An Update on African Swine Fever Virology.

Animal diseases constitute a continuing threat to animal health, food safety, national economy, and the environment. Among those, African swine fever (ASF) is one of the most devastating viruses affecting pigs and wild suids due to the lack of vaccine or effective treatment. ASF is endemic in countries in sub-Saharan Africa, but since its introduction to the Caucasus region in 2007, a highly virulent strain of ASF virus (ASFV) has continued to circulate and spread into Eastern Europe and Russia, and most recently into Western Europe, China, and various countries of Southeast Asia. Given the importance of this disease, this review will highlight recent discoveries in basic virology with special focus on proteomic analysis, replication cycle, and some recent data on genes involved in cycle progression and viral-host interactions, such as I215L (E2 ubiquitin-conjugating enzyme), EP402R (CD2v), A104R (histone-like protein), QP509L, and Q706L (RNA helicases) or P1192R (Topoisomerase II). Taking into consideration the large DNA genome of ASFV and its complex interactions with the host, more studies and new approaches are to be taken to understand the basic virus-host interaction for ASFV. Proteomic studies are just paving the way for future research.

Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line-A Potential Methodology for the Production of a Vaccine Candidate.

African swine fever (ASF) is a fatal viral disease of domestic swine and wild boar, considered one of the main threats for global pig husbandry. Despite enormous efforts, to date, neither the classical vaccine formulations nor the use of protein subunits proved to be efficient to prevent this disease. Under this scenario, new strategies have been proposed including the development of disabled infectious single cycle (DISC) or replication-defective mutants as potential immunizing agents against the ASF virus (ASFV). In this study, we describe the methodology to generate an ASFV-DISC mutant by homologous recombination, lacking the A104R gene, which was replaced by the selection marker (GUS gene). The recombinant viruses were identified when the infected cells acquired a blue color in the presence of X-Gluc (100 µg/mL), which is the substrate for the GUS gene. Since these viral particles result from loss-of-function mutations, being unable to replicate, helper-cell lines expressing the viral pA104R protein were produced. Vero and COS-1 cell lines were transfected by different methods, both physical and chemical, in order to stably express the ASFV-pA104R. Best results were obtained by using Lipofectamine 2000 and Nucleofection methodology of Vero with the pIRESneo vector and by using Flp-FRT site-directed recombination technology system in Flp-In CV-1 cells (transformed COS-1 cells with a single integration site in a transcriptional active region). In order to ensure an efficient and stable integration of the viral ORF on the host cellular genome, the maintenance of the insert was verified by PCR and its expression by immunofluorescence and immunoblot analysis. Although the isolation of the recombinant virus was not achieved, the confirmation of ASFV-ΔA104R sequence, and the detection of the recombinant mutant through three passages, suggest that this approach is feasible and could be a potential strategy to generate safe and efficient DISC vaccine candidates.

African swine fever virus replication events and cell nucleus: New insights and perspectives.

African swine fever (ASF) is currently matter for major concerns in global swine industry as it is highly contagious and causes acute fatal haemorrhagic fever in domestic pigs and wild boar. The absence of effective vaccines and treatments pushes ASF control to relay on strict sanitary and stamping out measures with costly socio-economic impacts. The current epidemic scenario of fast spreading throughout Asiatic countries impels further studies on prevention and combat strategies against ASF. Herein we review knowledge on African Swine Fever Virus (ASFV) interactions with the host cell nucleus and on the functional properties of different viral DNA-replication related proteins. This entails, the confirmation of an intranuclear viral DNA replication phase, the characterization of cellular DNA damage responses (DDR), the subnuclear compartments disruption due to viral modulation, and the unravelling of the biological role of several viral proteins (A104R, I215 L, P1192R, QP509 L and Q706 L), so to contribute to underpin rational strategies for vaccine candidates development.

The QP509L and Q706L superfamily II RNA helicases of African swine fever virus are required for viral replication, having non-redundant activities.

African swine fever virus is complex DNA virus that infects pigs with mortality rates up to 100% leading to devastating socioeconomic effected in the affected countries. There is neither a vaccine nor a treatment to control ASF. African swine fever virus genome encodes two putative SF2 RNA helicases (QP509L and Q706L). In the present study, we found that these two RNA helicases do not share a common ancestral besides sharing a sequence overlap. Although, our phylogenetic studies revealed that they are conserved among virulent and non-virulent isolates, it was possible to observe a degree of variation between isolates corresponding to different genotypes occurring in distinct geographic regions. Further experiments showed that QP509L and Q706L are actively transcribed from 4 h post infection. The immunoblot analysis revealed that both protein co-localized in the viral factories at 12 h post infection, however, QP509L was also detected in the cell nucleus. Finally, siRNA assays uncover the relevant role of these proteins during viral cycle progression, in particular, for the late transcription, genome replication, and viral progeny (a reduction of infectious particles up to 99.4% when siRNA against QP509L was used and 98.4% for siRNA against Q706L). Thus, our results suggest that both helicases are essential during viral infection, highlighting the potential use of these enzymes as target for drug and vaccine development against African swine fever.

African swine fever virus encodes for an E2-ubiquitin conjugating enzyme that is mono- and di-ubiquitinated and required for viral replication cycle.

African swine fever virus is the etiological agent of a contagious and fatal acute haemorrhagic viral disease for which there are no vaccines or therapeutic options. The ASFV encodes for a putative E2 ubiquitin conjugating enzyme (ORF I215L) that shows sequence homology with eukaryotic counterparts. In the present study, we showed that pI215L acts as an E2-ubiquitin like enzyme in a large range of pH values and temperatures, after short incubation times. Further experiments revealed that pI215L is polyubiquitinated instead of multi-mono-ubiquitinated and Cys85 residue plays an essential role in the transthioesterification reaction. In infected cells, I215L gene is transcribed from 2 hours post infection and immunoblot analysis confirmed that pI215L is expressed from 4 hpi. Immunofluorescence studies revealed that pI215L is recruited to viral factories from 8 hpi and a diffuse distribution pattern throughout the nucleus and cytoplasm. siRNA studies suggested that pI215L plays a critical role in the transcription of late viral genes and viral DNA replication. Altogether, our results emphasize the potential use of this enzyme as target for drug and vaccine development against ASF.

Sodium phenylbutyrate abrogates African swine fever virus replication by disrupting the virus-induced hypoacetylation status of histone H3K9/K14.

African swine fever virus (ASFV) causes a highly lethal disease in swine for which neither a vaccine nor treatment are available. Recently, a new class of drugs that inhibit histone deacetylases enzymes (HDACs) has received an increasing interest as antiviral agents. Considering studies by others showing that valproic acid, an HDAC inhibitor (HDACi), blocks the replication of enveloped viruses and that ASFV regulates the epigenetic status of the host cell by promoting heterochromatinization and recruitment of class I HDACs to viral cytoplasmic factories, the antiviral activity of four HDACi against ASFV was evaluated in this study. Results showed that the sodium phenylbutyrate fully abrogates the ASFV replication, whereas the valproic acid leads to a significant reduction of viral progeny at 48h post-infection (-73.9%, p=0.046), as the two pan-HDAC inhibitors tested (Trichostatin A: -82.2%, p=0.043; Vorinostat: 73.9%, p=0.043). Further evaluation showed that protective effects of NaPB are dose-dependent, interfering with the expression of late viral genes and reversing the ASFV-induced histone H3 lysine 9 and 14 (H3K9K14) hypoacetylation status, compatible to an open chromatin state and possibly enabling the expression of host genes non-beneficial to infection progression. Additionally, a synergic antiviral effect was detected when NaPB is combined with an ASFV-topoisomerase II poison (Enrofloxacin). Altogether, our results strongly suggest that cellular HDACs are involved in the establishment of ASFV infection and emphasize that further in vivo studies are needed to better understand the antiviral activity of HDAC inhibitors.

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription.

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (-78.3%), and reduced transcription of a late viral gene (-47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV.IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication.

African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro. Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (-66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF.

In vitro antiviral activity of fluoroquinolones against African swine fever virus.

African swine fever (ASF) is a viral swine disease against which neither an effective vaccine nor a treatment is available. The antiviral effect of thirty fluoroquinolones on the infectivity of African swine fever virus (ASFV) was screened in vitro. There was a severe reduction of the cytopathic effect in ASFV-infected Vero cells when exposed to six independent fluoroquinolones, or to some of their combinations, from an early phase of infection. Moreover, after 7-day treatments, ASFV genome could not be detected by PCR, and the culture supernatants were unable to infect new cell cultures. Pulsed field gel electrophoresis (PFGE) analysis revealed a diminished viral DNA replication without identifiable genome fragmentation in cells exposed to fluoroquinolones. In parallel, altered patterns of viral protein synthesis were observed from early infection. The overall results indicate that bacterial topoisomerase inhibitors interfere with the ASFV replication cycle probably by targeting a putative ASFV-topoisomerase II, opening a new window for antiviral treatments.

Novel multiregion hybridization assay for the identification of the most prevalent genetic forms of the human immunodeficiency virus type 1 circulating in Portugal.

The most efficient method for HIV-1 genetic characterization involves full-genome sequencing, but the associated costs, technical features, and low throughput preclude it from being routinely used for the analysis of large numbers of viral strains. Multiregion hybridization assays (MHA) represent an alternative for a consistent genetic analysis of large numbers of viral strains. Classically, MHA rely on the amplification by real-time PCR of several regions scattered along the HIV-1 genome, and on their characterization with clade-specific TaqMan probes (also known as hydrolysis probes). In this context, the aim of our study was the development of a technical variant of an MHA (vMHA(B/G/02)) for genotyping the most prevalent genetic forms of HIV-1 circulating in Portugal. Different sets of primers were designed for universal and clade-specific amplifications of several sections of the viral genome: gag, pol(Pr), pol(RT), vpu, env(gp120), and env(gp41). vMHA(B/G/02) was implemented using a real-time PCR-based approach, with detection dependent on the use of SYBR Green I. As an alternative, a technically less demanding strategy based on conventional PCR and agarose gel analysis of the reaction products was also developed. This method performed with overall good sensitivity and specificity (>91%) when a convenience sample of 45 plasma-derived HIV-1 strains was analyzed. Apart from the detection of subtype B, G, CRF02_AG, and CRF14_BG viruses, several unique B/G recombinant were also detected. Curiously, recombinant viruses including CRF02_AG sequences were not detected in the group of samples analyzed.

Species Composition and WNV Screening of Mosquitoes from Lagoons in a Wetland Area of the Algarve, Portugal.

The aim of this study was to evaluate mosquito abundance, species diversity, larval and adult population dynamics in seven lagoons integrated in the wetland coastal system of the Algarve, Portugal, in the summer of 2007, as well as the screening of these for West Nile virus (WNV). WNV has been isolated from mosquitoes in this region, in the summer of 2004, next to the putative area of infection of two linked human WN cases. Adult mosquitoes were collected with CDC traps baited with CO(2), and potential breeding sites were surveyed for immature stages. Morphological identification of 1,432 adult mosquitoes and 85 larvae revealed the presence of 10 species: Anopheles atroparvus, Anopheles algeriensis, Coquillettidia richiardii, Culex modestus, Culex pipiens, Culex theileri, Culex univittatus, Culiseta longiareolata, Aedes caspius, and Aedes detritus. Adult mosquito peak densities were recorded in July, contrasting with null larval breeding in the same month in the surveyed biotopes. Most abundant species were C. pipiens (52%), C. theileri (29%), and A. caspius (11%). Lagoon Salgados and Quinta das Salinas, exhibited the highest similarity of culicid fauna, despite being most distant from each other, Female mosquitoes (1,249 specimens) screened by RT-PCR, did not reveal WNV products. However, previous detection of WNV activity in this area, susceptible to re-introductions, demands for continued vigilance.

Mosquito surveys and West Nile virus screening in two different areas of southern Portugal, 2004-2007.

Longitudinal mosquito surveys were carried out in southern Portugal from 2004 to 2007, in a wetland area (Comporta, District of Setúbal) and around the perimeter of a dam irrigation plant that created the largest artificial lake in Europe, 250 km(2) (Alqueva, Districts of Evora and Beja). Our aim was to study the diversity, abundance, and seasonal dynamics of mosquitoes, comparing these two different areas, to screen mosquitoes for West Nile Virus (WNV), an arboviral agent already detected in Portugal, because these areas are populated with abundant avian fauna. Monthly collections of adult mosquitoes were carried out by Centers for Disease Control light-traps with CO(2) and by indoor resting collections. Mosquitoes were identified and screened for arboviruses by reverse transcriptase (RT)-polymerase chain reaction directed toward amplification of a 217-bp fragment of the NS5 gene. Mosquito peak densities were observed in July-August in Comporta and May-June, with a plateau in July-October, in Alqueva. However, densities were far higher in Comporta area (220,821 specimens) than in Alqueva area (9442 specimens), with a clear difference in species distribution, as in Comporta the predominant species was Culex theileri (85%), followed by Aedes caspius (6%), Anopheles atroparvus (4%), and Culex pipiens sensu latu (s.l.) (3%), whereas in Alqueva the predominant species was Cx. pipiens s.l. (56%), followed by An. atroparvus (18%), Cx. theileri (14%), and Culiseta longiareolata (9%). Female mosquitoes (8842 in 175 pools) of the species Ae. caspius, An. atroparvus, Culex mimeticus, Cx. pipiens Sensu latu (s.l.), Cx. theileri, and Culex univittatus were screened and found to be negative for WNV genomic RNA. Although there was no detection of WNV sequences in mosquitoes, vigilance should continue as the circulation of virus has been previously detected more than once in Portugal, in humans, animals, and mosquitoes, and in other surrounding Mediterranean countries.

Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal.

BACKGROUND: Culex pipiens L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground vs. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise Cx. pipiens collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group. RESULTS: Autogeny and stenogamy was evaluated in the F1 progeny of 145 individual Cx. pipiens females. Bayesian clustering analysis based on the genotypes of 13 microsatellites revealed two distinct genetic clusters that were highly correlated with the alternative traits that define pipiens and molestus. Admixture analysis yielded hybrid rate estimates of 8-10%. Higher proportions of admixture were observed in pipiens individuals suggesting that more molestus genes are being introgressed into the pipiens form than the opposite. CONCLUSION: Both physiological/behavioural and genetic data provide evidence for the sympatric occurrence of molestus and pipiens forms of Cx. pipiens in the study area. In spite of the significant genetic differentiation between forms, hybridisation occurs at considerable levels. The observed pattern of asymmetric introgression probably relates to the different mating strategies adopted by each form. Furthermore, the differential introgression of molestus genes into the pipiens form may induce a more opportunistic biting behaviour in the latter thus potentiating its capacity to act as a bridge-vector for the transmission of arboviral infections.