As stated previously,various blood-sucking insects, such as mosquitoes are known to transmit viral and bacterial infections, as well as stable flies (Stomoxys calcitrans) and horse flies (Tabanidae) can transmit virus via being eaten by pigs during feeding or bites. Therefore, blood-sucking fling insects may play a role in pathogen transmission within farms. However, the flies caught in our study were M. domestica and Drosophila spp., belong to non-blood sucking species. At present, no information exists about transmission of ASFV by these fly species. Additionally, prior studies that have noted the correlation between vector abundance and disease occurrence . Hence, one possible route of ASFV introduction into farms might be via the flies that have traveled or been introduced from infected farms. This mode of transmission in domestic pigs could explain the increasing number of outbreaks observed during the summer months in domestic pigs in China in 2018–2019. It is unlikely that this route is the principal mode of viral transmission within pig herds, but it is possible mechanism for initial entry of the virus into a pig population on a farm.
In this study, another important finding was that we did not detect ASFV genes from Drosophila flies (n = 3), this may be due to the small sample size. Meanwhile, ASFV genes in M. domestica flies were detected using nest PCR not conventional PCR, indicating that the DNA content of ASFV from M. domestica flies is relatively low. Therefore, preliminary speculation was that flies were not biological host of ASFV, since ASFV replication within them is not continuous [11,12,13],when ASF viruses were ingested by M. domestica flies along with decaying food, or were mechanical transmitted by M. domestica flies after released into the environment in pig farm caused by infected pigs. However, this does not mean that ASFV is inactivated from all flies in proximity to pig farms in China. Although it is to be determined whether the ASFV from the flies represents an active infection or a dietary/environmental origin, it is worth considering that M. domestica flies may potentially contaminate pig-feeding sources or accidentally be ingested by pigs via carrying the virus on/in their bodies.
Moreover, discovery of a novel genotype XXIV that was reported for the first time in soft ticks in Mozambique, highlights the diversity of ASFV variants found in the sylvatic cycle . In addition, it has previously been shown that the virus circulating in Sardinia has undergone genetic variation in two genome regions, B602L and EP402R. This variant has rapidly replaced the earliest viruses, perhaps because of some selective advantage, although clinical data suggest that Sardinian ASFV had no changes in virulence . These studies highlight the epidemiological importance of biting vector transmission and the sylvatic cycle in harboring and disseminating new and existing virus strains, and broaden our understanding of the potential ecological and biological drivers affecting ASFV genetic variability present in the environment.
Generally, conserved open reading frames encode structural proteins; transcription factors, such as mammary gland specific nuclear factor (MGF) implicated in interferon type 1 (IFN I) immune response; or proteins involved in nucleotide metabolism, DNA repair, and viral replication, such as A240L [15, 16]. An inner envelope component, the protein p17 (encoded by D117L), is required for the assembly of the capsid layer on the membrane [17, 18]. A179L, the viral Bcl-2 homolog of ASFV, interacts with pro-apoptotic Bcl-2 family proteins to inhibit apoptosis, and affects autophagy by interacting with Beclin1 through its BH3 homology domain . This domain displays major variability with five amino acid substitutions, four of which are in a continuous sequence. The degree of variation significantly differs from the other ASFV genes examined from M. domestica flies. Also, the other differences mainly display in the hydrophilicity of the BH3 binding groove and key conserved structural hallmarks of A179L-BH3 interaction features. The biological significance of these variations is not clear because we have not isolated the virus from these flies due to experiment condition limitation. Therefore, it is not clear whether or not the changes to protein structures interfere with their functions, the antigenic properties of A179L or affecting viral virulence, or more characteristics of non-blood sucking flies in transmission of ASFV.
As previously reported, ASFV A179L also inhibited autophagy by binding Beclin1 in the arthropod hosts [19, 20]. Protein interaction analysis using STING tool revealed that Bcl-2, the A179L homolog, can directly interact with Beclin1 in pig internal environment, whereas more regulatory proteins such as BAD and BID also participate in the regulation of the pig immune system. Concurrently, the results of A179L transfection and fluorescent qPCR verification showed that that over-expression of ASFV A179L from M. domestica flies and Georgia/2007 correlate with transcription levels of autophagy and apoptosis. qPCR results showed that Bcl-2 transcription was higher after 36 h of transfection of A179L obtained from both flies and pigs, compared with those of Bcl-2 at 24 h and 30 h, meanwhile, Beclin-1 transcription of autophagy gene keeping decreased all the time. Numerous large DNA viruses to subvert programmed cell death-based host defense systems typically use structural and functional homologs of Bcl-2. The A179 L protein is expressed in cells in both early and late stages post-infection with ASFV, but is not packaged into virus particles [18, 21]. This suggests that A179 L may be involved in inhibiting apoptosis throughout infection, but not during the earliest stages when viral cores enter the cytoplasm. Furthermore, apoptotic regulation by ASFV A179L from pigs and M. domestica flies need to be studied over time to understand the differences in apoptotic pathways and mechanisms.
We observed an independent cluster in our analysis as a result of variations in the ASFV A179L gene of M. domestica flies. Interestingly, many ASFV genes from M. domestica flies collected in two pig farms have genetic variations similar to the A179L, forming independent branches. Most of them encoded proteins involved in immunosuppression (Unpublished data, personal communication with professor Zeliangchen research team). The biological characteristics of the variations is not clear because live virus isolation is not attempted. Fly sample size and pig farm number with ASFV infection are limited in this study, but the authors propose that DNA variation of non ASFV p72 gene from M. domestica flies gives us a new insight on the potential risks to public health caused by gene recombination of ASFV in the environment, together with genetic variation and transmission of ASFV by non-blood sucking flies, which need to be carefully considered. In the future, further studies including increasing the research of other pathogens harbored by flying insects in farms of negative for ASF, samples size for M. domestica and Drosophila flies as well as other non-blood sucking fly species, especially considering that functional identification of other ASFV proteins derived from flying insects instead of primarily focused on single detection analysis of ASFV p72 gene from environmental samples. Furthermore, our study highlights the need for investigating the potential risk of ASFV harbored by non-blood sucking flies involved in spreading into the local domestic cycle and international value chains of pork product. These factors are important indicators of pathogen transmission by non-blood sucking flying insects.