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Molecular Epidemiology of Porcine Cytomegalovirus (PCMV) in Sichuan Province, China: 2010–2012

Porcine cytomegalovirus (PCMV) is an immunosuppressive virus that mainly inhibits the immune function of the macrophage and T-cell lymphatic systems, and has caused huge economic losses to the porcine breeding industry. Molecular epidemiological investigation of PCMV is important for prevention and treatment, and this study is the first such investigation in Sichuan Province, Southwest China. A PCMV positive infection rate of 84.4% (865/1025) confirmed that PCMV is widely distributed in Sichuan Province. A phylogenetic tree was constructed based on the PCMV glycoprotein B gene (gB) nucleotide and amino acid sequences from 24 novel Sichuan isolates and 18 other PCMV gB sequences from Genbank. PCMV does not appear to have evolved into different serotypes, and two distinct sequence groups were identified (A and B). However, whether PCMV from this region has evolved into different genotypes requires further research. Analysis of the amino acid sequences confirmed the conservation of gB, but amino acid substitutions in the major epitope region have caused antigenic drift, which may have altered the immunogenicity of PCMV.


Introduction
Porcine cytomegalovirus (PCMV) belongs to the genus Cytomegalovirus, subfamily Betaherpesvirinae, family Herpesviridae [1], and is an icosahedral virus with a double-stranded linear DNA genome. The virus particle diameter is 150-200 nm. PCMV is distributed globally, with reported cases in Britain, Japan, Germany, and the United States [2]. Rapidly progressive consumptive coagulopathy has been observed frequently in swine-to-baboon renal xenotransplantation, and there is also a risk of zoonotic transfer of PCMV in swine-to-human xenotransplantation. However, the risk of PCMV infection for human recipients needs further research [3,4,5].
PCMV induces fatal systemic infections in young animals, and can cause death in piglets and embryos. Infected animals show clinical symptoms of inclusion body rhinitis, pneumonia, and dysplasia, as well as disease of the nervous system. Similar to human cytomegalovirus (HCMV) and other cytomegaloviruses, lifelong latent infection often occurs in recovered pigs [6,7,8]. PCMV is highly host specific, and is unable to replicate in rabbits, rats, chicken embryos, or in a guinea pig cell culture system. Efforts to study PCMV have been hindered by its specificity for porcine epithelial cells. Cross-infection of PCMV and other viruses often occurs in immunosuppressed swine, and a variety of bacterial infections can occur following PCMV infection [9]. Therefore, PCMV has been recognized as a serious hazard for the pig breeding industry.
Currently, research on PCMV is lacking. Previous work has primarily concentrated on the DNA polymerase (DPOL) gene, glycoprotein B (gB) gene, and the ORF 40-like protein gene, and sequences for these regions are available in GenBank. Genetic diversity has been noted in the DPOL and gB genes of different PCMV strains, resulting in antigenic variation; however, there is no evidence to confirm that PCMV has evolved significantly different serotypes or genotypes [9,10,11].
The gB gene is highly conserved within the Herpesviridae family, and is widely used in species identification and phylogenetic analysis of herpesvirus species [9,12,13]. Several PCMV sequences from China are available in GenBank, but no systematic genome analysis of Chinese strains has been performed. In this study, we report the detection and characterization of PCMV based on sequencing and phylogenetic analysis of the gB nucleotide and amino acid sequences from a large quantity of samples collected from major breeding bases and rural farms in Sichuan Province, China.

Ethical Statement
Animal welfare standard in this study was established on the basis of internationally agreed and science based principles within the World Organisation for Animal Health (OIE). All experiments were carried out in accordance with China Animal Welfare Legislation and were approved by the Sichuan Agricultural University Committee on Ethics in the Care and Use of Laboratory Animals.

Samples
A total of 670 porcine serum samples, 322 pulmonary hilar lymph node, spleen, kidney, liver, tonsil, and brain tissue samples, and 33 semen samples were randomly sampled from 1025 piglets, nursery pigs, sows, and boars from major breeding bases and rural farms in different districts of Sichuan Province between August 2010 and July 2012 ( Figure 1).

DNA Extraction
PCMV genomic DNA was extracted from serum, semen, and tissue samples using a commercially available DNA extraction kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. The extracted DNA was quantitated by SmartSpec Plus Spectrophotometer (Hercules, CA, USA) and stored at 220uC until use in PCR reactions.
Amplification was carried out in PCR buffer containing 200 mM of each dNTP, 10 pmol of each primer, 1.0 U Taq DNA polymerase (Promega, Madison, WI, USA), and 1.5 mM MgCl 2, in a total volume of 25 mL. PCR was performed at 94uC for 3 min, followed by 35 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 35 s, and a final extension of 72uC for 8 min. PCR products were electrophoresed on a 1.5% agarose gel, followed by staining with ethidium bromide (Invitrogen, Carlsbad, CA, USA) and visualization under ultraviolet (UV) light on a Bio-Rad gel imaging system (Hercules, CA, USA).

Amplification of the PCMV gB Gene
PCMV genomic DNA was extracted from 24 antigen-positive samples as described above, and the primers P1:59-ATGA-CAGTGAGCAGTCGGAATTTATTCCGGAT-39 and P2:59-TCACACGTCCTCGGTGGATAGCTGC-39 were used to amplify the complete gB gene. The primers sets covered the entire coding region of the PCMV gB gene. PCR amplification was carried out as described above. The PCR products were electrophoresed on a 1.5% agarose gel, stained with ethidium bromide, and then visualized under UV light, as above. Cloning and Sequencing of the PCMV gB Gene The PCMV gB gene amplicons were gel-purified using a Gel Extraction Kit (Tiangen Biotech) and then cloned into pMD19-T Simple Vector (Takara, Dalian, China). The ligated products were transformed into Escherichia coli DH5a competent cells (Invitrogen). Colonies containing plasmids with an insert were identified by blue/white selection, and positive colonies were picked and grown in BL medium at 37uC for 8 hours. Plasmid DNA was then extracted using a Plasmid Mini Kit (Omega Bio-Tek, Norcross, GA, USA). Purified plasmid DNA was used as a template for sequencing (Invitrogen, Shanghai, China).

Phylogenetic Analyses
The amplified PCMV gB nucleotide and deduced amino acid sequences were assembled using LaserGene (DNAstar, Madison, WI, USA) and compared with nucleotide and amino acid sequences of 18 PCMV gB genes available from GenBank. The sequence homology was confirmed using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/ ). Multiple alignment was performed by ClustalW analysis [14], and Molecular Evolutionary Genetics Analysis (MEGA) software version 5.0 was used to conduct phylogenetic analyses and produce the neighbor-joining (NJ) and maximum-likelihood (ML) trees [15]. All analyses were based on the 2583 bp PCMV gB gene complete sequences and carried out using the Kimura 2parameter model [16].
gB Nucleotide and Amino Acid Sequence Analyses The nonsynonymous to synonymous nucleotide substitution rates were estimated by the Synonymous Nonsynonymous Analysis Program (SNAP) [17]. The transition/transversion bias (R) and substitution rates were estimated by MEGA 5.0. The substitution pattern and rates were estimated under the Kimura 2parameter model, using nucleotide frequencies of A = 25.00%, T/ U = 25.00%, C = 25.00%, and G = 25.00%. For estimating ML values, a user-specified topology was used. The maximum Log likelihood values for this computation were 2713.948 and 2737.102. Codon positions included were 1 st +2 nd +3 rd +noncoding. All positions containing gaps and missing data were eliminated, and there were 2580 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5.0 [16,18]. The Data Analysis in Molecular Biology and Evolution program (DAMBE) was used to establish the plots of transition (s) and transversion (v) frequencies against the K80 distance, and to estimate the amino acid substitution propensity [19]. The gB protein phosphorylation sites were predicted using NetPhos 2.0 [20], analysis of the secondary structure was based on the Network Protein Sequence Analysis server (NPS@) [21], and transmembrane topology was analyzed using the Network server [22]. Three-dimensional (3-D) molecular structures were constructed by the Swiss-Model workspace (alignment mode) [23]. The T cell epitopes were predicted by NetChop 3.1 [24], and the image of the T cell epitopes was generated using Protean (DNAstar).

Accession Numbers
The novel PCMV gB nucleotide sequences have been submitted to NCBI GenBank and assigned accession numbers KC342266-KC342289. Further Herpesviridae glycoprotein B nucleotide sequences were obtained from GenBank (http://www.ncbi.nlm. nih.gov/nuccore), Specific accession numbers are given in Figure 2 and Figure 3.

PCMV Detection
The collection locations of the 1025 samples were distributed throughout 14 districts in Sichuan Province. Overall, the PCMV positive infection rate was 84.4% (865/1025), and the infection rate in different tissue samples was 91% (323/355). The rate of infection in serum samples was 80.9% (509/637), and was 100% (33/33) in semen samples. There were a further 25 samples that were putatively positive for PCMV but could not be confirmed, and were therefore not included in the positive rates of infection.

PCR Amplification, Sequence Alignment, and Phylogenic Analysis
PCRs performed with primers P1 and P2 amplified the expected 2580 bp complete PCMV gB gene fragment. The authenticity of the fragments cloned into the pMD19-T Simple Vector was confirmed by agarose gel electrophoresis and DNA sequencing. The 24 nucleotide and deduced amino acid sequences of the PCMV gB genes obtained from samples from 14 different districts of Sichuan Province were used to construct phylogenetic trees using the neighbor joining (NJ) and maximum likelihood (ML) methods. The nucleotide and amino acid sequence data of 18 PCMV and 29 Herpesviridae reference strains obtained from the GenBank database were used for comparison. Bootstrap values (.60) were obtained for both the NJ and ML trees.

Glycoprotein B Sequence Analyses
The 24 novel gB nucleotide sequences and their deduced amino acid sequences were also aligned. The maximum Kimura distance between the PCMV gB nucleotide sequences of the 24 novel Sichuan strains and the other global strains was 2.33%, in comparison with 2.13% between the gB nucleotide sequences of ten Chinese PCMV strains ( Figure 4) 22 of which were found in more than one sequence. Based on a set of codon-aligned gB nucleotide sequences, the dN/dS (nonsynonymous to synonymous substitution) rates among the global and Sichuan strains were 2.39 (0.82/0.6) and 1.82 (0.39/0.27) respectively. The maximum likelihood estimates of the substitution matrix were also calculated ( Table 2).
The 18 deduced glycoprotein B amino acid sequences from the global strains were aligned and analyzed along with the novel 24 Sichuan strains. The maximum index of substitution propensity was 0.9% between the Spanish 55b strain (accession no. AF268040.2), the B6 strain (accession no. AF268039.2), the Japanese OF-1 strain (accession no. AF268041.2), the Japanese Yamaguchi strain (accession no. AB771707.1, AB771708.1, AB771706.1), and the Swedish strain (accession no. AF394056,   (Table 3), including eight phosphorylation site substitutions. The 3-D conformation prediction showed that the substitutions in the loop region and b-turn altered not only the conformation of these regions, but also altered the conformation of the neighboring regions (Figure 7).

Discussion
PCMV is an economically important pathogen due to its immunosuppressive effect of inhibiting the immune function of T lymphocytes and macrophages, and PCMV-infected pigs are Table 3. Data from the 24 PCMV clinical isolates and prototype Sichuan strain (GenBank accession no. JN701021.1). Specimen  Category Amino acid substitutions   28  42  104 153 197 245 290 412 491 791 792 812 824 836 Chengdu KC342266-SCCD01 Hepar nursery swine

District Designation
Pulmonary hilar lymph node Pulmonary hilar lymph node p i g l e t -- easily infected by other pathogens [2,6,25,26]. To survey the prevalence of PCMV in Sichuan Province, China, and provide a foundation for its prevention, a large number of serum, semen, and tissues samples were collected from 14 different districts of the province. The sampling area covered the entire territory of Sichuan Province, including the Chengdu Plain and mountain region of Ganzi Prefecture. The PCMV infection in different tissues of the same pig has not been detected in this study, therefore, the distribution regulation of PCMV in animal body need further research. Nested PCR showed that PCMV infection was widespread in large-scale breeding bases and rural farms of Sichuan, and the antigen positive rate of 84.4% was close to the reported domestic and foreign rates of infection. According to the PCMV infection rates at different growth stages, piglets, nursery pigs, and expectant sows were more vulnerable to PCMV than boars. In this study, we could infer from clinical testing data that vertical transmission was the major transmission mode of PCMV. PCMV antigen detection results from different samples showed the virus is widely distributed in various organs of the body, and mainly affects the immune and respiratory systems. In this study, PCMV was detected for the first time in boar semen. The rate of PCMV  infection in sows from the same farm was very high; therefore, we speculate that PCMV may spread through the reproductive system, and it may be necessary to adopt quarantine measures for infected boars. Based on the results of clinical testing, PCMVinfected pigs were always co-infected with porcine reproductive and respiratory syndrome virus (PRRSV), transfusion transmitted virus (TTV1), and pseudorabies virus (PRV). In addition, Streptococcus, Haemophilus parasuis, Actinobacillus, and Pasteurella suis infections often occur following PCMV infection, making prevention and treatment more difficult. Therefore, the collaborative immune suppression mechanisms between PCMV and other immunosuppressive viruses are worthy of further study, and we believe that this avenue has great significance for prevention and control of immunosuppressive viruses.
PCMV glycoprotein B is an important transmembrane glycoprotein that plays a major role in the fusion and adhesion of the virus onto the membrane of host cells when the virus enters cells or transfers between cells [27]. Glycoprotein B also plays an important role in the induction of humoral and cellular immune responses. It is the target of cytotoxic T lymphocytes, and is recognized by natural killer cells that have been activated by lymphokine. Glycoprotein B induces protective immunity and has significant immunogenicity, therefore, further research could provide a new approach for the treatment and prevention of PCMV [28,29]. Molecular epidemiological analysis of PCMV based on full-length gB gene sequences from different strains and further gene molecular characterization analyses would help to devise a suitable strategy to prevent losses in production.
This study is the first molecular analysis of the full-length PCMV gB nucleotide sequence from PCMV strains isolated worldwide. The nucleotide and amino acid sequences of 24 new Sichuan strains and 13 global strains were aligned and analysed. Phylogenetic tree and homology analysis showed there was no significant variation between gB nucleotide and amino acid sequences from different PCMV strains, confirming that the gB gene sequences from Sichuan and global PCMV strains are highly conserved, and that the viral genome is stable. These factors are conducive to the development of measures for the prevention and treatment of the virus.
We determined that the homologies of gB nucleotide and amino acid sequences investigated in this study were higher than 98% and 97%, respectively. According to the ''75%/85% rule'', if more than 75% of the nucleotide sequence or more than 85% of the amino acid sequence is homologous, the sequences are considered to be from the same serotype [30,31]. This finding confirmed that globally, PCMV strains have not evolved into significantly different serotypes since PCMV was first isolated in 1955 in Britain.
Phosphorylation may affect the stability of the PCMV virus complex. The level of phosphorylation may adjust the binding energy between the viral proteins in the replication complex, and affect the interaction between the viral and host proteins. The results of 3-D conformation prediction showed that the phosphorylation site substitutions of serine, threonine, and tyrosine, at T 28 RY, S 153 RP, N 197 RY, T 290 RA, F 792 RS, Y 812 RD, I 824 RT, and A 836 RT (Figure 7), have likely altered the conformation of the PCMV gB protein. These amino acid substitutions were concentrated in the gB major epitope region in PCMV SC strains, and antigenic drift was affected by accumulation of point mutations among the surface proteins. Mutations in the epitope regions may change the tissue tropism of the virus, therefore, the conformational change in the b-turn and loop regions may have already lead to antigenic drift, changed the immunogenicity of the virus to the host, and affected the specific immune response in the organism. However, whether these changes have altered the capacity of gB for adhesion and penetration of the host cell membrane needs further investigation.

Conclusion
This is the first molecular epidemiological investigation of PCMV in Sichuan Province, China. The PCMV infection rate of 84.4% (865/1025) confirmed that PCMV has been widely distributed in southwest China. PCMV was detected for the first time in porcine semen, allowing us to conclude that it may spread through the reproductive system. The results of nucleotide and amino acid sequence analyses confirmed that the PCMV gB protein is conserved, but that amino acid substitutions in the gB epitope region may cause antigenic drift, and alter the immunogenicity of PCMV.

Ethical Approval
Animal welfare standard in this study was established on the basis of internationally agreed and science based principles within the World Organisation for Animal Health (OIE). All experiments were carried out in accordance with China Animal Welfare Legislation and were approved by the Sichuan Agricultural University Committee on Ethics in the Care and Use of Laboratory Animals.