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\begin{document}

\title{Genome analysis of Getah virus (GETV) isolated from a live swine
vaccine: The commercial live vaccines might be a potential route for
GETV transmission}



\author[1]{Feng Zhou}%
\author[1]{Aojie Wang}%
\author[1]{Lu Chen}%
\author[1]{Xingang Wang}%
\author[1]{Dandan Cui}%
\author[1]{Hongtao Chang}%
\author[2]{chuan-qing WANG}%
\affil[1]{Henan Agricultural University}%
\affil[2]{Henan Aricultural University}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\begin{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.%
\end{abstract}%



\sloppy


\textbf{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.

\textbf{KEY WORDS:} Porcine reproductive and respiratory syndrome (PRRS)
vaccine; Getah virus (GETV) contamination; Isolation;; Public health
security

Getah virus (GETV) belongs to the genus \emph{Alphavirus} within the
family \emph{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.

\textbf{Acknowledgements}

The study was supported by the National Key Research and Development
Program of China (NO 2016YFD0501100) and the Program for Science \&
Technology Innovation Research Team in the Universities of Henan
Province (NO 14IRTSTHN015)

\textbf{Conflict of interest statement}

The authors declare no conflict of interest.

\textbf{Ethics Statements}

All the sample collections were performed after obtaining verbal
approval of farm owner. This study was approved by the Ethics Review
Committee of the National Institute for Viral Disease Control and
Prevention, Chinese Center for Disease Control and Prevention. All the
animal studies were conducted in accordance with the principles of Henan
agricultural university Animal Care and Use Committee (IACUC).

\textbf{Data Availability Statement}

All data generated or analyzed during this study are included in the
article.

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