Discussion
In China, GETV was first identified in Hainan Province in southern China in 1964, and one GETV strain designated M-1 was isolated from wild-caught mosquitoes (Li et al., 1992). Since then, several investigators have demonstrated that GETV is present in mosquitoes in China with a wide distribution (Shi et al., 2019). Serum antibody against GETV can be detected in humans, pigs, horses, cows, sheep, kangaroos, and other animals (Zhang et al., 2016). Before 2016, studies about GETV were limited to serological investigations of the virus in human and animals, while the isolation and identification of the virus were limited only in mosquitoes in China(Shi et al., 2019; Li et al., 2019; Yang et al., 2018). There were few studies focus on pathogenic analysis of GETV on pig. In this study, GETV named HNJZ-S1 was isolated from pig herd for the first time in China. We characterized HNJZ-S1 by passage and plaque titer, whole genome sequencing, Electron microscopy and animal infection experiments with mice and piglet.
Severity of porcine reproductive and respiratory syndrome (PRRS) implicated co-infection with other pathogens in pig herd in China, normally found JEV, PRV, PRRSV, porcine circovirus, porcine parvovirus and CSFV. Therefore, our laboratory often performed multiplex RT-PCR or PCR assay to identify pathogens related to pig reproductive disorder. Recently our laboratory has used a pair of RT-PCR primers to detect the E2 gene of CSFV since 2007. Amplicon was expected to be 276 bp. However, from September 2016, samples have shown a nonspecific band around 1000 bp instead. Curiously we sequenced the fragment. The sequence was identical between 8147bp to 9469bp of the GETV genome. The results of the whole genomic sequence alignment showed that there is no homology between CSFV and GETV. It was an accident that the sequence of the pair primers matched both viral genomes by 96.45%. However, we did not find any one of normal testing pathogens, such as JEV, PRV, PRRSV, porcine circovirus, porcine parvovirus and CSFV by RT-PCR or PCR. We only detected one non-specific band using CSFV primers by RT-PCR. Therefore, abortion in this case may have been caused by something new pathogen. We purified virus using Marc-145 cells by single CPE picking and then perform the whole genomic sequencing. We named this strain HNJZ-S1 and uploaded it into GenBank accession KY3638. The data show the full-length genome sequence of HNJZ-S1was 11689 bp with homologous 97.4%–99.3% identical to the GETV strains available in GenBank (Table 1 ). Phylogenetic analysis showed that HNJZ-S1 was closely related to Japanese mosquito isolate 12IH26, horse isolates 14-I-605-C1 and 14- I-605-C2, and Chinese mosquito isolate HB0234 (Table 2 and Figure 4 ). Alignment of the isolate reported in this study with available GETV full-length genome sequences showed that the genetic conservation of GETV was higher than that of other single-stranded positive sense RNA viruses such as PRRSV and PEDV. The genomes of strains isolated over the past 20 years were highly similar. Although the results of this study showed that HNJZ-S1 was closely related to Chinese mosquito isolates, Japanese and Korean pig and horse isolates, whether mosquitoes can transmit GETV independently among the above three countries is a question worth further study. In addition, other routes of transmission should also be considered, such as air transport of viral vectors, migratory birds and other blood-sucking insects.
Given the clinical findings and HNJZ-S1 verification, we investigated the morphology of viral particle and performed pathogenicity. Our results showed that the GETV HNJZ-S1 strain could cause death of 3-day-old newborn mice (Figure 5 and 6 ) and 3-day-old piglets (Figure 7 ). No obvious lesions were observed in dying piglets except in the thinner intestinal wall. GETV nucleic acids were detected in the brain, spleen, lung, kidney, bladder, small intestine and rectum of dying piglets, and HNJZ-S1 obtained from piglet brain tissue. Animal experiments and studies reported that GETV mainly infects pregnant and newborn animals (Yage et al., 1987; Yang et al., 2018) coordinating with this study. Therefore, the study of human GETV infection should focus on serological investigations of pregnant women or newborns, rather than all people. This may help researchers to determine whether GETV poses a risk to humans. Furthermore, pig-derived GETV was described and isolated for the first time in China due to a nonspecific amplification event during CSFV RT-PCR detection in our laboratory, which indicated that the original CSFV amplification primers need to be optimized.
Our results confirmed the existence of GETV infection in Chinese pigs and provided valuable data for the studies on GETV pathogenicity, pathogenic mechanism, public health and might opened a new field for the study on GETV infection. China is the world’s largest pork producer and consumer and the possibility that human contact with GETV-infected pigs and their products may frequent. Therefore, the epidemiology of pig diseases in China is closely related to human health. This study is not only a warning for public health safety, but also takes a step toward understanding transmission of the virus.
We respect that there are several limitations to our study. Although GETV had lethal effects on newborn piglets as well as obvious pathogenicity in pregnant mice and newborn mice shown in this study, experiments are required to further elucidate the basis for pregnant sow. GETV has been studied for more than 60 years. Compared with other viruses of the same genus, however, much remains unknown regarding the transmission mechanisms and pathogenicity of GETV. A more comprehensive and in-depth understanding of this virus my help us to effectively prevent and control human and animal diseases caused by GETV.