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.