Isolation and Characterization of Swinepox Virus from Outbreak in Russia.

Swinepox virus (SWPV) is the only member of the Suipoxvirus genus of the Poxviridae family and is an etiologic agent of a worldwide disease specific for domestic and wild pigs. SWPV outbreaks are sporadically recorded in different regions of Russia. In 2013, an outbreak of the disease causing skin lesions was registered on a pig farm in Russia. The presence of SWPV in the scab samples was assessed by in-house real-time PCR, reference PCR amplification, and nucleotide sequencing of the viral late transcription factor-3 (VLTF-3) gene and was then confirmed by virus isolation. Thus, the in-house real-time PCR proposed in this study could serve as a useful tool for the rapid specific detection of the swinepox virus. In the study, it has been demonstrated for the first time that nasal and oral swabs can be used for PCR diagnosis of the disease and for swinepox virus isolation. Phylogenetic analysis revealed that the isolated virus was closely related to SWPV isolates registered in Germany, USA, and Brazil, and slightly differed from the Indian isolates. During experimental infection of pigs, a low pathogenicity of the Russian isolate was observed. Our data provides the first report on the isolation and characterization of swinepox virus in Russia.

Seroimmunotyping of African swine fever virus.

The extreme genetic and immunobiological heterogeneity exhibited by the African swine fever virus (ASFV) has been a significant impediment in the development of an efficacious vaccine against this disease. Consequently, the lack of internationally accepted protocols for the laboratory evaluation of candidate vaccines has become a major concern within the scientific community. The formulation of such protocols necessitates the establishment of a consensus at the international level on methods for the determination of homologous and heterologous isolates/strains of ASFV. The present article provides a comprehensive description of biological techniques employed in the classification of ASFV by seroimmunotypes. These techniques involve a holistic evaluation of ASFV isolates/strains based on their antigenic properties as determined by the hemadsorption inhibiting test (HAdI) using type-specific sera and an immunological test (IT) conducted on pigs inoculated with attenuated strains. The article outlines the methods for setting up the HAdI test, an IT on pigs, and the processes involved in the acquisition of type-specific serums for the HAdI test. It is pertinent to note that the definitive classification of seroimmunotype can only be ascertained after conducting an IT on pigs. The findings from the HAdI test or the phylogenetic analysis of the EP402R gene should be considered preliminary in nature.

Deletion of the CD2 Gene in the Virulent ASFV Congo Strain Affects Viremia in Domestic Swine, but Not the Virulence.

African swine fever (ASF) is an infectious disease that causes the most significant losses to the pig industry. One of the effective methods for combating this disease could be the development of vaccines. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. Deletion of the EP402R gene encoding a CD2-like protein led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. Here we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, in vivo, animals infected with ΔCongo-v_CD2v had a delay in the onset of the disease and viremia compared to animals infected with the parental strain. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background.

Comparison of Attenuated and Virulent Strains of African Swine Fever Virus Genotype I and Serogroup 2.

African swine fever (ASF) is a contagious disease of pigs caused by the ASF virus (ASFV). The main problem in the field of ASF control is the lack of vaccines. Attempts to obtain vaccines by attenuating the ASFV on cultured cell lines led to the production of attenuated viruses, some of which provided protection against infection with a homologous virus. Here we report on the biological and genomic features of the attenuated Congo-a (KK262) virus compared to its virulent homologue Congo-v (K49). Our results showed differences in in vivo replication and virulence of Congo-a. However, the attenuation of the K49 virus did not affect its ability to replicate in vitro in the primary culture of pig macrophages. Complete genome sequencing of the attenuated KK262 strain revealed an 8,8 kb deletion in the left variable region of the genome compared to the virulent homologue K49. This deletion concerned five genes of MGF360 and three genes of MGF505. In addition, three inserts in the B602L gene, genetic changes in intergenic regions and missense mutations in eight genes were detected. The data obtained contribute to a better understanding of ASFV attenuation and identification of potential virulence genes for further development of effective vaccines.

Analysis of gene expression in monocytes of immunized pigs after infection with homologous or heterologous African swine fever virus.

African swine fever is a deadly disease of pigs caused by the large DNA virus (ASFV). Despite intensive research, little is known about the molecular mechanisms of ASFV pathogenesis. Transcriptome analysis of host and viral genes in infected macrophages revealed changes in expression of genes involved in various biological processes, including immune response, inflammatory response and apoptosis. To understand the mechanisms of virus pathogenesis, we used transcriptome analysis to identify the differences in gene expression between peripheral blood monocytes (PBMCs) isolated from pigs immunized with attenuated Congo ASFV strain (KK262), and then infected in vitro with virulent homologous Congo strain (K49) or heterologous Mozambique strain (M78). We found that overexpression of IFN-γ was detected only in cells infected with M78, although the expression of interferon-stimulated genes was increased in both types of cells. In addition, up-regulation of pro-inflammatory cytokines and chemokines was found in PBMCs infected with the heterologous strain M78, in contrast to the cells infected with K49. These data may indicate the beginning of an early immune response in cells infected with a heterologous, but not homologous strain. Transcriptome analysis revealed down-regulation of genes involved in endocytosis and phagocytosis in cells infected with the K49 strain, but not in PBMCs infected with M78. On the contrary, we detected activation of endoplasmic reticulum stress response genes in cells infected with a homologous strain, but not in cells infected with a heterologous strain. This study is the first attempt to determine the differences in the response to ASF infection between homologous and heterologous strains at the cellular level. Our results showed that not only genes of the immune response, but also genes involved in endocytosis and cellular stress response may be important for the formation of cross-protective immunity. This data may be useful for vaccine development or testing of candidate vaccines.

Complete genome sequence of virulent genotype I African swine fever virus strain K49 from the Democratic Republic of the Congo, isolated from a domestic pig (Sus scrofa domesticus).

African swine fever is one of the most feared infectious diseases in the pig industry. African swine fever virus (ASFV) is an enveloped, cytoplasmic double-stranded DNA virus and the only member of the family Asfarviridae. Although ASFV is known to have been circulating on the African continent since at least 1921, little is known about the genetic characteristics of historical ASFV strains isolated in sub-Saharan Africa. The few complete ASFV genome sequences obtained from African historical isolates have demonstrated genetic diversity, but the available data are limited and insufficient for fully understanding the molecular evolution and continental spread of ASFV. Here, we report the complete genome sequence of the virulent ASFV strain K49, collected during an outbreak in the Belgian Congo (now the Democratic Republic of the Congo) in 1949. The complete genome sequence of ASFV strain K49 was determined using an Illumina HiSeq platform and is 189,523 bp in length with a mean GC content of 38.43%, with 189 genes annotated. This is the first reported complete genome sequence of an ASFV serogroup 2 isolate. Phylogenetic analysis demonstrated genetic divergence within genotype I, and strain K49 formed a separate branch from other ASFV genotype I isolates.

Immunobiological Characteristics of the Attenuated African Swine Fever Virus Strain Katanga-350.

The African swine fever virus (ASFV) is the cause of a recent pandemic that is threatening the global pig industry. The virus infects domestic and wild pigs and manifests with a variety of clinical symptoms, depending on the strain. No commercial vaccine is currently available to protect animals from this virus, but some attenuated and recombinant live vaccine candidates might be effective against the disease. This article describes the immunobiological characteristics of one such candidate-the laboratory-attenuated ASFV strain, Katanga-350-which belongs to genotype I. In this study, we assessed clinical signs and post-mortem changes, the levels of viremia and the presence of viral DNA caused by injection of ASF virus strains Katanga-350, Lisbon-57, and Stavropol 08/01. Intramuscular injection of this strain protected 80% of pigs from a virulent strain of the same genotype and seroimmunotype (Lisbon-57). At least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous (genotype II, seroimmunotype VIII) virulent strain (Stavropol 08/01). Virus-specific antibodies were detectable in serum and saliva samples between 8-78 days after the first inoculation of the Katanga-350 strain (the observational period). The results suggested that this strain could serve as a basis for the development of a recombinant vaccine against ASF viruses belonging to seroimmunotype I.

Growth Kinetics and Protective Efficacy of Attenuated ASFV Strain Congo with Deletion of the EP402 Gene.

African swine fever (ASF) is an emerging disease threat to the swine industry worldwide. There is no vaccine against ASF, and progress is hindered by a lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. We have previously demonstrated that homologous ASFV serotype-specific proteins CD2v (EP402R) and/or C-type lectin are required for protection against challenge with the virulent ASFV strain Congo (Genotype I, Serogroup 2), and we have identified T-cell epitopes on CD2v which may be associated with serotype-specific protection. Here, using a cell-culture adapted derivative of the ASFV strain Congo (Congo-a) with specific deletion of the EP402R gene (ΔCongoCD2v) in swine vaccination/challenge experiments, we demonstrated that deletion of the EP402R gene results in the failure of ΔCongoCD2v to induce protection against challenge with the virulent strain Congo (Congo-v). While ΔCongoCD2v growth kinetics in COS-1 cells and primary swine macrophage culture were almost identical to parental Congo-a, replication of ΔCongoCD2v in vivo was significantly reduced compared with parental Congo-a. Our data support the idea that the CD2v protein is important for the ability of homologous live-attenuated vaccines to induce protective immunity against the ASFV strain Congo challenge in vivo.

Identification and Characterization of Bluetongue Virus Serotype 14 in Russia.

This paper reports a case of bluetongue virus (BTV) infection in the Smolensk and Kaluga regions of Russia in 2011-2012. The virus was initially detected in heifers transferred in Russia from Germany through Poland and Belarus in 2011. On day 27 of quarantine, RNA and infectious viruses of BTV were detected in four heifers, but five were serologically positive. However, on day 3 before shipment, all heifers were seronegative and PCR-negative for BTV. Thus, a few animals from this consignment were viremic without any evident subclinical infection. Based on Seg-2 (VP2 gene) and Seg-5 (NS1 gene) sequencing, the recovered virus had 99.86-100% nucleotide identity with BTV-14-like viruses such as the vaccine BTV-14 strain RSArrrr/BTV 14 and the BTV-14 isolates detected in Lithuania and Poland in 2012. Subsequently, BTV-14 was also reported in local animals in two regions of Russia. During the monitoring survey, 1623 local animals within a 300-km radius were tested, of which 471 tested positive by ELISA and 183 by PCR for BTV-14 RNA. No other serotypes were identified in either imported or aboriginal animals within that radius. The Culicoides midges trapped at the site of the outbreak in May 2012 tested positive for the BTV-14 genome, indicating that the possible mechanism of spread most likely occurs via vector bites. However, further investigation is required to confirm this hypothesis, which would provide an improved understanding of the circulation and overwintering of BTV in northern latitudes.