Preliminary Analysis of African Swine Fever Virus Carriage and Transmission by Mosquitoes

Background Mosquitoes are important insect vectors, but whether they can carry and transmit African swine fever virus (ASFV) in large-scale pig farms in China is unknown. Results In this study, probe qPCR analysis was performed on mosquitoes from ve pig farms with ASF virus (ASFV). Analysis of ASFV in 463 mosquitoes yielded negative cycle threshold (CT) value), and detection remained negative after mixing samples from all ve pig farms. Conclusions Therefore,


Introduction
African swine fever (ASF) causes great economic losses to the pig industry due to its rapid spread, infectiousness, and high mortality. ASF was rst reported in 1921 in domestic pigs in Kenya, and it had spread to Europe in 1957, followed by Ukraine, Lithuania, Latvia, Russia, and various other countries between 2012 and 2017. Since August 2018, ASF has been prevalent in China, causing serious economic losses to the swine industry [1]. At present, no safe and effective vaccine for ASF has been successfully developed, and infection sources and transmission routes of ASF virus (ASFV) are many and complex due to its resistance to the external environment. It remains infectious for a long time in a variety of porcine products, arthropods, and contaminated environments, greatly increasing the di culty of its prevention and control [2]. Therefore, timely detection, containment of sources of infection, and blockage of transmission are important for preventing the spread of ASF. Soft ticks are the main vectors and reservoir hosts of ASFV, and ASF is mainly circularly transmitted in three ways; pig-pig, pig-soft tick, and pig-soft tick-wild boar contacts [3]. ASFV replicates in soft ticks that become a reservoir of the virus, causing infection by biting pigs or by feeding. Soft ticks facilitate viral replication, remain infected up to the fourth week after feeding, and can still infect pigs after 469 days of infection. However, although soft ticks is a potential source of ASFV infection, there is currently no evidence of a direct relationship between soft ticks and the transmission of ASFV in Europe [4,5]. Additionally, although soft ticks are distributed throughout China, they have great geographical environmental limitations compared with other sources of vector-borne transmissions such as mosquitoes.
As the climate in southern China gets hotter and the season for active mosquitoes arrives, mosquitoes may carry and transmit ASFV in pig farms, but this has not been proven. Mosquitoes are known to transmit as many as 80 diseases through blood feeding, including Japanese encephalitis virus, West Nile virus, dengue virus, Zika virus, or yellow fever virus [6]. The occurrence of ASF is highly seasonal in Europe, and mainly endemic from July to September, which may be related to the frequent activity of blood-feeding insects (e.g., ies, mosquitoes) in summer [7]. One research group captured 15 different species of insects (including house ies and mosquitoes) and identi ed nucleic acids from house ies and mosquitoes in an epidemiological survey following an ASF outbreak [8]. These studies indicated the possibility of mosquito transmission of ASFV, but there are no relevant reports in China. Therefore, in this study, we measured CT values for ASFV by probe qPCR in mosquitoes captured from ve large-scale pig farms with ASF, or suspected of having ASF, in Jiangsu Province, China. We investigated the possible mosquito carriage and transmission of ASFV, and the potential threat to large-scale pig farms. The results could inform the development of strategies for the prevention and control of ASF in pig farms.

Mosquito sample collection
A total of 463 mosquitoes were captured from pigpens in ve large-scale pig farms with ASF, in Jiangsu Province (Table 1). 50−100 mosquitoes were captured using mosquitoes trap cage from each pig farm, the mosquitoes were identi ed by stereo microscope. After a week of collection, all mosquitoes were cleaned using 75% ethanol for 5 min, rinsed twice in sterile phosphate-buffered saline (PBS), and immediately placed in liquid nitrogen for storage. (50 pmol/µL) 5'-GATACCACAAGATC (A/G) GCCGT-3', probe 5'-CCACGGGAGGAATACCAACCCAGTG-3' (5 pmol/µL). after which the remaining mosquitoes were mixed prior to DNA extraction. Samples were considered positive with a CT value ≤ 35, potentially positive with a CT value between 35 and 40, and negative with no CT value. For potentially positive samples the ampli cation curve was inspected, and if logarithmic ampli cation was observed samples were presumed positive, but they were presumed negative if ampli cation was not logarithmic. DNA was re-extracted and probe qPCR assays were performed on these suspicious samples, and if the repeated ampli cation curves showed logarithmic ampli cation, samples were considered positive sample, but they were considered negative if ampli cation was not logarithmic. The positive control was standard ASFV strain, and the negative control was sterilized distilled water without DNA.

Identi cation of mosquito species
In this study, mosquito species were determined by stereomicroscopy, and the results revealed most of them to be Culex pipiens pallens (Fig. 1), there were only a few Anopheles and Aedes mosquitoes (3 Anopheles, 5 Aedes) in accordance with the distribution of common mosquitoes in Jiangsu Province [9]. In the following study, we discarded Anopheles and Aedes mosquitoes due to the small numbers.

ASFV detection by probe qPCR
probe qPCR was performed on 463 mosquitoes from ve pig farms, CT value of positive control was 21.54, while the mosquitoes in all ve farms gave negative results (no CT values; Fig. 2A). This may be because a single mosquito carrying a small amount of virus could not be detected. Thus, the remaining mosquitoes from the same pig farm were mixed and DNA was extracted, but the results remained negative (no CT values; Fig. 2B).

Discussion
ASFV, a complex, cytoplasmic double-stranded DNA virus 175−215 nm in diameter, is the only DNA virus that uses arthropods as biological vectors [10]. At present, our understanding of ASFV is not comprehensive, and many aspects including virulence, pathogenesis and transmission mechanism remain poorly understood. The development of safe and effective ASVF vaccines may take a long time, hence understanding potential vectors is essential for preventing epidemic outbreaks and detecting potential epidemics. Thus, it is necessary to investigate the possibility of mosquitoes as ASFV carriers and vectors.
In this study, ve pig farms in Jiangsu Province with ASF, or suspected of having ASF, were selected, 463 mosquitoes were captured, and they were con rmed to be Culex pipiens pallens by stereomicroscopic observation. A previous study counted the constituent ratio of mosquitoes in Jiangsu Province from April to November and found that Culex pipiens pallens was the dominant species, with indoor captures accounting for 75.07−92.86% of all mosquitoes [9], consistent with the results of the present study.
A number of criteria are usually met when judging whether or not mosquitoes are virus vectors; (1) viruses can be isolated from mosquitoes; (2) under experimental conditions, mosquitoes can be infected after feeding on blood of the viral host; (3) mosquitoes can transmit the virus by biting other mosquitoes during the experiment; (4) mosquitoes feed on blood from natural hosts and this can be proven [11]. If mosquitoes meet the above four criteria, mosquitoes can be considered a vector of the virus. The mosquitoes detected in the present study yielded negative results for ASFV, and did not satisfy criteria 1.
There have been very few studies on the potential role of mosquitoes in ASFV transmission, but one failed to validate the vector competence of mosquitoes and horse ies for the virus [12]. A recent study detected low viral doses in mosquitoes collected from an infected farm in Estonia, but virus isolation was unsuccessful, and the researchers believe that the viral DNA found in mosquitoes is likely to be due to general contamination of the environment by ASFV, but whether mosquitoes have transmission capacity requires further study [13]. Along with the present work, none of the above reports could con rm the role of mosquitoes as vectors in transmitting ASFV. Epidemiological surveys in China have shown that among the 155 ASFV outbreaks in which the epidemic source has been identi ed, 100 outbreaks have been caused by the allocation and transportation of pigs and their products, and the transmission of viruses by related personnel and vehicles, accounting for ~ 64.5% [14].
The transmission of ASF mainly depends on its strong resistance, since the virus can survive in the external environment and various biological media for a long time. Vehicles, personnel, tools and swill are the main sources of virus transmission. Therefore, we speculate that mosquitoes are not heavily involved in ASFV transmission, but the possibility of mechanical transmission cannot be ruled out. Large-scale pig farms still need to follow normal mosquito control procedures when formulating strategies for the prevention and control of ASF, but new virus-speci c measures are not needed. Rather, attention should be payed to disinfection and epidemic prevention management related to personnel and vehicles entering and leaving farms. Availability of data and materials The datasets used and analyzed during the current study available from the corresponding author on reasonable request.