C-reactive protein, haptoglobin and Pig-Major acute phase protein profiles of pigs infected experimentally by different isolates of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiologic agent of PRRS, one of the most important diseases in swine worldwide. In the present work, the effects of different PRRSV strains were tested on a piglet experimental model to study the induced acute phase response. For this purpose, pigs (n=15 for each group) were intranasally inoculated with one of five PRRSV strains (isolates EU10, 12, 17, 18 from genotype 1 and isolate JA-142 from genotype 2). The acute phase response was monitored by measuring acute phase proteins (APPs). Specifically, the serum concentration of haptoglobin (Hp), C-reactive protein (CRP) and Pig-Major Acute Protein (Pig-MAP) was determined at 0, 3, 6, 9, 12, 15, 18 and 21 days p.i. Clinical signs and growth performance were also monitored during the experiment. All animals became viremic after inoculation during the study period. The APP response was heterogeneous and dependent on the strain, being strains EU10, EU 18 and JA-142 those that induced the highest response and the strongest clinical signs. In general, Hp was the most sensitive biomarker for PRRSV infection, CRP behaved as moderate and Pig-MAP was the less responsive during the course of PRRSV experimental infection. Hp and CRP were significantly discriminatory between infected and control pigs, but not Pig-MAP.

Comparison of two real-time RT-PCR assays for differentiation of C-strain vaccinated from classical swine fever infected pigs and wild boars.

Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.

Comparison of the immunoperoxidase monolayer assay and three commercial ELISAs for detection of antibodies against porcine circovirus type 2.

The aim of this study was to compare and correlate antibody titres against porcine circovirus type 2 (PCV2) in porcine sera (n = 1270) obtained by immunoperoxidase monolayer assay (IPMA) with the results of three commercial ELISAs (designated E1, E2 and E3). The correlation between IPMA and ELISA results was excellent (r(2) ≥ 0.90). Compared to IPMA, E2 had the highest sensitivity (93.0%), followed by E3 (90.1%) and E1 (85.0%); the specificity was 100% for all tests. All three commercial ELISAs had predictive values similar to those of IPMA and could be used to monitor antibody responses against PCV2 infection and/or vaccination.

Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia.

Porcine circovirus type 2 (PCV2) causes some of the most significant economic losses in pig production. Several multisystemic syndromes have been attributed to PCV2 infection, which are known as PCV2-associated diseases (PCVDs). This study investigated the origin and evolution of PCV2 sequences in domestic pigs and wild boars affected by PCVDs in Croatia. Viral sequences were recovered from three wild boars diagnosed with PCV2-systemic disease (PCV2-SD), 63 fetuses positive for PCV2 DNA as determined by PCR, 14 domestic pigs affected with PCV2-SD (displaying severe interstitial nephritis) and five domestic pigs with proliferative and necrotising pneumonia. Seventeen complete PCV2 genomes were recovered. Phylogenetic and evolutionary analyses based on median-joining phylogenetic networks, amino acid alignments and principal coordinate analysis were performed using complete genomes, as well as complete and partial ORF sequences for ORF1 and ORF2. Two of the 17 PCV2 sequences belonged to PCV2a, 14 to PCV2b and one was unclustered. PCV2b was the predominant genotype in Croatia and has been linked to international trade as a route of introduction. Correlation between particular viral strains with PCVDs is lacking.

Analysis of ORF5 and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes 1 and 2 retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates.

UNLABELLED: Recombination is currently recognized as a factor for high genetic diversity, but the frequency of such recombination events and the genome segments involved are not well known. In the present study, we initially focused on the detection of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) isolates by examining previously published data sets of ORF5 sequences (genotypes 1 and 2) obtained worldwide. We then examined full-length genome sequences in order to determine potential recombination breakpoints along the viral genome. For ORF5, 11 sets of genotype 1 sequences from different geographical areas, including 2 Asian, 1 American, and 7 European regions, and three sets of genotype 2, including sets from China, Mexico, and the United States, were analyzed separately. Potential recombination breakpoints were detected in 10/11 genotype 1 sets, including 9 cases in which the clustering of at least one isolate was different before and after the breakpoints. In genotype 2, potential breakpoints and different tree clustering of at least one strain before and after the breakpoint were observed in 2 out of 3 sets. The results indicated that most of the ORF5 data sets contained at least one recombinant sequence. When the full-length genome sequences were examined, both genotype 1 and 2 sets presented breakpoints (10 and 9, respectively), resulting in significantly different topologies before and after the breakpoints. Mosaic genomes were detected in genotype 1 sequences. These results may have significant implications for the understanding of the molecular epidemiology of PRRSV. IMPORTANCE: PRRSV is one of the most important viruses affecting swine production worldwide, causing big economic losses and sanitary problems. One of the key questions on PRRSV arises from its genetic diversity, which is thought to have a direct impact on immunobiology, epidemiology, diagnosis, and vaccine efficacy. One of the causes of this genetic diversity is recombination among strains. This study provides evidence that recombinant PRRSV isolates are common in most of the countries with significant swine production, especially PRRSV genotype 1. This observation has implications in the proper characterization of PRRSV strains, in the future development of phylogenetic studies, and in the development of new PRRSV control strategies. Moreover, the present paper emphasizes the need for a deeper understanding of the mechanisms and circumstances involved in the generation of genetic diversity of PRRSV.

Development of a suspension microarray for the genotyping of African swine fever virus targeting the SNPs in the C-terminal end of the p72 gene region of the genome.

African swine fever virus (ASFV) causes one of the most dreaded transboundary animal diseases (TADs) in Suidae. African swine fever (ASF) often causes high rates of morbidity and mortality, which can reach 100% in domestic swine. To date, serological diagnosis has the drawback of not being able to differentiate variants of this virus. Previous studies have identified the 22 genotypes based on sequence variation in the C-terminal region of the p72 gene, which has become the standard for categorizing ASFVs. This article describes a genotyping assay developed using a segment of PCR-amplified genomic DNA of approximately 450 bp, which encompasses the C-terminal end of the p72 gene. Complementary paired DNA probes of 15 or 17 bp in length, which are identical except for a single nucleotide polymorphism (SNP) in the central position, were designed to either individually or in combination differentiate between the 22 genotypes. The assay was developed using xMAP technology; probes were covalently linked to microspheres, hybridized to PCR product, labelled with a reporter and read in the Luminex 200 analyzer. Characterization of the sample was performed by comparing fluorescence of the paired SNP probes, that is, the probe with higher fluorescence in a complementary pair identified the SNP that a particular sample possessed. In the final assay, a total of 52 probes were employed, 24 SNP pairs and 4 for general detection. One or more samples from each of the 22 genotypes were tested. The assay was able to detect and distinguish all 22 genotypes. This novel assay provides a powerful novel tool for the simultaneous rapid diagnosis and genotypic differentiation of ASF.

Genotypic shift of porcine circovirus type 2 from PCV-2a to PCV-2b in Spain from 1985 to 2008.

Changes in porcine circovirus type 2 (PCV-2) genotypes were evaluated before, during and after outbreaks of post-weaning multisystemic wasting syndrome (PMWS) in (1) a retrospective study using pig sera collected in Spain from 1985 to 2008 and (2) a longitudinal study using pig sera collected from two farms in Spain over periods of 7 and 14 years. In both studies, there was a rapid genotypic shift from PCV-2a to PCV-2b that was related to the peak of PMWS epizootics in Spain and the appearance of PMWS on the two farms studied longitudinally.

Age-related tissue distribution of swine Torque teno sus virus 1 and 2.

Torque teno viruses (TTVs) are small, non-enveloped viruses with a circular single-stranded DNA genome, belonging to the family Anelloviridae. In swine, two genetically distinct species have been identified, Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2). The aim of the present work was to study the tissue distribution of TTSuV1 and TTSuV2 in pigs of different ages, including foetuses at the second and last thirds of gestation, and animals at 5 days and 5, 15 and 24 weeks of age. Investigated tissues included brain, lung, mediastinal and mesenteric lymph nodes, heart, liver, spleen, kidney and bone marrow. Viral DNA from tissue extractions were tested by a comparative PCR for the presence of TTSuVs. Overall, TTSuV1 and TTSuV2 species were found in all tissues tested, with variations depending on age, and following similar infection dynamics in all tissues, increasing progressively in prevalence and virus load over time. The highest prevalence was found at 5 weeks of age and maintained afterwards, and the highest loads of virus in the different tissues were seen in the oldest animals (15 and 24 weeks of age). No animals were negative to TTV, including foetuses. In conclusion, the present study indicated that swine TTSuV1 and TTSuV2 can be found virtually in all body tissues of the pig. Both swine TTV species were present in high levels in almost all older animals, while viral negative tissues were only found in 5-week-old and 5-day-old pigs, and foetuses.

Sow vaccination modulates the colonization of piglets by Haemophilus parasuis.

Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs and a colonizer of the upper respiratory tract of healthy pigs. A good balance between colonization and immunity is important to avoid a disease outbreak. This work studied the colonization of H. parasuis in healthy piglets coming from vaccinated and non-vaccinated sows. Piglets from vaccinated sows had higher IgG levels at early time points and subsequently were colonized later and to a lower degree than piglets from non-vaccinated ones. The variability of H. parasuis isolates was investigated by 2 genotyping methods: enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST). A high turnover of strains was found in both groups of piglets, with few strains found on more than one sampling occasion. We found a higher number of H. parasuis strains (16 strains) within a given farm than previously thought. Overall, more H. parasuis diversity was found in piglets from non-vaccinated sows than in those from vaccinated sows. These results indicate that vaccination of sows in a farm delays the colonization of piglets and reduces the carriage and heterogeneity of H. parasuis strains.

A proposal on porcine circovirus type 2 (PCV2) genotype definition and their relation with postweaning multisystemic wasting syndrome (PMWS) occurrence.

Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS). Despite first sequencing studies did not find any association between PCV2 sequences and PMWS occurrence, recent works have suggested the opposite. In the present study, 87 open reading frame 2 (ORF2) sequences obtained from pigs with different clinical conditions and coming from farms with different PMWS status were analyzed. Results further confirmed the existence of two genogroups and the definition of two PCV2 genotypes (1 and 2) is proposed. All sequences included in genotype 1 came from pigs from PMWS affected farms, while all sequences obtained from non-PMWS affected farms corresponded to genotype 2. Moreover, infection of single pigs from PMWS affected farms harbouring both genotypes is described. Present results suggest that PCV2 genotype 1 may potentially be more pathogenic than PCV2 genotype 2.

Molecular evolution of porcine circovirus type 2 genomes: phylogeny and clonality.

Porcine circoviruses (PCVs) type 1 (PCV1) and type 2 (PCV2) show high levels of nucleotide similarity, but PCV1 is considered non-pathogenic and PCV2 has been associated with several disease outcomes in pigs, mainly postweaning multisystemic wasting syndrome (PMWS). After exploring different topologies of the origin of PCVs, it was concluded that PCV1 and PCV2 seem to have a common origin. On the other hand, PCV2 could be divided into two groups (1 and 2) and eight clusters (1A to 1C and 2A to 2E), but none of those was apparently associated with disease status or geographic area. When phylogenetic trees constructed with the whole PCV2 genome, the cap or the rep genes were compared, some incongruence was identified. The possible existence of recombination was evaluated and cluster 1B was found to have a possible recombinant origin. Selective pressure was detected in all parts of the PCV2 genome, especially in the rep gene. Finally, the cap gene was the more suitable phylogenetic and epidemiological marker for PCV2, despite the fact that the virus can undergo recombination mainly within the first part of the rep region.

Interferon alpha therapy in haemophilic patients with chronic hepatitis C: a French multicentre pilot study of 58 patients.

BACKGROUND AND OBJECTIVES: Information about the long-term efficacy of interferon alpha (interferon-alpha) in haemophilic patients with chronic hepatitis not co-infected with the human immunodeficiency virus (HIV-1) is still limited. Previous studies seemed to indicate a low rate of response. The aim of this study was to evaluate the safety and long-term efficacy of interferon treatment in multi-transfused haemophiliacs. METHODS: Fifty-eight haemophiliacs were scheduled to receive 3 MU of interferon-alpha 2b three times a week for 12 months. The patients were followed up for at least 24 months post-treatment. Response was assessed by measurements of serum hepatitis C virus (HCV) RNA. RESULTS: Twenty-four patients (41.4%) dropped out. Except for seven patients, the symptoms that led to interrupting interferon treatment would probably not have resulted in the same decision in non-haemophilic patients. One patient developed an inhibitor to the deficient clotting factor without haemorrhagic consequences. In an intent to treat, the sustained virological response rate was 14%. However, when considering only the 34 patients who received the full treatment, HCV-RNA was cleared in eight patients (23%). CONCLUSIONS: This study suggests that multi-transfused haemophiliacs with chronic hepatitis not co-infected with HIV-1 respond to prolonged treatment with interferon-alpha in a similar proportion to that observed in non-haemophiliacs. There was a high rate of patients who did not complete the interferon-alpha treatment, and this seems to be characteristic of this patient population.

Molecular and morphologic approaches to discrimination of variability patterns in chub mackerel, Scomber japonicus.

The systematic status and the evolutionary biology of chub mackerel (Scomber japonicus) in the South West Atlantic Ocean is confusing with an unknown degree of genetic differentiation and reproductive isolation between units. Simultaneous genetic and morphologic analyses were made on 227 fish collected from two areas of the South West Atlantic Ocean and one from the Mediterranean Sea. The genetic analysis was based on 36 protein-coding loci, 16 of which were variable. The morphologic analyses include six morphometric length measurements and a meristic character. Correspondence between genetic and morphologic variability patterns indicates isolated Mediterranean and Southwest Atlantic subgroups of S. japonicus and, less clearly, possible additional divergence in two regional stocks within the latter group. The most conservative approach to management is to manage the stocks independently of one another.

Hepatitis C virus among blood donors: follow-up study.

BACKGROUND: The exact significance of antibodies to hepatitis C virus (HCV) in blood donors remains unknown. Confirmatory tests of anti-HCV-reactive serum and HCV RNA by polymerase chain reaction (PCR) are used to refute a large proportion of false-positive results. STUDY DESIGN AND METHODS: Ninety-two blood donors who were anti-HCV reactive in a first-generation enzyme-linked immunosorbent assay (ELISA) were reevaluated 10 months later with a second-generation ELISA (ELISA-2) as well as with second-generation recombinant immunoblot assay (RIBA-2) and by PCR. RESULTS: Twenty-five (43.9%) of the 57 ELISA-2-positive donors were confirmed as positive by RIBA-2; of these, 84 percent were HCV RNA positive in PCR. Of the 57 who were still anti-HCV positive, 46 were followed up and tested again in the same manner 2 years after the first screening. At that time, the pattern was little changed: 94 percent of RIBA-2- and PCR-positive donors remained positive. Of RIBA-2- and PCR-positive blood donors, 62 percent had abnormal alanine aminotransferase levels in at least one of the three evaluations. Among the anti-HCV-positive donors confirmed by RIBA-2, 60 percent, versus 12.6 percent in the control group, had a significantly (p < 0.001) more frequent risk factor for HCV infection, due to parenteral exposure to blood. CONCLUSION: These data confirm a good correlation between RIBA-2 reactivity and the detection of HCV RNA in a population of anti-HCV-positive blood donors.

Serological responses to different genotypes of hepatitis C virus in France.

The relationship between hepatitis C virus (HCV) genotypes and antibody status was studied in 104 chronic non-A, non-B hepatitis patients and asymptomatic HCV-infected blood donors. On the basis of amplification of the nonstructural protein 3 (NS3) coding region by PCR and hybridization with specific probes, 55 and 42 patients were identified as being infected with type I and type II, respectively, according to the classification by H. Okamoto, K. Kurai, S. Okada, K. Yamamoto, H. Lizuka, T. Tanaka, S. Fukuda, F. Tsudaand, and S. Mishiro (Virology 188:331-341, 1992). All samples were tested for antibodies to 5.1.1, C-100, C-33, and C-22 proteins by a second-generation recombinant immunoblot assay. Among 97 patients with known HCV genotypes, 31 of 42 patients infected with type II and 24 of 55 infected with type I had antibodies against all four antigens (P < 0.01). In the type II-infected group, more patients had detectable antibodies to 5.11, C-33, and C-22 proteins than in the type I group (P < 0.05). No difference was found in the serological response to C-100 between the two groups.