ABSTRACT
We reported a GETV strain, named GETV-V1, was isolated from a commercial
modified live vaccine (MLV) against porcine reproductive and respiratory
syndrome virus (PRRSV), which is widely used on pigs in China. Further
results showed that nine batches of MLV vaccine (three batches per year)
from the same manufacturer between 2015 and 2017 were all positive for
GETV. The complete genomes of the isolate were sequenced and it was
found that the genomes of GETV-V1 had the highest similarities with a
strain 16-I-674, which was isolated from the sick horses in Japan. This
is the first report of GETV contaminant in live swine vaccine in China.
Our finding demonstrates that vaccination of the commercial live
vaccines might be contaminated and became a potential new route for GETV
transmission in swine, pointing to the need for more extensive
monitoring of the commercial live vaccines in China.
KEY WORDS: Porcine reproductive and respiratory syndrome (PRRS)
vaccine; Getah virus (GETV) contamination; Isolation;; Public health
security
Getah virus (GETV) belongs to the
genus Alphavirus within the family Togaviridae and is a
mosquito-transmitted arbovirus (Berge et al., 1975) that can infect many
vertebrates and arthropods (Doherty et al., 1966). The first GETV strain
was isolated from Culex in Malaysia in 1955(Go et al., 2014).
The prototype virus strain was MM2021
(Elisberg et al., 1963). Serological survey of GETV showed that GETV
antibodies have been identified in humans, pigs, cattle, horses, goats,
rabbits, kangaroos, chickens, foxes and some wild birds in many
countries within Europe, Asia and Oceania.
GETV can
cause reproduction disorders and
fetal death in pigs, as well as body rash, leg edema and fever in
horses. The potential risk to animal health posed by GETV must not be
overlooked (Li et al., 1992; Kurogi et al., 1975; Doherty et al., 1972;
Tajima et al., 2014; Yago et al., 1987; Fukunaga et al., 2000)
The first GETV strain in China was isolated from mosquitoes in Hainan
province in 1964. Since then, GETVs have been isolated from mosquitoes
in more than 10 provinces in China, including Hainan, Shanghai, Sichuan,
Yunnan, Hubei and Gansu (Yang et al., 1984; Li et al., 2017; Li et al.,
2017; Li et al., 1992).
Porcine
reproductive and respiratory syndrome virus (PRRSV) and GETV are
different but common viruses
associated
with reproduction disorders in sows, and both are capable of growing in
Marc-145 cells. PRRSV modified
live vaccine (MLV) is widely used in China. In October 2017, in titer
test of the PRRSV MLV vaccine used by a pig farm where abortion in a
pregnant sow occurred, we found that the
caused cytopathic effect (CPE) by
the supernatant of vaccine on Marc-145 cells was significantly different
from that of the previously vaccinated PRRSV strain. Similarly, CPE was
also observed in other cell lines, including Vero, PK-15, BHK-21 and
human hepatocellular carcinoma HepG-2
cells.
Therefore, exogenous virus was detected by reverse
transcription-polymerase chain reaction (RT-PCR) using specific primers.
The results demonstrated that GETV
were positive. Because the pig farm had also been vaccinated with
classical swine fever virus, Japanese encephalitis virus, porcine
parvovirus and PRRSV. We suspected that GETV might be a contaminant in
these commercial vaccines. As expected,
a GETV strain was isolated from
the commercial PRRS MLV vaccine following isolation, passage,
purification by plaque assay and observation by transmission electron
microscopy. This virus was designated as GETV-V1. In this study, we
characterized the isolated GETV strain and the genome data of this newly
identified GETV strain.
Further RT-PCR
results showed that
the commercial PRRS MLV vaccine
was positive for GETV, including nine batches of vaccine (three batches
per year) from the same manufacturer between 2015 and 2017.
Immunofluorescence assay (Figure 1) showed that Marc-145 cells infected
with the GETV isolate reacted specifically with GETV-specific monoclonal
antibody (produced by our laboratory).
The
fairly uniform morphological appearance of GETV indicated that the
diameter of the virus was 60–80 nm and close to spherical in shape. The
fiber protein was clearly visible on the membrane (Figure 2). These data
demonstrated that GETV-V1 was isolated from a contaminated live swine
vaccine.
Excluding the poly(A) tail, the
complete genome sequence of GETV-V1 was 11,689 nucleotides (nt) in
length with a 5′-UTR of 78 nt and 3′-UTR of 402 nt (Table 1). The genome
was similar to that of previously reported GETV strains. Sequence
alignment of GETV-V1 with all GETV sequences available in GenBank
revealed that GETV-V1 shared the highest identity (98.9%) with 16-I-674
(isolated from a horse in Japan in 2016), 98.1% identity with Chinese
strains JL1707, JL1708, 12IH26 and Japanese strain 14-I-605-C2.
Phylogenetic analysis showed that
GETV-V1, together with HB0234, JL1707, JL1708, HNJZ-S1 and HNJZ-S2
strains, was clustered into the Japanese GETV branch (Fig.3).
Interestingly, although the Chinese reference strains were close to
GETV-V1 geographically and temporally, they were phylogenetically much
closer to each other, compared with GETV-V1. These results
revealed that the GETV strains
circulating in China are genetically diverse, thus providing a
potential
platform
for evolution.
Analysis using MegAlign software showed that GETV-V1 had 36 unique
nucleotide mutations in its genome, including 35 nucleotide
substitutions and 1 nucleotide insertion (Table 2). Interestingly, only
15 nucleotide mutations resulted in amino acid substitutions.
These mutations with the
nucleotide insertion in the 3′ UTR of the genome could be a unique
hallmark of GETV-V1.
In summary, we isolated and characterized one GETV strain from a
commercial live pig vaccine produced in China. Complete genome analysis
showed that GETV-V1 share high identity with GETV isolates HNJZ-S1,
14-I-605-C1 and 16-I-599, and these strains are pathogenic to pigs or
horses (Nemoto et al., 2016). This study firstly
reported for the contamination
with GETV in live attenuated PRRS vaccines in China, implying that
monitoring of exogenous virus in live vaccines for pigs needs to be
improved. Furthermore, we should also pay more attention to the
potential public health effect of vaccines contaminated with this virus.