Development of optimized protocol for culturing African swine fever virus field isolates in MA104 cells.

The goal of this study was to identify a candidate commercial cell line for the replication of African swine fever virus (ASFV) by comparing several available cell lines with various medium factors. In the sensitivity test of cells, MA104 and MARC-145 had strong potential for ASFV replication. Next, MA104 cells were used to compare the adaptation of ASFV obtained from tissue homogenates and blood samples in various infectious media. At the 10th passage, the ASFV obtained from the blood sample had a significantly higher viral load than that obtained from the tissue sample (P = 0.000), exhibiting a mean cycle threshold (Ct) value = 20.39 ± 1.99 compared with 25.36 ± 2.11. For blood samples, ASFV grew on infectious medium B more robustly than on infectious medium A (P = 0.006), corresponding to a Ct value = 19.58 ± 2.10 versus 21.20 ± 1.47. African swine fever virus originating from blood specimens continued to multiply gradually and peaked in the 15th passage, exhibiting a Ct value = 14.36 ± 0.22 in infectious medium B and a Ct value = 15.42 ± 0.14 in infectious medium A. When ASFV was cultured from tissue homogenates, however, there was no difference (P = 0.062) in ASFV growth between infectious media A and B. A model was developed to enhance ASFV replication through adaptation to MA104 cells. The lack of mutation at the genetic segments encoding p72, p54, p30, and the central hypervariable region (CVR) in serial culture passages is important in increasing the probability of maintaining immunogenicity when developing a vaccine candidate.

L'objectif de cette étude était d'identifier une lignée cellulaire commerciale candidate pour la réplication du virus de la peste porcine africaine (ASFV) en comparant plusieurs lignées cellulaires disponibles et différents milieux. Lors du test de sensibilité des cellules, MA104 et MARC-145 présentaient un fort potentiel pour la réplication d'AFSV. Par la suite, les cellules MA104 ont été utilisées pour comparer l'adaptation d'ASFV obtenu d'homogénats de tissus et d'échantillons de sang dans différents milieux. Au dixième passage, l'ASFV obtenu de l'échantillon de sang avait une charge virale significativement plus élevée que celle obtenue de l'échantillon de tissu (P = 0,000), avec une valeur seuil moyenne de cycles (Ct) de 20,39 ± 1,99 comparativement à 25,36 ± 2,11. Pour les échantillons sanguins, l'ASFV a poussé sur le milieu B de manière plus robuste que sur le milieu A (P = 0,006), ce qui correspond à une valeur Ct de 19,58 ± 2,10 versus 21,20 ± 1,47. L'ASFV provenant des échantillons sanguins continua de se multiplier graduellement et atteignit un pic au 15e passage, avec une valeur Ct de 14,36 ± 0,22 dans le milieu B et une valeur Ct de 15,42 ± 0,14 dans le milieu A. Toutefois, lorsque l'ASFV fut cultivé à partir des homogénats de tissus, il n'y avait pas de différence (P = 0,062) dans la croissance d'ASFV entre les milieux A et B. Un modèle a été développé pour augmenter la réplication d'ASFV par adaptation aux cellules MA104. L'absence de mutation au segment génétique codant pour p72, p54, p30, et la région hypervariable centrale (CVR) dans des passages en série en culture est importante en augmentant la probabilité de maintenir une immunogénicité lors du développement d'un vaccin candidat.(Traduit par Docteur Serge Messier).

The Isolation and Replication of African Swine Fever Virus in Primary Renal-Derived Swine Macrophages.

African swine fever virus (ASFV) causes hemorrhagic disease in domestic pigs by replicating mainly in monocyte/macrophage lineages. Various primary cells including pulmonary alveolar macrophages have been used for the propagation of ASFV on this account. However, ethical constraints and consistency problems exist as it is necessary to harvest same phenotype of primary cells in order to continue a study. We suggested renal-derived swine macrophages as a novel primary cell candidate to address these issues. These primary cells proved to be permissive to both cell adapted ASFV and a wild-type ASFV. Compared to the commercial cell line MA-104, the renal-derived macrophages were more suitable to isolate the field virus. The consistent molecular characteristics of the renal-derived macrophages were demonstrated by immunocytochemistry with antibodies against macrophage cell surface markers including CD163, CD172a, and Iba-1. Viral protein p30 and p72 expression in ASFV infected macrophages was confirmed by immunocytochemistry by use of specific monoclonal antibodies. We observed increase of cell-free viral DNA and infectious virus titer in infected cell supernatant in successive days-post-infection. These results demonstrated that primary renal-derived swine macrophages are useful for ASFV isolation and propagation in terms of cell phenotypes, susceptibility to the virus, and virus production.

Influenza A Viruses of Swine (IAV-S) in Vietnam from 2010 to 2015: Multiple Introductions of A(H1N1)pdm09 Viruses into the Pig Population and Diversifying Genetic Constellations of Enzootic IAV-S.

Active surveillance of influenza A viruses of swine (IAV-S) involving 262 farms and 10 slaughterhouses in seven provinces in northern and southern Vietnam from 2010 to 2015 yielded 388 isolates from 32 farms; these viruses were classified into H1N1, H1N2, and H3N2 subtypes. Whole-genome sequencing followed by phylogenetic analysis revealed that the isolates represented 15 genotypes, according to the genetic constellation of the eight segments. All of the H1N1 viruses were entirely A(H1N1)pdm09 viruses, whereas all of the H1N2 and H3N2 viruses were reassortants among 5 distinct ancestral viruses: H1 and H3 triple-reassortant (TR) IAV-S that originated from North American pre-2009 human seasonal H1, human seasonal H3N2, and A(H1N1)pdm09 viruses. Notably, 93% of the reassortant IAV-S retained M genes that were derived from A(H1N1)pdm09, suggesting some advantage in terms of their host adaptation. Bayesian Markov chain Monte Carlo analysis revealed that multiple introductions of A(H1N1)pdm09 and TR IAV-S into the Vietnamese pig population have driven the genetic diversity of currently circulating Vietnamese IAV-S. In addition, our results indicate that a reassortant IAV-S with human-like H3 and N2 genes and an A(H1N1)pdm09 origin M gene likely caused a human case in Ho Chi Minh City in 2010. Our current findings indicate that human-to-pig transmission as well as cocirculation of different IAV-S have contributed to diversifying the gene constellations of IAV-S in Vietnam. IMPORTANCE: This comprehensive genetic characterization of 388 influenza A viruses of swine (IAV-S) isolated through active surveillance of Vietnamese pig farms from 2010 through 2015 provides molecular epidemiological insight into the genetic diversification of IAV-S in Vietnam after the emergence of A(H1N1)pdm09 viruses. Multiple reassortments among A(H1N1)pdm09 viruses and enzootic IAV-S yielded 14 genotypes, 9 of which carried novel gene combinations. The reassortants that carried M genes derived from A(H1N1)pdm09 viruses became predominant, replacing those of the IAV-S that had been endemic in Vietnam since 2011. Notably, one of the novel reassortants likely caused a human case in Vietnam. Given that Vietnam is the second-largest pig-producing country in Asia, continued monitoring of IAV-S is highly important from the viewpoints of both the swine industry and human public health.