). Aeromonads were isolated from 44 (22%) of the meat samples, and 1 (0.5%) from the blood samples. Seroprevalence by indirect ELISA-based WC antigen was estimated as 68% in cattle, 44% in buffaloes, 60% in goats, and 30% in pigs. OMP-based ELISA yielded a seroprevalence of 56%, 48%, 52%, and 22% in cattle, buffaloes, goats, and pigs, respectively. The results revealed that OMP-based ELISA and WC-based ELISA were in agreement with one another. Isolation along with high seropositivity demonstrates the presence of foodborne Aeromonas spp. in the Nagpur city of Central India

Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek&rsquo;s disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology