Judith Galeota

Porcine Reproductive and Respiratory Syndrome Virus: Description of Persistence in Individual Pigs upon Experimental Infection

ABSTRACT We studied the persistence of porcine reproductive and respiratory syndrome virus (PRRSV) in individual experimentally infected pigs, during a period of up to 150 days postinfection (dpi). The results of this study suggest that the persistence of PRRSV involves continuous viral replication but that it is not a true steady-state persistent infection. The virus eventually clears the body and seems to do it in most of the animals by 150 dpi or shortly thereafter. High genetic stability was seen for several regions of the persistent PRRSVs genome, although some consistent mutations in the genes of envelope glycoproteins and M protein were also observed.

Duration of Infection and Proportion of Pigs Persistently Infected with Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Understanding the dynamics of porcine reproductive and respiratory syndrome virus (PRRSV) persistence in individual pigs is essential to the development of successful control programs. The objectives of this study were to investigate the proportion of inoculated pigs that become persistently infected with PRRSV and the duration of their infection. Additionally, different diagnostic techniques that detect persistent infections were compared. Twenty-eight 35-day-old pigs were inoculated with PRRSV. Serum and tonsil biopsy samples were collected on days 0, 7, 14, and 28 and then approximately monthly thereafter until day 251 postinoculation (p.i.). Tonsil, lymph node, and lung samples were collected following euthanasia on day 251 p.i. Virus was isolated from serum and tonsil biopsy samples that had been collected through days 28 and 56 p.i., respectively. Viral RNA was detected by reverse transcription (RT)-PCR in serum and tonsil biopsy samples that had been collected through day 251 p.i., although no serum samples collected from days 84 to 196 p.i. were positive and the presence of infectious PRRSV was not detected by swine bioassay of tissue samples collected at necropsy. The results confirmed that RT-PCR is more sensitive than virus isolation in identifying PRRSV-infected pigs. Six pigs that were persistently infected through days 225 or 251 p.i. remained seropositive throughout the study, although one pig had an enzyme-linked immunosorbent assay sample-to-positive ratio that was only slightly above the cutoff value of 0.40. Twenty of 28 tonsil biopsy samples collected on day 84 p.i. were positive by RT-PCR compared to only 1 positive biopsy sample out of 28 collected on day 119 p.i. The studys results indicate that most pigs clear PRRSV within 3 to 4 months, but that some may remain persistently infected for several months.

Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype.

Summary. Although live-attenuated vaccines have been used for some time to control clinical symptoms of the porcine reproductive and respiratory syndrome (PRRS), the molecular bases for the attenuated phenotype remain unclear. We had previously determined the genomic sequence of the pathogenic PRRSV 16244B. Limited comparisons of the structural protein coding sequence of an attenuated vaccine strain have shown 98% homology to the pathogenic 16244B. Here we have confirmed the attenuated phenotype and determined the genomic sequence of that attenuated PRRSV vaccine and compared it to its parental VR-2332 and the 16244B strains. The attenuated vaccine sequence was colinear with that of the strain 16244B sequence containing no gaps and 212 substitutions over 15,374 determined nucleotide sequence. We identified nine amino acid changes distributed in Nsp1β, Nsp2, Nsp10, ORF2, ORF3, ORF5 and ORF6. These changes may provide the molecular bases for the observed attenuated phenotype.

A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus

Abstract The nucleotide sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) was determined. Transfection of MARC-145 cells with capped in vitro transcripts derived from a full-length cDNA clone of the viral genome resulted in infectious PRRSV with growth characteristics similar to that of the parental virus. Primer extension analysis revealed that during replication, the viral polymerase corrected the two nonviral guanosine residues present at the 5′ terminus of the transfected transcripts. Animal studies showed that the cloned virus induced hyperthermia, persistent viremia, and antibody response, similar to that observed with the parental virus. Contact transmission occurred rapidly within 3 days of introduction of naı̈ve pigs into the group of clone virus-inoculated pigs. These results suggest that the cloned virus retains the in vivo virulence and contagion properties of the parental virus, thus, providing the background for reverse genetics manipulation in systematic examination of attenuation and virulence phenotypes.

Detection of Chronic Wasting Disease in the Lymph Nodes of Free-Ranging Cervids by Real-Time Quaking-Induced Conversion

ABSTRACT Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of deer, elk, and moose, is the only prion disease affecting free-ranging animals. Since the disease was first identified in northern Colorado and southern Wyoming in 1967, new epidemic foci of the disease have been identified in 20 additional states, as well as two Canadian provinces and the Republic of South Korea. Identification of CWD-affected animals currently requires postmortem analysis of brain or lymphoid tissues using immunohistochemistry (IHC) or an enzyme-linked immunosorbent assay (ELISA), with no practical way to evaluate potential strain types or to investigate the epidemiology of existing or novel foci of disease. Using a standardized real-time (RT)-quaking-induced conversion (QuIC) assay, a seeded amplification assay employing recombinant prion protein as a conversion substrate and thioflavin T (ThT) as an amyloid-binding fluorophore, we analyzed, in a blinded manner, 1,243 retropharyngeal lymph node samples from white-tailed deer, mule deer, and moose, collected in the field from areas with current or historic CWD endemicity. RT-QuIC results were then compared with those obtained by conventional IHC and ELISA, and amplification metrics using ThT and thioflavin S were examined in relation to the clinical history of the sampled deer. The results indicate that RT-QuIC is useful for both identifying CWD-infected animals and facilitating epidemiological studies in areas in which CWD is endemic or not endemic.

Evidence for the Localization of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Antigen and RNA in Ovarian Follicles in Gilts

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.

Natural Cases of 2009 Pandemic H1N1 Influenza A Virus in Pet Ferrets

Respiratory swab samples were collected from 5 pet ferrets (Mustela putorius furo) exhibiting influenza-like illness. The ferrets represented 3 households in 2 states. In each case, the owners reported influenza-like illness in themselves or family members prior to the onset of a similar illness in the ferrets. Realtime reverse transcription polymerase chain reaction assays designed for the detection of the 2009 H1N1 Influenza A virus were conducted in the state animal health laboratories. The assays included detection of the matrix gene of Influenza A virus and neuraminidase gene specific for 2009 H1N1 virus. Samples were positive for both screening assays. The samples were confirmed positive by the National Veterinary Services Laboratories. The history of illness in family members prior to illness in the ferrets suggests that Influenza A virus was transmitted from humans to the ferrets.

Ring test evaluation of the repeatability and reproducibility of a Porcine reproductive and respiratory syndrome virus oral fluid antibody enzyme-linked immunosorbent assay

The precision of a Porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody enzyme-linked immunosorbent assay (ELISA) was evaluated by calculating reliability coefficients for assay repeatability (within laboratory) and assay reproducibility (between laboratories). Randomly ordered oral fluid samples of known (n = 39) and unknown (n = 224) PRRSV antibody status were tested in 12 diagnostic laboratories. Each laboratory tested the samples twice, first using an antibody ELISA kit and reagents provided to them (phase 1) and then using an ELISA kit and reagents configured in their respective laboratory (phase 2). Repeatability (within laboratory) reliability coefficients calculated using results from samples of known PRRSV antibody status ranged from 0.724 to 0.997 in phase 1 and from 0.953 to 0.998 in phase 2. Reproducibility (between laboratories) reliability coefficients were calculated for 3 conditions: case 1—samples of unknown status (n = 224); case 2—samples of known status (n = 39), and case 3—all samples (n = 263). Among the 3 cases, reliability coefficients ranged from 0.937 to 0.964 in phase 1 and from 0.922 to 0.935 in phase 2. For case 3, it was estimated that 96.67% of the total variation in phase 1 and 93.21% in phase 2 could be attributed to the oral fluid samples themselves. Overall, the PRRSV oral fluid antibody ELISA was highly repeatable and reproducible. The current study supports the routine use of this test in laboratories providing diagnostic service to pig producers.

Genomic analysis of the differential response to experimental infection with porcine circovirus 2b

Porcine circovirus type 2 (PCV2) is the etiological agent of a group of associated diseases (PCVAD) that affect production efficiency and can lead to mortality. Using different crossbred lines of pigs, we analyzed host genetic variation of viral load, immune response and weight change following experimental infection with a PCV2b strain (n = 386). Pigs expressed variation in the magnitude and initiation of viremia and immune response recorded weekly until 28 days post-infection. A higher viral load was correlated with weight gain (r = -0.26, P < 0.0001) and presence of PCV2-specific antibodies (IgM, r = 0.26-0.34, P < 0.0001; IgG, r = 0.17-0.20, P < 0.01). In genome-wide association analyses of the responses at different time points, the proportions of phenotypic variation explained by combined effects of 56 433 SNPs were 34.8-59.4% for viremia, 10.1-59.5% for antibody response and 5.6-14.9% for weight change. Relationships between genomic prediction of overall viral load and weight gain during the first weeks of challenge were negative (-0.21 and -0.24 respectively, P < 0.0001). Individuals that carried more favorable alleles across three SNPs on SSC9 (0.60 Mb) and SSC12 (6.8 and 18.2 Mb) partially explained this relationship, having lower viral load (P < 0.0001); lower viremia at day 14 (P < 0.0001), day 21 (P < 0.01) and day 28 (P < 0.05) and greater overall average daily gain during infection (ADGi ; P < 0.01), ADGi at week 3 (P < 0.001) and week 4 (P < 0.01). These additive genetic relationships could lead to molecular solutions to improve animal health and reduce production costs.

Herpesvirus Infections in Rock Hyraxes (Procavia Capensis)

Seven juveniles and 3 adults from a closed group of 19 rock hyraxes (Procavia capensis) housed in a zoos indoor rock exhibit died or were euthanized after developing blepharoconjunctivitis and orofacial ulcers over a 2-week period. Histopathologic examination of dermal ulcers and ulcerated tongues revealed amphophilic to basophilic intranuclear inclusion bodies in epithelial cells bordering ulcers. Epithelial cells with inclusion bodies were often characterized by cytomegaly and karyomegaly, and many cells had formed syncytia. Examination of inclusion bodies in tongue epithelium by transmission electron microscopy revealed icosahedral nucleocapsids, approximately 80–95 nm in diameter, with morphologic features consistent with herpesvirus. Cytopathic effect (CPE) typical of alphaherpesvirus infection was seen in bovine turbinate, equine dermal, and Vero cell monolayers after inoculation with homogenates of the skin lesions, but CPE was not seen after inoculation onto Madin-Darby canine kidney or swine testicle cell monolayers. Polymerase chain reaction analysis using degenerate primers that targeted a portion of the herpesvirus polymerase gene generated a product of approximately 227 base pairs. The product was cloned, sequenced, and then analyzed using BLAST. At the nucleotide level, there was 86%, 77%, and 76% shared identity with Eidolon herpesvirus 1, Human herpesviruses 1 and 2, and Cercopithecine herpesvirus 2, respectively. Herpesvirus infections in rock hyraxes have not been characterized. The data presented in the current study suggest that a novel alphaherpesvirus caused the lesions seen in these rock hyraxes. The molecular characteristics of this virus would tentatively support its inclusion in the genus Simplexvirus.