Pathogenesis and antigenic characterization of a new East European subtype 3 porcine reproductive and respiratory syndrome virus isolate

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is divided into a European and North American genotype. East European PRRSV isolates have been found to be of the European genotype, but form different subtypes. In the present study, PRRSV was isolated from a Belarusian farm with reproductive and respiratory failure and designated "Lena". Analyses revealed that Lena is a new East European subtype 3 PRRSV isolate. The main purpose of this investigation was to study the pathogenesis and antigenic characteristics of PRRSV (Lena). Results: Obvious clinical and virological differences were observed between the animals inoculated with a recent European subtype 1 PRRSV isolate (Belgium A) and animals inoculated with PRRSV (Lena). Three out of six pigs inoculated with PRRSV (Belgium A) had anorexia and low fever at 3, 4 and 5 days post-inoculation (dpi). High fever, anorexia and depression were prominent signs in most pigs inoculated with PRRSV (Lena) between 2 and 28 dpi. Four pigs out of ten died during the experiment. Arcanobacterium pyogenes was isolated from lungs of one animal that died, and Streptococcus suis was isolated from lungs of one animal that was euthanized. The difference in viral titres in sera from PRRSV (Belgium A) and PRRSV (Lena)-infected pigs was statistically significant (p < 0.05) at 7, 10, 14 and 21 dpi. The highest viral titres in sera ranged from 10(4.8) to 10(6.1) TCID50/ml for PRRSV (Lena) whereas they ranged from 10(3.1) to 10(4.8) TCID50/ml for PRRSV (Belgium A). The replication of PRRSV (Lena) was further studied in depth. Viral titres ranged from 10(2.5) TCID50/100 mg to 10(5.6) TCID50/100 mg in nasal secretions between 3 and 14 dpi and from 10(2.8) TCID50/100 mg to 10(4.6) TCID50/100 mg in tonsillar scrapings between 3 and 21 dpi. High viral titres were detected in lungs (10(2.3)-10(7.7) TCID50/g tissue), tonsils (10(2.0)-10(6.2) TCID50/g tissue) and inguinal lymph nodes (10(2.2)-10(6.6) TCID50/g tissue) until 35, 28 and 35 dpi, respectively. To examine the antigenic heterogeneity between the East European subtype 3 isolate Lena, the European subtype 1 strain Lelystad and the North American strain US5, sets of monospecific polyclonal antisera were tested in immunoperoxidase monolayer assays (IPMAs) with homologous and heterologous viral antigens. Heterologous antibody titres were significantly lower than homologous titres (p = 0.01-0.03) for antisera against PRRSV (Lena) at all sampling time points. For antisera against PRRSV (Lelystad) and PRRSV (US5), heterologous antibody titres were significantly lower than homologous titres at 14 and 21 dpi (p = 0.01-0.03) and at 10 and 14 dpi (p = 0.04), respectively. Conclusions: Lena is a highly pathogenic East European subtype 3 PRRSV, which differs from European subtype 1 Lelystad and North American US5 strains at both the genetic and antigenic level

In vitro susceptibility of six isolates of equine herpesvirus 1 to acyclovir, ganciclovir, cidofovir, adefovir, PMEDAP and foscarnet

International audienc

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3+CD8high cells starting from 14 dpi. Although CD3+CD8high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3+CD8high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages

Outcome of experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses

Background: Porcine circovirus type 1 (PCV1) has been described as a non-cytopathic contaminant of the PK-15 cell line. Several experimental infections with PCV1 failed to reproduce disease in pigs. Therefore, PCV1 is generally accepted as non-pathogenic to pigs. To our knowledge, nothing is known about the outcome of PCV1 infections in porcine foetuses. This was examined in the present study. Results: Nine foetuses from three sows were inoculated at 55 days of gestation: three with 10(4.3) TCID50 of the PCV1 cell culture strain ATCC-CCL33, three with 10(4.3) TCID50 of the PCV1 field strain 3384 and three with cell culture medium (mock-inoculated). At 21 days post-inoculation, all 6 PCV1-inoculated and all 3 mock-inoculated foetuses had a normal external appearance. Microscopic lesions characterized by severe haemorrhages were observed in the lungs of two foetuses inoculated with CCL33. High PCV1 titres (up to 10(4.7) TCID50/g tissue) were found in the lungs of the CCL33-inoculated foetuses. All other organs of the CCL33-inoculated foetuses and all the organs of the 3384-inoculated foetuses were negative (< 10(1.7) TCID50/g tissue) by virus titration. PCV1-positive cells (up to 121 cells/10 mm(2) in CCL33-inoculated foetuses and up to 13 cells/10 mm(2) in 3384-inoculated foetuses) were found in the heart, lungs, spleen, liver, thymus and tonsils. PCR and DNA sequencing of Rep recovered CCL33 or 3384 sequences from CCL33- or 3384-inoculated foetuses, respectively. Conclusions: From this study, it can be concluded that cell culture PCV1 can replicate efficiently and produce pathology in the lungs of porcine foetuses inoculated at 55 days of foetal life

Genome sequences of two pseudorabies virus strains isolated in Greece

Pseudorabies virus (species Suid herpesvirus 1) belongs to the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae, and is the causative agent of an acute and frequently fatal disease that affects mainly pigs. Here, we report the genome sequences of two strains of this virus isolated in Greece in 2010

Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution

<p>Abstract</p> <p>Background</p> <p>Throughout the history of human influenza pandemics, pigs have been considered the most likely "mixing vessel" for reassortment between human and avian influenza viruses (AIVs). However, the replication efficiencies of influenza viruses from various hosts, as well as the expression of sialic acid (Sia) receptor variants in the entire porcine respiratory tract have never been studied in detail. Therefore, we established porcine nasal, tracheal, bronchial and lung explants, which cover the entire porcine respiratory tract with maximal similarity to the <it>in vivo </it>situation. Subsequently, we assessed virus yields of three porcine, two human and six AIVs in these explants. Since our results on virus replication were in disagreement with the previously reported presence of putative avian virus receptors in the trachea, we additionally studied the distribution of sialic acid receptors by means of lectin histochemistry. Human (Siaα2-6Gal) and avian virus receptors (Siaα2-3Gal) were identified with <it>Sambucus Nigra </it>and <it>Maackia amurensis </it>lectins respectively.</p> <p>Results</p> <p>Compared to swine and human influenza viruses, replication of the AIVs was limited in all cultures but most strikingly in nasal and tracheal explants. Results of virus titrations were confirmed by quantification of infected cells using immunohistochemistry. By lectin histochemistry we found moderate to abundant expression of the human-like virus receptors in all explant systems but minimal binding of the lectins that identify avian-like receptors, especially in the nasal, tracheal and bronchial epithelium.</p> <p>Conclusions</p> <p>The species barrier that restricts the transmission of influenza viruses from one host to another remains preserved in our porcine respiratory explants. Therefore this system offers a valuable alternative to study virus and/or host properties required for adaptation or reassortment of influenza viruses. Our results indicate that, based on the expression of Sia receptors alone, the pig is unlikely to be a more appropriate mixing vessel for influenza viruses than humans. We conclude that too little is known on the exact mechanism and on predisposing factors for reassortment to assess the true role of the pig in the emergence of novel influenza viruses.</p

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 theB646L(p72) gene, nucleotide alignment of the intergenic region (IGR) betweenI73RandI329Lgenes and translation of the central variable region (CVR) coded byB602Lgene. 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 theB646L(p72) gene, the viruses clustered into ASFV p72 genotype II. The viruses characterized in this study lacked a GAATATATAG fragment between theI173Rand theI329Lgenes and were classified as IGR I variants. Furthermore, the tetrameric amino acid repeats within the CVR of theB602Lgene 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

Plasma membrane cholesterol is required for efficient pseudorabies virus entry

AbstractAlphaherpesviruses comprise closely related viruses of man and animal, including herpes simplex virus, varicella-zoster virus and pseudorabies virus (PRV). Here, using methyl-beta-cyclodextrin and fluorescently tagged PRV, we directly show that depletion of cholesterol from the plasma membrane of host cells significantly reduces PRV entry. Cholesterol depletion did not reduce PRV attachment, but stalled virus particles at the plasma membrane before penetration of the cell. Cholesterol depletion results in destabilization of lipid raft microdomains in the plasma membrane, which have been shown before to be involved in efficient entry of different viruses. A significant fraction of PRV virions appears to localize juxtaposed to GM1, a lipid raft marker, during entry. Together, these data indicate that cholesterol and possibly cholesterol-rich lipid rafts may be important during PRV entry

Antigenic differences among porcine circovirus type 2 strains, as demonstrated by the use of monoclonal antibodies

Journal of General Virology 2008, Vol. 89:pp 177–187This study examined whether antigenic differences among porcine circovirus type 2 (PCV-2) strains could be detected using monoclonal antibodies (mAbs). A subtractive immunization protocol was used for the genotype 2 post-weaning multisystemic wasting syndrome (PMWS)-associated PCV-2 strain Stoon-1010. Sixteen stable hybridomas that produced mAbs with an immunoperoxidase monolayer assay (IPMA) titre of 1000 or more to Stoon-1010 were obtained. Staining of recombinant PCV-2 virus-like particles demonstrated that all mAbs were directed against the PCV-2 capsid protein. Cross-reactivity of mAbs was tested by IPMA and neutralization assay for genotype 1 strains 48285, 1206, VC2002 and 1147, and genotype 2 strains 1121 and 1103. Eleven mAbs (9C3, 16G12, 21C12, 38C1, 43E10, 55B1, 63H3, 70A7, 94H8, 103H7 and 114C8) recognized all strains in the IPMA and demonstrated neutralization of Stoon-1010, 48285, 1206 and 1103, but not VC2002, 1147 and 1121. mAbs 31D5, 48B5, 59C6 and 108E8 did not react with genotype 1 strains or had a reduced affinity compared with genotype 2 strains in the IPMA and neutralization assay. mAb 13H4 reacted in the IPMA with PMWS-associated strains Stoon-1010, 48285, 1206 and VC2002, and the porcine dermatitis and nephropathy syndrome-associated strain 1147, but not with reproductive failure-associated strains 1121 and 1103. mAb 13H4 did not neutralize any of the tested strains. It was concluded that, despite the high amino acid identity of the capsid protein (¢91 %), antigenic differences at the capsid protein level are present among PCV-2 strains with a different genetic and clinical background