In this study, we published the results o f sequencing, analyzing and comparing the genome o f the attenuated PRRSV strain Hanvetl.vn compared with the original pathogenic strain 02HY..
Trang 1Vietnam Journal o f Biotechnoỉogy 20(2): 245-252, 2022
H A N V E T 1 V N S T R A IN C O M P A R E D W IT H O R IG IN A L V IR U L E N T 0 2 H Y
S T R A IN O F T H E P O R C IN E R E P R O D U C T IV E A N D R E S P IR A T O R Y S Y N D R O M E
V IR U S
Nguyên Thi Nga1, Ha Thi Thu2, Nguyên Thi Hoa2, Vu Thi Hien2, Nguyên Thu Trang3, Nguyên Thanh Ba3, Tran Van Khanh3, Nguyên Huu Vu3, Dong Van Quyen2, To Long Thanh4, Dinh Duy Khang2H
1Ịnstitute o f Science and Technology, Mỉnỉstry o f Public Security, 47 Pharn Van Dong Road, Cau Giay District, Hanoi, Vietnam
:ỊnstituteofBiotechnology, Vietnam Academy o f Science and Technology, 18 Hoang Quoc VietRoad, Cau Giay District, Hanoi, Vietnam
}Hanvet Pharmaceutỉcal Co., Ltd, 88 Truông Chinh Road, Dong Da District, Hanoi, Vietnam 4National Center fo r Veterỉnary Diagnostics, MARD's Department o f Animal Health, 15/78 Giai Phong Road, Dong Da District, Hanoi, Vietnam
To whom correspondence should be addressed E-mail: khangdd.ibt@gmail.com
Received: 01.11.2021
Accepted: 21.01.2022
SUMMARY
The attenuated porcine reproductive and respiratory syndrome virus (PRRSV) strain Hanvetl vn was developed by Hanvet Pharmaceutical Co., Ltd by inoculating the vimlent strain 02HYon Marc-
145 cells for 80 generations and used to produce PRRS vaccine In this study, we published the results
o f sequencing, analyzing and comparing the genome o f the attenuated PRRSV strain Hanvetl.vn compared with the original pathogenic strain 02HY The genomes o f strains Hanvetl.vn and 02HY have 8 reading frames, coding for 8 non-structural and structural proteins: N SPla, N SPlb, GP2, GP3, GP4, GP5, MP, NP After sequencing and translating into proteins, the gene sequence o f each open reading trame (ORF) o f strain Hanvetl.vn was compared with the sequence o f pathogenic strain 02HY to tĩnd nucleotide and amino acid changes The results showed that the Hanvetl vn pathogenic strain genome (Genbank Accession KU842720) when compared with the pathogenìc strain 02HY genome (Submission2490633) had89 nucleotide mutations that changed 51 amino acids in 7 ORFs and 7 proteins, respectively Particularly, ORF6 encoding for the M protein is completely unchanged The size o f each reading trame is also exactly the same It showed that there were no insertion and deletion (Indel) mutations in the ORFs o f the attenuated strain after 80 generations o f inoculation There was a change in the genome that made the sừain Hanvetl.vn become attenuated, but the gene encoding for the GP5 protein that induces the production o f neutralizing antibodies only changed two nucleotides at position 471 (A->G), causing the TCA codon to tum into a TCG codon This is a silent mutation and both codons code for the amino acid Serine (S) The second mutation at position 587 (A->T) causes Glutamine (Q) to transíorm into Leucine (L) However, this modiíĩcation does not belong to the GP5 antigenic epitopes In clonclusion, after 80 passages, despite changes occurred in genes o f Hanvetl.vn strain for becoming an attenuated strain, the GP5 protein o f the attenuated strain did not change its antigenic amino acids.
Keywords: Hanvetl.vn attenuated strain, 02HY virulent steain, genome sequence, nucleotide and amino acid changes, GP5 antigen epitope.
Trang 2Porcine Respiratory and Reproductive
Syndrome (PRRS) is a contagious disease in pigs
of all ages The disease caused by PRRSV has a
complicated course, difficult to control, and
witha high morbidity rate The emergence of
highly virulent PRRSV sừains in China in 2006-
2007 caused a series of PRRSV outbreaks in
Asia (Tian et ai; 2007) The PRRS epidemic has
killed millions of pigs since 2006 (Zhou, Yang,
2010) Highly pathogenic strains of PRRSV have
also caused outbreaks in other countries in the
Asian region The íírst case o f PRRS appeared in
Vietnam in early 2007 (Feng et al; 2008;
Metwallyet al; 2010) then spread to other
Southeast Asian countries such as Laos,
Philippines, Cambodia etc (Jantafongeí ai;
2010; Ni et al; 2012; Tomimbeneeí al; 2015)
In Vietnam, from 2007 untilnow, PRRS has
occurred in many provinces and cities
throughout the country, causing heavy losses to
the livestock industry (Pham Van Son, 2018)
There have been many vaccines produced for
disease prevention, including the attenuated
PRRS vaccine that is being prioritized for
development and use in China and Vietnam
because of its superior protection compared to
other kinds of vaccines In Vietnam, in order to
prevent diseases, many integrated measures have
been directed by the Ministry of Agriculture and
Rural Development, in which the use o f vaccines
is the top priority
There were two strains to be attenuated for
the use as vaccines including the attenuated
PRRSV strain Hanvetl.vn by the Hanvet
Pharmaceutical Company Limited and the KTY-
PRRS-06 strain by the Vietnam National
University o f Agriculture (Trinh DinhThaue? al;
2018), respectively The attenuated PRRSV
strain Hanvetl.vn used in the production of
PRRS vaccine by the Hanvet Pharmaceutical
Company Limited was created by 80 generations
of continuous subculture on Marc-145 cells
In this paper, we present the results of
sequencing, analysis and comparison of changes
in the genome betvveenthe attenuated virus strain
Hanvetl.vn and the original pathogenic strain 02HY in order to evaluate the genetic changes related to the virulence and immunogenicity of the vaccine
MATERIALS AND METHODS
Materials
The attenuated vaccine strain Hanvetl.vn and the virulent strain 02HY are provided by Hanvet Pharmaceutical Company Limited cDNA synthesis kit: SuperSciptTM First- StrDNA Synthesis System for RT-PCR (Invitrogen) Cloning Kit: TA cloning Kit (Invitrogen) Gene sequencing kit: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) High purity Chemicals used in molecular biology research are provided by companies such as Sigma, Merck, Invitrogen, include: Taq DNA polymerase, Yeast extract, Tryptone, Agar, Amp (Ampicillin), X-gal, EDTA (Ethylenediaminetetraacetic acid), SDS (Sodium dodecyl sulphate), Chloroíòrm, Trizol, Sodium acetate, Ethanol, NaCl, Tris-HCl,
Ethylenediaminetetraacetic acid)
Methods
Genetic sequences of North American PRRSV genotypes circulating in Vietnam and the region with numbers FJ393456, FJ393457, FJ393458, FJ393459, FJ394029 and attenuated vaccine strain RespPRRS were used to design primer pairs to amplity the whole genomes of Hanvetl.vn and 02HY viruses Using GeneDoc 2.7 software to compare and design primer pairs for gene amplitìcation
Ampliíication and cloning of 15 gene segments belonging to the attenuated strain Hanvetl.vn and pathogenic strain 02HY were caưied out as described previously (Nguyên Thi
Nga et al; 2018) The sequencing reaction of
clones was períbrmed using the BigDye™ Terminator v3.1 Cycle Sequencing Kit from Applied Biosystems, using two forward and reverse primers M13F and M13R Automated sequencing on the ABI 3100 System and
Trang 3Vietnam Journal o f Biotechnology 20(2): 245-252, 2022
sequence analysis using BioEdit and DNA Star
software were carried out Comparison of ORFs
and proteins for mutations was períòrmed by the
PC/Gene software program Protein epitopes
were analyzed using the Predicting Antigenic
ịhttp://imed med ucm es/Tools/antigenic.pl).
RESULTS AND DISCUSSION
RESULTS
Ampliíication and cloning of genesegments of
the PRRSV genome
After extraction of total RNA ữom infected
viral fluid of the Marc-145 cells, RNA was
converted to cDNA using Invitrogen's
SuperSciptTMFirst-StrDNA Synthesis kit
cDNA was used as template strand to ampliíy
gene segments of PRRSV attenuated Hanvetl.vn
and virulent strain 02HY by PCR with 15
speciíic primer pairs designed so that the
ampliííed gene íragments have verlapping
regions for easily reassembled into a complete
genome To facilitate the storage and sequencing
of genes, after purification, the PCR Products
were ligated to pCR2.1 cloning vector,
transformed into E coli cells with Top F' strain
The recombinant plasmids cariying amplifíed
gene ữagments from the PRRSV genome were
screened and examined by cutting with the
restriction enzyme EcoBl The gene fragment
from the recombinant plasmid was separated from the vector with the same size as the PCR product ligated to the cloning vector (Nguyên
Thi Nga et al., 2018).
Sequencing and analyzing the genome of the
pathogenic strain 02HY
Recombinant plasmidsinserted with PCR Products were puriTied and sequenced using an automated gene sequencer (ABI 3100) The resulting data were processed and analyzed using DNAStar and GeneDoc software After sequencing, assembly and annotation, we obtained the attenuated Hanvetl vn genome with
8 reading frames, encoding for 8 proteins (GenBank accession number: KU842720) Similarly, strain 02FIY also has 8 reading frames, coding for 8 proteins (Submission: 2490633) The length of each reading írame of the attenuated strain ITanvetl.vn iscompletely unchanged compared to the original pathogenic strain 02HY The number o f nucleotides and amino acids of each reading írame of the attenuated strain Hanvetl.vn and pathogenic strain 02HY are shown in Table 1
The results in Table 1 showed that there were
no deletion and insertion (Indel) mutations in the open reading frames of strain Hanvetl.vn after
80 generations of inoculation
Table 1 Number of nucleotides and amino acids of each reading trame of the attenuated strain Hanvetl vn and the virulent parent strain 02HY.
nucleotides
amino acids
protein
Genomic changes in the H anvetl.vn strain
compared with pathogenic 02HY strain
The changes in nucleotide sequences and
amino acid sequences in each ORF/protein of the attenuated strains of Hanvetl.vn compared to its virulent strain 02HY are shown in Tables 2 and
3, respectively
Trang 4The attenuated strain Hanvetl.vn had 89
point mutations in all reading írames that made
the changes in 51 amino acids Among 89 point-
mutations there were missense mutations
(leading to change of amino acid they encode
for) and silent mutations (no change of amino
acid) as seen in the attenuated strain Hanvetl.vn
No nonsense mutations causing stop codons
were found ORF5 encoding for the GP5 protein
containing neutralizing antibody-producing
epitopes had two-nucleotide changes at position
471 (A->G) and 587 (A->T) The change o f TCA
to TCG (A->G) is a silent mutation causing no
change of amino acid Serine (S) The
muation(A->T) had made a change from Glutamine (Q) to Leucine (L) (Figs 1 and 2)
Table 4 shows epitope amino acids in the epitopicstrecht in the GP5 polypeptide of the attenuated Hanvetl.vn sừain The amino acid change at position 196 (L196Q) occuưed in this attenuated strain was outside the GP5 antigen epitope region (Table 4) Thus, it can be concluded, after 80 passages, although the genes of Hanvetl.vn strain have some changes to become attenuated compared to the pathogenic 02HY strain during the attenuation, butthe GP5 epitope amino acids responsible for the production of neutralizing antibodies were not aữected
H A M V _ O K F 5 _ N
0 2 H Y _ O R F 5 _ N
11AN V_0R I-'S_N
0 2 K Y 0RF5 N
HANV’ DRF5 N
02KY_O RF$_N
HAMV_ORF5_N
Ũ2HY_ORF5_N
H A M V ORF5 N
Ũ2HYjORFS N
HAHV_0RF5_N
Q2KY_ORF5_N
ỈỈA&IV_jDRF5 N
0 2KY_QRF5_N
HAM^/I(0 R F 5 N
02 KY ORF$ N
 T G T T G G G G A A G T G C T T G A C C G C G T G C T G T T G C T C G C G A T T G C T T T T T T T ™5Q
I M u I n u m m 11! I u m 1 M H 11 m i M M I i n M H n I
A T G T T G G G G A A G T G C T T G A C C G C G T G C T G T T G C T C G C G A T T G C T T T T T T T - 5 0
G T G G T G T A T C G T G C C G T Ĩ C T À T C T T G C T G T G C T C G r C A A C G C C A G C G A C A - 1 0 0
I u 11 u M í m m M I i I n 1 1 1 ũ ! I í n I m H 1 1 1 ! ị I ỉ I i 11
G 7 G G T G T A T C G T G C C G T T C T A T C T T G C T G T G C T C G T C A A C G C C A G C G A C A -iồo
A C A A C A G C T C r C A T A T T C A G T T G A T t T A T A A C T T G A C G C T A T G T G A G C T G “ 1 5 0
! i : u M u III ĩ 1 i 11111 m 11 h 111 m m n u III i 1111II
A C A A C A G C T C T C A T A T 7 C A G T T G A T T T A T A A C T T G A C G C T A T G T G A G C T G - 1 5 0
Ằ A T G G C A C A G À T T G G C T G G C A C A A A À T T T T G A C T G G G C Ằ G r G G A ữ A C T T T - 2 0 0
u u I M u I M M l í u i ỉ u u 1 m M I I I M H! I I IM M H i 11
A A T G G C A C A G A T T G G C T G G C A C A A A A T T T T G A Ủ T G G G C A G T G G A G A C T T T - 2 0 0
T G 7 C A T C T T C C C C G T G T T G A C T C A C A T T G T T T C C T A T G O G G C A C T C A C C A - 2 5 0
1 I i I i H I ! ỉ í H H I H m I M I 1! M I M M ỉ m M m Ị i I 1 í ị !
r G T C A T C T T C C C C G T G T T G A C T C A C A T T G T T T C C T A T G G G G C A C T C A C C A - 2 5 0
C C A G C C A 7 T T C C T T G A C A C A G T T G 6 T C T G G C C A C T G T G T C C A C C G C C G G A - 3 0 0
m i u u II m i I i m I m u M i m n u i m M 1 H M 1 M í
C G A G C C A T T T C C T T G A C A C A G T T G G r C T G G C C A C T G T G T C C A C C S C C G G A - 3 0 0
t A T T A T C A C G Ổ G C G G T A T G T C T T G A G T A G C A T T T A C G C A Ổ T C t G T G C T C T - 3 5 0
H u i I M M I i i ỉ ! H 1 IM H I II H MI III II í M 1 I n M m u
r A T T A r C A C G G G C G G T A T G T C T T G A G T A G C A T T T A C G C A G r C T G T G C T C T - 3 5 0
G G C T G C G C T G A T T T G C T T T G T C A T T A G G C T T G C G A Ã G A A C T G C A T G T C C T - 4 0 0
m u m i MI 1 m m m 1 11III m ! M 1111 M m Ị í m 11
G G C T G C G C T G A T T T G C T r r G T C A T T A G G C T T G C G M G A A C r G C A T G T C C T “ 4 0 0
HAHV_0RF5_N
Ũ2HY_ORF5Jf
H A W _ O R F 5 _ N
Ũ 2 K Y O R f 5 _ N
- G G C G C r A C T C T T G T A C C A G Ằ Ĩ Ẫ T A C C A A C T r C C T T C T G G A C A C T A A G G G C - 4 5 0
Hì m 11 m ĩ u MH m IIM u u M u m m M ì u n li M
- G G C G C T A C T C T T G T A C C Ầ G A T A Ĩ A C C A A C T r C C T T C T G G A C A C l 'A A G G G C - 4 5 0
471
- A G A C T C T A T C e T T G G C G G T C G C C C G r C A T T G T G G A G A A A A G G G G T A A G G T “ 5 0 0
I I M M ! I ị M 1 i M í M I Ị I 1 I í M I M I I 1 M m M I M 11 M M I
- A G A C T C T A T C G T T G G C G G T C A C C C G T C A T T G T G G A G A A A A G G G G T A A G G t - 5 0 0
H A N V J 3 R F 5 N
02HYJDRF6_N
0 2 B Y O R F 5 N
- T G A G G T C G A A G G T C A C C T G A T C G A C C T C A Ẵ G A G Ằ G T T G T G C T T G A T G G T T - 5 5 0
m M ỉ I M I I I M II 11 ỉ I II M M H M I I M I M M M I M n M M
- T G A G G T C G A A G G T C A C C T G A T C G A C C T C M G A G A G r T G T G C T T G A T G G T T ~ 5 5 p
sạ?
“ C C G C G G C A A C C C C T T T A A C C A G A G T T T C A G C G G A A C T A T G G G G T C G T C T C ~ < S ũQ
IH M H m m M IM i M M M m M ! M M I I M 1 I II I III M
- C C G C G G C A A C C C C T T T A A C C A G A G T r T C A G C G G M C A A T G G G G T C G T C T C - ê ũ O
Figure 1 Comparison of the nucleotide sequence of the ORF5 gene of the attenuated strain Hanvetl vn with that of the 02HY strain The attenuated strain Hanvetl.vn appeared with 2 point mutations at positions 471 and 587.
Trang 5Vietnam Journal o f Biotechnology 20(2): 245-252, 2022
Table 2 Number of nucleotides and amino acids changed in each ORF of the Hanvetl vn attenuated strain compared with the 02HY pathogenic parent strain.
nucleotides -chanaes in each
acids changes in
Table 3 Identilý of nucleotide and amino acid sequences of hlanvetlvn attenuated strain compared with 02HY pathogenic strain.
in the two strains
HÃNV_ORF5_ P - MLGK'CLTftCCCSRI,I,FI,WCI¥PFYI,AVLV|.lASDIWSSHIQlIYWI.'rLCEI, - 5 0
m 1111 i l i 11) 11111 m 11 í M I I 1111 m I M í ? I I I i í I I I 1 1
0 2 HY_ORF5_ P - MLGKCLTACCCsRLLFLWCIVPF YXiAVLVNASDI® 8 s tí l ũ l l ĩĩỉiTLC EIi - 5 0
HANV_ORF5_P - N<5rEMIAQỈlFDWAVETFVĩFPVLTHXVSYQALrTSHFLDTVGLATVSrAG - 1 0 0
111 i 1111( 1 I I I i I m I f I I I 1111 1111I I i 1 1 I I 1 1 I I I I f I I 111
ó 2 HY ORF5 p - KGTDWXAQNFDKAVETFVIPPVLTHIVS YGALT TSHPLDTVGLATVSrAG - 1 0 0
HAHV _ORF5 _p -
02HY_ORF5_P -
HRSV QEF5 p
02HY ORF5_P
-YYHẸRYVLSSIYAVCALAALICFVĨRLAKNCMSWRYSCTRYTHFLLDrKE - 1 5 0
1 1 1 1 i 1 1 1I I1 1 1! 1 1 1 1 1 1 1 1 1 1 1 m II m II m 1 1 m ! I m I (
YYHGRYVPSSIYAVCALAAĩ IcrrcR U U Q lC M S M R rS CTRYTKFĨ.Ĩ.ĐTKG - 1 5 0
RLYRMRSPVIVEKRGKVEVEGHLI DLKRVVLDGSAATPLTRVSAElĩíGRl - 2 0 0
I I I í 1111 í i I I I i 11 m i M I 111111111111111 i 11I I 11 I I 11 RL¥RWSFVWFJ«GỈOTWEGHLíDLKRWLOGS»TP'L'ĩiWSAEữWGRJ, - 2 0 0
Fỉgtire 2 Comparison of the amino acid sequence of the GP5 protein of the attenuated strain Flanvet1 vn with that of iie 02HY strain The silent mutation at nucleotide position 471 does not change the amino acid Serine The missense mutation at position 587 causes Glutamine (Q) to transíorm into Leucine (L), however this amino acid is outside the antigenic epitope region (Table 4).
Table 4 GP5 protein epitopes of the attenuated strain Hanvetl vn
Number starting
position
position
YAVCALAALICFVIRLAK
129
Trang 6In addition to imported vaccines,
domestically researched and manufactured
vaccines have also played an important role in
the prevention of PRRSV in Vietnam The
Hanvet Pharmaceutical Co., Ltd has succeeded
in creating an attenuated strain Hanvetl vn by 80
generations o f inoculation on Marc-145 cells and
using this strain to produce vaccine against
porcine reproductive and respiratory syndrome
virus
Sequencing and analyzing the genome and
íinding genetic changes of the attenuated strain
Hanvetl.vn compared with the original
pathogenic strain 02HY, in this study, have
provided important iníòrmation for the
monitoring of genetic changes following to
decide saíety and protective immunogenicity of
vaccines during production The results of
comparing nucleotide and amino acid sequences
of all 8 reading frames of the attenuated strain
Hanvetl.vn with the original pathogenic strain
02HY showed that there were 89 nucleotide
mutations that changed 51 amino acid mutations
scattered in seven openreading frames and seven
proteins, respectively However, ORF6 encoding
for the M protein is completely unchanged In the
open reading írames, only silent mutations and
missense mutations appeared, no nonsense
mutations generating stop codons The size of
each reading írame is also exactly the same It
showeđ that there were no insertion and deletion
(Indel) mutations in the open reading írames
(ORFs) of the attenuated strain after 80
generations of inoculation
There was a change in the genome that made
the Hanvetl.vn strain become attenuated, but the
gene encoding the GP5 protein changed only two
nucleotides at position 471 (A->G) causing the
TCA codon to tum into TCG This is a silent
mutation and both codons code for the amino
acid Serine (S) The mutation at position 587 (A-
>T) causes Glutamine (Q) to tum into Leucine
(L) However, this modiíication does not belong
to the GP5 antigenic epitopes Thus, it can be
said that, after 80 passages in Marc-145 cell line,
although the genes of strain Hanvetl.vn have changed to become an attenuated strain, the gene encoding GP5 protein has not change the immunogenicity - GP5 is an essential protein playing an important role in stimulating the immune response to generate PRRSV-
neutralizing antibodies (Popescu et al., 2017)
Thereíòre, the conservation of the antigenicity of the GP5 proteinoí the attenuated strain Hanvetl.vn compared with the original pathogenic strain 02HY is decisive for the eíĩectiveness of the vaccine
In addition to the attenuated strain generated
by the Hanvet Pharmaceutical Company Limited, the another attenuated strain named KTY-PRRS-06 has also been recently succeeded
by the Vietnam National University of Agriculture using the serial passages for 90 generations on Marc-145 cell line The attenuated strain causes cytophathic effect (CPE)
12 hours aíter iníection and completely damage the cells after 48 hours The attenuated strain KTY-PRRS-06 is not pathogenic when administered to pigs and induces a speciíĩc antibody response against PRRSV The authors compared the ORF5 sequence of the attenuated strain KTY-PRRS-06 with the pathogenic strain and found 13 different positions in the nucleotide sequence and 8 different positions in the amino
acid sequence (Trinh DinhThaueí al., 2018)
Thus, the attenuated strain KTY-PRRS-06 after
90 generations of inoculation on the Marc-145 cells appeared more point mutations than the attenuated strain Hanvetl.vn In the work by Trinh DinhThau et al (2018), the authors did not analyze each mutation in the ORF5 gene and did not clariíy whether these mutations affect the ability to produce PRRSV-neutralizing antibodies belonging to the epitopes
Allende et al., 2000 sequenced and analyzed
the variation in the genome of the attenuated RespPRRS strain used in PRRS vaccine production and compared with the virulent parent strain 16244B The attenuated strain RespPRRS was created by subculture of 25 generations on MA-104 cells at 35-37°C, then 20 generations in the same cell line but at 3 l°c The
Trang 7Vietnam Journal o f Biotechnology 20(2): 245-252, 2022
results shovved that there were no Indel mutations
in the genome and discovered 212 point
mutations in the whole genome Thus, through
55 generations of inoculation on M A-104 cells
vvith 25 generations at 35-37°C and 20
generations at 3 1 ° c , the authors created an
attenuated strain with a higher number of point
mutations compared with our study (212
mutations compared with 89 mutations in our
study)
The authors also identiíĩed the changes of 9
amino acids located on 7 proteins N spip, Nsp2,
NsplO, ORF2, ORF3, ORF5, ORF6 and
suggested that the modiíications of these amino
acids may provide the molecular bases for the
observed attenuated phenotype
CONCLUSSION
In this study, the genome of the attenuated
PRRSV sừain Hanvetl.vn was sequenced,
analyzed and compared with the genome of the
parental pathogenic strain 02HY to find out the
eenetic variation of the attenuated strain
Hanvetl.vn after 8 0 passages onMarc-145 cells
The results showed that the genomes of strains
Hanvetl.vn and 02HY have 8 reading írames,
coding for 8 non-structural and structural
proteins: N SPla, NSPlb, GP2, GP3, GP4, GP5,
MP, NP After sequencing and translating into
proteins, the gene sequence o f each open reading
frame (ORF) of strain Hanvetl vn was compared
with the sequence o f pathogenic strain 02HY to
find nucleotide and amino acid changes The
results showed that, after 80 generations of
inoculation, in the genome o f strain Hanvetl.vn,
there were 89 nucleotide mutations that changed
51 amino acids in seven open reading írames and
seven proteins respectively Remarkably, ORF6
encoding for the M protein is completely
unchanged
The size of each reading frame is also exactly
the same It showed that there were no insertion
and deletion (Indel) mutations in the open
reading frames (ORFs) of the attenuated strain
aíter 80 generations of inoculation There was a
change in the genome that made the strain
Hanvetl.vn become attenuated, but the gene encoding the GP5 protein that induces the production of the neutralizing antibodies only changed two nucleotides at position 471 (A-
>G)is a silent mutation causing no change in amino acid Serine (S) The mutation at position
587 (A->T) changed Glutamine (Q) to Leucine (L), but this Leucine amino acid change is outside of the GP5 antigenic epitopes It is concluded, after 80 passages, the Hanvetl.vn strain have changed at some sites compared to the pathogenic strain to become an attenuated strain, but the gene encoding GP5 protein responsible for production of the neutralizing antibodies has not been altered
Acknowledgments: The work was carried out
with ịìnanciaỉ support within the framework o f the research project o f the Institute o f Bỉotechnoỉogy, Vietnam Academy o f Science and Technology.
REFERENCES
Allende R, Kutish GF, LaegreidW, LuZ, Lewis
TL, Rock DL, FriesenJ, Galeota JA, Doster
AR, Osorio FA (2000)Mutations in the genome o f porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype Arch
G'ro/145:l 149-1161.
Feng Y, Zhao T, Nguyên T, Inui K, Ma Y, Nguyên
TH, Nguyên v c , Liu D ,B u i QA, To LT,W ang
c , Tian K, Gao GF (2008) Porcine reproductive and respiratory syndrome virus variants, Vietnam and China, 2007 Emerg Infect Dis 14:1774-1776.
Jantafong T, Sangtong p, Saenglub w , Mungkundar
c , Romlamduan N, Lekchareonsuk c , Lekcharoensuk
p (2015) Genetic diversity o f porcine reproductive and respiratory syndrome virus in Thailand and Southeast Asia from 2008 to 2013 VetM icrobioỉnô:
229-238 Metvvally s, Mohamed F, Faaberg K, Burrage T, Prarat M, Moran K, Bracht A, Mayr G, Beminger M, Koster L, To TL, Nguyên VL, Reising M, Landgraf J, Cox Lubroth J, Carrillo c (2010) Pathogenicity and molecular characterization o f emerging porcine reproductive and respiratory syndrome viras in Vietnam in 2007 TransboundEmerg Dis 57: 315-
329
Trang 8Nguyên TN, Ha TT, Nguyên TH, Vu TH, Tran Thi TH,
Tran VK, Nguyên TB, Nguyên HV, Dong VQ, To LT,
Dinh DK (2018) Sequencing and analysis for complete
genome o f attenuated Hanvetl vn strain use for vaccine
production against porcine reproductive and
respiratory syndrome Vieừiam JBiotech 16: 51-57.
N i J, Yang s, Bounlom D, Yu X, Zhou z , Song J,
Khamphouth V, Vatthana T, Tian K (2012)
Emergence and pathogenicity o f highly pathogenic
Porcinereproductive and respiratory syndrome virus
in Vientiane, Lao People's Democratic Republic J
Vet Diagn Invest 24: 349-354.
Popescu LN, Trible BR, Chen N, RowlandRR (2017)
GP5 o f porcine reproductive and respiratory
syndrome virus (PRRSV) as a target for homologous
and broadly neutralizing antibodies Vet Microbiol
209:90-96.
Pham v s (2018) Research on creating master seed
virus and trial production o f inactivated vaccines
against reproductive and respiratory syndrome in pigs
from virus isolates in Vietnam PhD dissertation
Vietnam National University o f Agriculture.
Trinh DT, Nguyên TL, Nguyên BH, Nguyên HN, Le Huynh TP, Nguyên VT, Nguyên VT, Pham NT, Pham
HN, Huynh Thi ML, Nguyên TH (2018) Study on attenuated porcine reproductive and respiratory syndrome virus from highly pathogenic virus strain J ofV et Sci25: 5-16.
Tian K, Yu X, Zhao T, Leng Y, Cao z , Wang c, Hu
Y, Chen X, Hu D, Tian X, Liu D, Zhang s, Deng X, Ding Y, Yang L, Zhang Y, Xiao H, Qiao M, Wang B, Hou L, Wang X, Yang X, Kang L, Sun M, Jin p, Wang s, Kitamura Y, Yan J, Gao GL (2007) Emergence o f fatal PRRSV variants: unparalleled outbreaks o f atypical PRRS in China and molecular dissection o f the unique hallmark PLoS One 2:e526.
Tomimbene B, Lrossard JP, Chhim V, Som s, Guitian
J, Drew TW (2015) Emergence o f highly pathogenic porcine reproductive and respiratory syndrome (HP- PRRS) in medium-scale swine farms in southeastem Cambodia Prev VetMed 118: 93-103.
Zhou L, Yang H (2010) Porcine reproductive and respiratory syndrome in China Vi rua Res 154: 31-37.