Genetic profile of African swine fever virus responsible for the 2019 outbreak in northern Malawi.

BACKGROUND: African swine fever (ASF) is an infectious transboundary animal disease which causes high mortality, approaching 100% in domestic pigs and it is currently considered as the most serious constraint to domestic pig industry and food security globally. Despite regular ASF outbreaks within Malawi, few studies have genetically characterized the causative ASF virus (ASFV). This study aimed at genetic characterization of ASFV responsible for the 2019 outbreak in northern Malawi. The disease confirmation was done by polymerase chain reaction (PCR) followed by molecular characterization of the causative ASFV by partial genome sequencing and phylogenetic reconstruction of the B646L (p72) gene, nucleotide alignment of the intergenic region (IGR) between I73R and I329L genes and translation of the central variable region (CVR) coded by B602L gene. RESULTS: All thirteen samples collected during this study in Karonga district in September 2019 were ASFV-positive and after partial genome sequencing and phylogenetic reconstruction of the B646L (p72) gene, the viruses clustered into ASFV p72 genotype II. The viruses characterized in this study lacked a GAATATATAG fragment between the I173R and the I329L genes and were classified as IGR I variants. Furthermore, the tetrameric amino acid repeats within the CVR of the B602L gene of the 2019 Malawian ASFV reported in this study had the signature BNDBNDBNAA, 100% similar to ASFV responsible for the 2013 and 2017 ASF outbreaks in Zambia and Tanzania, respectively. CONCLUSIONS: The results of this study confirm an ASF outbreak in Karonga district in northern Malawi in September 2019. The virus was closely related to other p72 genotype II ASFV that caused outbreaks in neighboring eastern and southern African countries, emphasizing the possible regional transboundary transmission of this ASFV genotype. These findings call for a concerted regional and international effort to control the spread of ASF in order to improve nutritional and food security.

Detection of Equine Herpesvirus (EHV) -1, -2, -4 and -5 in Ethiopian Equids with and without Respiratory Problems and Genetic Characterization of EHV-2 and EHV-5 Strains.

Infections with equine herpesviruses (EHVs) are widespread in equine populations worldwide. Whereas both EHV-1 and EHV-4 produce well-documented respiratory syndromes in equids, the contribution of EHV-2 and EHV-5 to disease of the respiratory tract is still enigmatic. This study describes the detection and genetic characterization of EHVs from equids with and without clinical respiratory disease. Virus-specific PCRs were used to detect EHV-1, -2, -4 and -5. From the total of 160 equids with respiratory disease, EHV-5 was detected at the highest prevalence (23.1%), followed by EHV-2 (20.0%), EHV-4 (8.1%) and EHV-1 (7.5%). Concurrent infections with EHV-2 and EHV-5 were recorded from nine (5.2%) diseased horses. Of the total of 111 clinically healthy equids, EHV-1 and EHV-4 were never detected whereas EHV-2 and EHV-5 were found in 8 (7.2%) and 18 (16.2%) horses, respectively. A significantly higher proportion of EHV-2-infected equids was observed in the respiratory disease group (32/160, 20.0%; P = 0.005) compared to those without disease (8/111; 7.2%). EHV-2-positive equids were three times more likely to display clinical signs of respiratory disease than EHV-2-negative equids (OR 3.22, 95% CI: 1.42-7.28). For EHV-5, the observed difference was not statistically significant (P = 0.166). The phylogenetic analysis of the gB gene revealed that the Ethiopian EHV-2 and EHV-5 strains had a remarkable genetic diversity, with a nucleotide sequence identity among each other that ranged from 94.0 to 99.4% and 95.1 to 100%, respectively. Moreover, the nucleotide sequence identity of EHV-2 and EHV-5 with isolates from other countries acquired from GenBank ranged from 92.9 to 99.1% and 95.1 to 99.5%, respectively. Our results suggest that besides EHV-1 and EHV-4, EHV-2 is likely to be an important contributor either to induce or predispose equids to respiratory disease. However, more work is needed to better understand the contribution of EHV-2 in the establishment of respiratory disease.

Equine Herpesvirus-1 Myeloencephalopathy, an Emerging Threat of Working Equids in Ethiopia.

Although equine herpesvirus myeloencephalopathy (EHM) is a sporadic and relatively uncommon manifestation of equine herpesvirus-1 (EHV-1), it has the potential for causing devastating outbreaks in horses. Up till now, there were no reported EHM outbreaks in donkeys and mules. This study describes the isolation and molecular characterization of EHV-1 from clinically EHM-affected horses (n = 6), mules (n = 3) and donkeys (n = 82) in Ethiopia during outbreaks from May 2011 to December 2013. The incidence of EHM cases was higher from April to mid-June. EHM in donkeys was more severe and death without clinical signs of paralysis, and recumbency was frequently observed. The main age of affected equines ranged from 7 to 10 years (n = 51; 56.0%), and females (n = 58; 63.7%) were more affected than males. The incidence of neuropathogenic (D752 ) and non-neuropathogenic (N752 ) variants of EHV-1 from EHM-affected equines in Ethiopia was assessed by sequencing the DNA polymerase gene (ORF30) of the EHV-1 isolates. The results indicated that from the total of 91 clinically affected equines, 90 (98.9%) of them had an ORF30 D752 genotype. An ORF30 N752 variant was only found in one donkey. Analysis of ORF68 as grouping marker for geographical differences showed that the Ethiopian EHV-1 isolates belong to geographical group 4. Due to the fatal nature of EHV-1 in donkeys, it would be interesting to examine the pathogenesis of EHM in this species. At present, there is no vaccine available in Ethiopia, and therefore, outbreaks of EHV-1 should be controlled by proper management adaptations. In addition, it is important to test the efficacy of the commercial vaccines not only in horses, but also in donkeys and mules.

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei (Boone), and the outcome for virus and cell.

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)-killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV-killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV-killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV-killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post-inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post-inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV-killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular-sensing molecules.

Differences in uptake and killing of pathogenic and non-pathogenic bacteria by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone).

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non-pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi-granulocytes) have the main function in phagocytosis of both pathogenic and non-pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non-virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post-inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non-virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.

Sensitivity of porcine epidemic diarrhea virus (PEDV) to pH and heat treatment in the presence or absence of porcine plasma.

Emergence of porcine epidemic diarrhea virus (PEDV) resulted in massive neonatal mortality in the North-American and Asian pork industry. Measures to prevent its geographical spread are of utmost importance to safeguard susceptible porcine populations. The major infection route is direct or indirect faecal-oral contact. Adequate biosafety measures should be in place at all levels of the swine production chain, including feed and feed ingredients. Present study aimed to investigate the sensitivity of PEDV to thermal inactivation at neutral and alkaline pH in presence or absence of porcine plasma. Cell culture medium and porcine plasma at different pH (7.2, 9.2, 10.2) and temperature conditions (4 °C, 40 °C, 44 °C, 48 °C) were inoculated to a final titer of 5.5 log10 TCID50 PEDV/ml, incubated for up to 120 min and the residual infectivity was determined by endpoint dilution assay. Irrespective of presence of plasma, PEDV was not sensitive to pH 7.2-10.2 at 4 °C. At moderate temperatures (≥40 °C), both alkaline pH and presence of plasma potentiated thermal inactivation. Inactivation of 8 log10 TCID50/ml plasma within 30 min (8D value<30 min) by moderate pH and temperature would denote potential industrial processing conditions that ensure safety towards PEDV while limiting denaturation of bioactive components. Virus-spiked plasma required heat treatment of 40 °C and alkalinization to pH 9.2 to achieve 8 log10 reduction within such time. At pH 10.2 and 48 °C, the 8D value was 4.6 min in plasma and 15.2 min in MEM. Here we propose heat-alkalinity-time (HAT) pasteurization as a highly efficient method to inactivate PEDV during industrial processing of porcine plasma.

Porcine reproductive and respiratory syndrome virus infection is associated with an increased number of Sn-positive and CD8-positive cells in the maternal-fetal interface.

It is already known that porcine reproductive and respiratory syndrome virus (PRRSV) infection in lungs changes a local cell pattern and cytokine profile. However, there is no information about cellular and immunological events upon PRRSV infection in the maternal-fetal interface yet. The altered number and/or function of macrophages and NK cells in the maternal-fetal interface during infection may have a functional importance for virus replication. In addition, local cellular and immunological disbalance may also disrupt fragile homeostasis and contribute to the PRRSV-related reproductive disorders. Sialoadhesin (Sn)-positive macrophages are target cells for PRRSV and Sn overexpression has been observed upon chronic inflammatory and infectious diseases. It is also known that mouse Sn-positive macrophages in lymph nodes are able to closely interact with and activate NK cells in response to viral particles. Therefore, the main purpose of the present study was to examine if PRRSV infection is associated with altered Sn expression on endometrial and placental macrophages. In addition, CD8-positive cells (porcine endometrial NK cells were previously described as CD8(+)CD3(-) cells) were localized and quantified in the PRRSV-positive and control tissues. Tissue samples were obtained from three PRRSV-inoculated and three non-inoculated control sows at 100 days of gestation. Real-time RT-PCR showed a clear upregulation of Sn mRNA expression in the PRRSV-positive endometrium/placenta (p<0.05). Sn-, CD163- and CD14-specific immunofluorescence stainings revealed that PRRSV-inoculated sows had a significantly higher number of Sn-positive macrophages in the endometrium and placenta due to de novo Sn expression on local CD163-positive macrophages. Along with the increased number of Sn-positive macrophages an increased number of CD8-positive cells, which were mostly CD3-negative, was observed in the PRRSV-positive endometrium. The effects of the observed cellular changes on virus replication and potential contribution to placental damage and reproductive disorders are discussed.

Purification of white spot syndrome virus by iodixanol density gradient centrifugation.

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60,000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80,000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1,000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.

Porcine sialoadhesin: a newly identified xenogeneic innate immune receptor.

Extracorporeal porcine liver perfusion is being developed as a bridge to liver allotransplantation for patients with fulminant hepatic failure. This strategy is limited by porcine Kupffer cell destruction of human erythrocytes, mediated by lectin binding of a sialic acid motif in the absence of antibody and complement. Sialoadhesin, a macrophage restricted lectin that binds sialic acid, was originally described as a sheep erythrocyte binding receptor. Given similarities between sialoadhesin and the unidentified macrophage lectin in our model, we hypothesized porcine sialoadhesin contributed to recognition of human erythrocytes. Two additional types of macrophages were identified to bind human erythrocytes-spleen and alveolar. Expression of sialoadhesin was confirmed by immunofluorescence in porcine tissues and by flow cytometry on primary macrophages. A stable transgenic cell line expressing porcine sialoadhesin (pSn CHO) bound human erythrocytes, while a sialoadhesin mutant cell line did not. Porcine macrophage and pSn CHO recognition of human erythrocytes was inhibited approximately 90% by an antiporcine sialoadhesin monoclonal antibody and by human erythrocyte glycoproteins. Furthermore, this binding was substantially reduced by sialidase treatment of erythrocytes. These data support the hypothesis that porcine sialoadhesin is a xenogeneic receptor that mediates porcine macrophage binding of human erythrocytes in a sialic acid-dependent manner.

Impact of a novel inactivated PRRS virus vaccine on virus replication and virus-induced pathology in fetal implantation sites and fetuses upon challenge.

Preventing congenital infection is important for the control of porcine reproductive and respiratory syndrome (PRRS). Recently, in our laboratory, an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine has been developed. Promising results in young pigs encouraged us to test the vaccine potency to prevent congenital infection. In the present study, the performance of this experimental inactivated vaccine was investigated in pregnant gilts. An advanced protocol was used to test the PRRSV vaccine efficacy. This protocol is based on recent insights in the pathogenesis of congenital PRRSV infections. Three gilts were vaccinated with an experimental PRRSV 07V63 inactivated vaccine at 27, 55, and 83 days of gestation. Three unvaccinated gilts were included as controls. At 90 days of gestation, all animals were intranasally inoculated with 10(5) tissue culture infectious dose 50 (TCID(50)) of PRRSV 07V63. Twenty days postchallenge animals were euthanized and sampled. The vaccinated gilts quickly developed virus neutralizing (VN) antibodies starting from 3 to 7 days postchallenge (1.0 to 5.0 log2). In contrast, the unvaccinated gilts remained negative for VN antibodies after challenge. The vaccinated gilts had shorter viremia than the control gilts. Gross pathology (mummification) was observed in 8% of the fetuses from vaccinated gilts and in 15% of the fetuses from unvaccinated gilts. The number of fetuses with severe microscopic lesions in the fetal implantation sites (a focal detachment of the trophoblast from the uterine epithelium; a focal, multifocal, or full degeneration of the fetal placenta) was lower in the vaccinated (19%) versus unvaccinated (45%) gilts (P < 0.05). The number of PRRS-positive cells in the fetal placentae was higher in unvaccinated versus vaccinated gilts (P < 0.05). In contrast, the number of PRRS-positive cells in the myometrium/endometrium was higher in vaccinated versus unvaccinated gilts (P < 0.05). Fifty-seven percent of the fetuses from the vaccinated gilts and 75% of the fetuses from the unvaccinated gilts were PRRSV-positive. In conclusion, implementation of the novel experimental inactivated PRRSV vaccine primed the VN antibody response and slightly reduced the duration of viremia in gilts. It also reduced the number of virus-positive fetuses and improved the fetal survival, but was not able to fully prevent congenital PRRSV infection. The reduction of fetal infection and pathology is most probably attributable to the vaccine-mediated decrease of PRRSV transfer from the endometrium to the fetal placenta.

Micro-dissecting the pathogenesis and immune response of PRRSV infection paves the way for more efficient PRRSV vaccines.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important infectious pathogen in pigs worldwide nowadays. Due to its genetic drift and increasing power to escape from immunity, PRRSV becomes more and more difficult to control. Based on a better knowledge of PRRSV, its interaction with the host cell, the macrophage, its pathogenesis and the immunity against this virus, new vaccines can now be constructed. This research-based development of new generation vaccines will allow swine industry to face the devastating consequences of PRRSV infections in the future. The present review summarizes the present knowledge on the pathogenesis, the immune response and the research-based vaccine development.

Cell tropism and entry of porcine circovirus 2.

Porcine circovirus 2 (PCV2) may induce reproductive failure (return to oestrus, embryonic death, mummification, weak- and stillborn piglets) and postweaning multisystemic wasting syndrome (PMWS). Furthermore, it may modulate the immunity in such a way that it aggravates the outcome of many bacterial and viral infections. In the present paper, the cellular tropism and entry of PCV2 are described and linked with the pathological and clinical consequences.

Complete genome characterization of a East European Type 1 subtype 3 porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of major economic importance that causes reproductive and respiratory problems in pigs. PRRSV strains are divided into European (Type 1) and North-American (Type 2) genotypes. Within the European PRRSV genotype, three subtypes have been delineated. Full genome sequences for North American and European subtype 1 strains have been described. Here, the first complete genomic characterization of a European subtype 3 strain (Lena) is described. Amplification of Orf1a and Orf1b fragments was achieved using a set of degenerate oligonucleotides. Using RT-PCR with Lena-specific primers, the full length sequence (15001 nt) was obtained. Alignment of Lena with European subtype 1 reference strain Lelystad showed variation over the entire length (84% identity/89% similarity at amino acid level) with the most variation in Orf1a (Nsp2/NSP2) with a deletion of 29 amino acids. Phylogenetic relationships using different Orfs supported Lena's genetic distinction from European subtype 1 strains. The availability of the European subtype 3 PRRSV full genome may be important for the understanding of PRRSV evolution and the more pronounced pathogenic nature of Lena.

Equine alphaherpesviruses (EHV-1 and EHV-4) differ in their efficiency to infect mononuclear cells during early steps of infection in nasal mucosal explants.

Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ(1) 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium. Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM.

Genetic characterization of African swine fever viruses from a 2008 outbreak in Tanzania.

Outbreaks of African swine fever (ASF) have been reported in the past from several countries in sub-Saharan Africa. The aim of this study was to genotype ASF viruses (ASFVs) from the 2008 outbreak in Morogoro and Dar es Salaam regions of Tanzania. Tissue samples from domestic pigs that died as a result of severe haemorrhagic disease were collected and analysed with PCR and genome sequencing methods using ASFV-specific primer sets. Nucleotide sequence data were obtained for the B646L (p72), E183L (p54) and the variable region of the B602L gene sequences. Phylogenetic analyses based on DNA sequences showed that the 2008 Tanzanian isolates belonged to p72 genotype XV and clustered together with those derived from the 2001 outbreak in Tanzania. Analysis of the tetrameric amino acid repeat regions within the variable region of the B602L gene showed that the repeat signature of the 2008 Tanzanian ASFV was unique and contained three novel tetramers (U = NIDT/NTDT and X = NTDI). Epidemiological investigation suggested that transportation of live pigs continues to play an active role in the epidemiology of ASF in Tanzania. It is recommended that future control of ASF spread in Tanzania should focus on the early detection and confirmation of the disease, prompt institution of quarantine measures, culling and proper disposal of infected and in-contact animals and decontamination of affected premises.

Role of group A p21-activated kinases in the anti-apoptotic activity of the pseudorabies virus US3 protein kinase.

The alphaherpesvirus US3 kinase is a conserved multifunctional serine/threonine kinase that plays a role in several processes, including modulation of the actin cytoskeleton, egress of virus particles from the nucleus and inhibition of apoptosis. However, the mechanisms used by the US3 protein to exert its functions remain poorly understood. Recently, we identified the group A p21-activated kinases PAK1 and PAK2 as important effectors in the US3-mediated cytoskeletal rearrangements. Here, we investigated if group A PAKs are also involved in the anti-apoptotic properties of US3. Infection experiments using a group A PAK inhibitor pointed at a moderate role for group A PAKs in the anti-apoptotic properties of US3. Furthermore, infection assays using wild type and US3null PRV in wild type MEF, PAK1(-/-) MEF and PAK2(-/-) MEF indicated that PAK2 does not play a role in US3-mediated inhibition of apoptosis during infection, whereas PAK1 plays a significant, yet limited role. Experiments in US3-transfected MEF using staurosporine as apoptosis trigger confirmed these observations. These results show that PAK1 plays a significant, yet limited, role in the anti-apoptotic activity of US3.

Pathologic and virologic findings in mid-gestational porcine foetuses after experimental inoculation with PCV2a or PCV2b.

Two major genotypes of porcine circovirus type 2 (PCV2) have been described: PCV2a and PCV2b. Previous studies mainly used PCV2a to experimentally reproduce reproductive failure in sows. This study aims to determine the clinical and virological outcome of surgical inoculation of 55-day-old immuno-incompetent porcine foetuses with PCV2a or PCV2b. Twelve foetuses were inoculated with PCV2: three with the post-weaning multisystemic wasting syndrome (PMWS)-associated PCV2a strain Stoon-1010, three with the reproductive failure-associated PCV2a strain 1121, three with the PMWS-associated PCV2b strain 48285 and three with the porcine dermatitis and nephropathy syndrome-associated PCV2b strain 1147. Four foetuses were mock-inoculated with cell culture medium. At 21 days post-inoculation eleven out of twelve PCV2-inoculated foetuses were oedematous and had distended abdomens, whereas one had a normal external appearance. All PCV2-inoculated foetuses had haemorrhages and congestion in internal organs and an enlarged liver. High PCV2 titres (>10(4.5)TCID(50)/g tissue) were found in all PCV2-inoculated foetuses, especially in the heart, spleen and liver. High numbers of PCV2-infected cells (>1000 infected cells/10mm(2) tissue) were observed in the hearts. PCR and DNA sequencing of the capsid gene recovered pure PCV2a and pure PCV2b sequences from PCV2a- and PCV2b-inoculated foetuses, respectively. All mock-inoculated and the remaining foetuses were normal in appearance and were PCV2 negative in virus titrations and indirect immunofluorescence stainings. The present study shows that PCV2a and PCV2b induce similar gross pathological lesions and replicate to similar high titres in organs of 55-day-old immuno-incompetent porcine foetuses.

Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants.

Equine herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (N-->D) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D(752) or N(752), to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N(752) isolates and at 24 h p.i. for all D(752) isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D(752) isolates compared with N(752) isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals.

Detection of truncated circular DNA species in Escherichia coli with a PCV2-containing plasmid with a single PCV2 origin of replication.

OBJECTIVE: Porcine circovirus type 2 (PCV2) is a small circular single-stranded DNA virus that causes postweaning multisystemic wasting syndrome in pigs. Cloning of the PCV2 genome in a plasmid allows the construction of infectious clones. Our objective was to clone single PCV2 genomes from an isolate containing a mixture of strains, in a plasmid in order to obtain pure PCV2 strains. METHODS: PCR amplification of PCV2 genomes and cloning followed by restriction enzyme analysis and sequencing. Transfection and PCV2 titration on PK-15 cells. RESULTS: Single-copy PCV2 genomes from three Belgian PCV2 strains were cloned. Unexpectedly, agarose gel analysis revealed that additional circular DNA species were generated in Escherichia coli. Restriction enzyme analysis and sequencing showed that the circular DNA species were truncated and derived from the plasmid containing the PCV2 genome. Mutagenesis of the PCV2 replicase gene abolished the formation of these DNA species. The infectious clones were transfected in PK-15 cells and pure PCV2 viral strains were obtained. CONCLUSION: Infectious clones were obtained that can be used for antigenic mapping and mutagenesis. In addition, our findings suggest that the replicase protein was expressed in E. coli and involved in the generation of the truncated DNA species.

Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV1 strains during the acute phase of infection in horses.

Equine herpesvirus 1 (EHV1) replicates in the respiratory tract of horses, after which infected leukocytes transport virus throughout the body, resulting in abortion or nervous system disorders. Two EHV1 strains circulate in the field: neurovirulent and non-neurovirulent. To investigate differences in replication in the upper respiratory tract (URT), an experimental inoculation study in ponies was performed with both strains. Two groups of six ponies, were inoculated intranasally with 10(6.5) TCID(50) of either strain. Clinical signs, nasal shedding and viremia were evaluated. At early time points post-inoculation (pi), one pony of each group was euthanized. Tissues were collected for titration and immunostainings. Number and size of EHV1-induced plaques were calculated, and individual EHV1-infected cells were quantified and characterized. Inoculation with either strain resulted in nasal shedding and replication in several tissues of the URT. Both strains replicated in a plaquewise manner in epithelium of the nasal mucosa, but replication in epithelium of the nasopharynx was largely limited to non-neurovirulent EHV1. Plaques were never able to cross the basement membrane, but individual infected cells were noticed in the connective tissue of all examined tissues for both strains. The total number of these cells however, was 3-7 times lower with non-neurovirulent EHV1 compared to neurovirulent EHV1. CD172a(+) cells and CD5(+) lymphocytes were important target cells for both strains. Interestingly, in lymph nodes, B-lymphocytes were also important target cells for EHV1, irrespective of the strain. Viremia was detected very early pi and infected cells were mainly CD172a(+) for both strains. In summary, these results are valuable for understanding EHV1 pathogenesis at the port of entry, the URT.