Specific detection and differentiation of classic goose parvovirus and novel goose parvovirus by TaqMan real-time PCR assay, coupled with host specificity

Background Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. Results After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/μl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. Conclusions We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.


Background
Waterfowl parvoviruses, including goose parvoviruses (GPVs), Muscovy duck parvoviruses (MDPVs) and the variant viruses of GPVs and MDPVs, were renamed as Anseriform dependoparvovirus 1 by the International Committee on Taxonomy of Viruses (ICTV) and have been assigned to the genus Dependoparvovirus in subfamily Parvovirinae under family Parvoviridae based on similarities in phylogenetic properties (https://talk.ictvonline.org/taxonomy/). These viruses contain a linear, single-stranded DNA genome (approximately 5.1 kb in length). Both the 5′-terminal and 3′terminal ends of these viruses have two inverted terminal repeats (ITR) forming a hairpin structure. There are two main open reading frames (ORFs). The left ORF encodes the non-structural protein (NS) responsible for both viral replication and regulation. The right ORF encodes the structural proteins VP1, VP2 and VP3. The VP2 and VP3 contain the same carboxyl-terminal portion as VP1 in these viruses [1][2][3].
GPV infection, also known as Derzsy's disease in Europe, was described in China by Professor Fang in the early 1960s [4]. The virus mainly affects goslings and Muscovy ducklings that are less than one-month-old. Muscovy duck parvovirus infection, also known as "three-week" disease in China, was initially described by Professor Lin in our laboratory in the early 1990s [5]. In contrast to GPV, MDPV infection occurs only in Muscovy ducklings and is characterized by watery diarrhoea, wheezing, and locomotor dysfunction. Both GPVs and MDPVs infections are widespread in China, causing huge economic loss due to the high mortality and morbidity within waterfowl husbandry industries.
Genomic comparison of GPV (strain B, GenBank accession number U25749) and MDPV (strain FM, GenBank accession number U22967) [2] indicated 82.1% nucleotide similarity at the genome level. Furthermore, these strains shared 83.0 and 90.6% nucleotide and amino acids similarity at the NS level and 81.5 and 87.6% nucleotide and amino acids at the VP1 level, respectively. The high similarity at the nucleotide and amino acids level between GPVs and MDPVs may cause false positive results due to MDPV contamination when using a GPV-specific diagnosis method for Muscovy ducklings.
Real-time PCR is an extremely useful tool that has been widely used for viral diagnostic applications. The TaqMan probe, which was designed to bind to a specific region of the target DNA, has shown improved specificity when distinguishing between closely related strains with high nucleotide similarity [6][7][8]. The TaqManbased real-time PCR method (TaqMan qPCR) has been used for GPV detection; Woźniakowski et al. [9] established TaqMan qPCR for both classic GPV and MDPV that targeted the ITR region of the viruses. Confusion when calculating results may occur because the genomes of GPVs and MDPVs share two ITR repeat regions. Additionally, mutations and deletions in the ITR repeat regions were found recently, which may cause false negative results [10][11][12]. Recently, novel GPVs (designated as N-GPVs) causing short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks were found in China [13][14][15]. Niu X et al. [16] and Wang J et al. [17] proposed a TaqMan-based real-time PCR method for the specific detection of N-GPV. The VP3 gene of N-GPV was chosen as the target gene for primer-pairs and probe design, but this only detected N-GPV, not classic GPV (cGPV). Here, we report on the development of a specific TaqMan qPCR for both cGPVs and N-GPVs, which targets the NS differences between GPVs (including cGPVs and N-GPVs) and MDPVs. Based on the host specificity of cGPV (geese, Muscovy ducks, swans and Anser cygnoides) and N-GPV (Cherry Valley ducks, Peking ducks and Mule ducks), our TaqMan qPCR can be used for the specific differentiation of cGPV and N-GPV, coupled with host specificity. sequence. Thus, the probe (FAM-5′-ACCTGGTAATTGTTCYTGC TTCTCT-3′-Eclipse) was designed with a degenerate base (C/T = Y), which allowed the designed GPV-qP probe to cover 33 of 37 (89.19%) isolates. When the 17 MDPV isolates were compared at position 1649-1673, 8 of 15 (53.33%) shared AGAAAACCCGTGGGGACTATCAG GT, 4 of 15 (26.67%) shared AGAAAACCCGTCGGGAC-TATCAGGT, 2 of 15 (13.33%) shared AGAAAA CCCGTGGGGAGTATCAGGT and 1 of 15 (6.67%) shared AGAAAACTCGTGGGGACTATCAGGT. These data showed significant differences between GPVs and MDPVs within the probe design region. The primers GPV-qF and GPV-qR and the TaqMan probe GPV-qP variations are listed in Table 1.

Real-time PCR
The CalQplex software (Mastercycler ep realplex, Eppendorf, Germany) automatically uses the Ct values from plasmid pT-G serial dilutions to calculate the standard curve of the TaqMan real-time PCR assay. The results show Ct values as a function of the amount of different copies of DNA. The standard curve of the assay showed linearity with a slope of − 3.344, Y-intercept of 37.19, efficiency of 99% and R2 of 0.999 (Fig. 1). The detection limit was assessed at 5.02 × 10 1 copies/μl (Fig. 2). For the specificity analysis, both cGPV and N-GPV produced strong fluorescent signals. No cross-reactivity was detected with other pathogens (i.e., E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV]) or cDNA (i.e., DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV) (Fig. 3). For intra-assay variability, low SD values (ranging from 0.11 to 0.55) were observed for each dilution mean and the CVs ranged from 0.58 to 1.74%; for inter-assay variability, low SD values (ranging from 0.12 to 0.75) were observed for each dilution mean and the CVs ranged from 0.66 to 2.37% (listed in Table 2).

Clinical samples application
TaqMan qPCR and cPCR were simultaneously performed on clinical samples. The results are summarized in Table 3. The frequency of GPVs (including cGPVs and N-GPVs) was determined to be 37 and 32% by Taq-Man qPCR and cPCR, respectively. As summarized in Table 4, for 25 Mule duck embryos, 3 embryos (12%) and 2 embryos (8%) tested positive using the TaqMan qPCR and cPCR methods, respectively. For newly hatched Mule ducklings, 5 ducklings (20%) and 3 embryos (12%) tested positive using the TaqMan qPCR and cPCR methods, respectively. Moreover, all cPCR samples tested positive when using the TaqMan qPCR.

Discussion
Real-time PCR technology has proven beneficial for studying the role of viral reactivation, which can help clarify the progression of disease. In contrast to conventional PCR, fluorescence intensity during each PCR cycle is used to quantify real-time PCR amplified products. Currently, there are two major types of real-time PCRs based on fluorescent dye and specificity: double-stranded DNAintercalating dye (e.g., SYBR Green I, Eva Green) and HybProbe-based real-time PCR (e.g., TaqMan-probe, MGB-probe). TaqMan-probe is a representative of the hydrolysis type and is designed to bind to a specific site of the target DNA; this probe has shown improved specificity in distinguishing between closely related strains [6][7][8].
In this study, the real-time PCR probe we designed indicated that 21 of 37 sequences (53.76%) shared the "AGA-GAAGCAGGAACAATTACCAGGT" sequences. These 21 sequences all belonged to the classic-GPV group. Twelve of thirty-seven sequences (32.43%) shared the "AGAGAAG-CAGGAACAATTACCAGGT" secxquence. These 12 sequences all belonged to the N-GPV group. We designed two probes, one (designated as GPV-qP0) was synthesized with "ACCTGGTAATTGTTCCTGCTTCTCT" and the other (designated as GPV-qP) was synthesized with "ACCTGGTAATTGTTCYTGCTTCTCT" using a degenerate base (C/T = Y). After optimizing the real-time PCR, both probes could be used for the quantification of classic GPV and N-GPV, sharing the same detection limit of 5.02 × 10 1 copies/μl. To cover the most frequently occurring GPV, the GPV-qP was then chosen as the TaqMan probe for the present research.
In this study, a total of 52 NS gene sequences (37 GPVs and 15 MDPVs) were compared for primer and probe design. Previous studies showed that NS genes shared characteristic variations between GPVs and MDPVs that could be used to design more precise primers and probes [18,19]. Using a similar strategy, a TaqMan real-time PCR for the detection and quantification of GPV was developed and Previous studies provided evidence that cGPV could spread via vertical transmission in geese [20,21]. Similarly, there was possible vertical transmission of N-GPV between breeder Cherry Valley and Pekin ducks to their ducklings [22,23]. Classic MDPV shared the same phenomenon of possible vertical transmission, similar to our recent work [18]. In this study, we demonstrated that N-GPV appeared to possible vertically transfer from breeder Mule ducks to ducklings. Thus, future countrywide surveillance in Mule ducks should be enhanced.

Conclusions
Based on the characteristic variable regions of NS genes in GPVs and MDPVs, we developed a specific detection of cGPV and N-GPV by TaqMan real-time PCR assay. Moreover, cGPV and N-GPV could be distinguished using the assay coupled with host specificity. Furthermore, our results demonstrated that N-GPV may be able to transmit vertically from breeding Mule ducks to ducklings.

Primers and probe selection
Previous studies demonstrated that the NS gene homology between GPVs (cGPVs and N-GPVs) and MDPVs ranged from 80.8 to 83.4% and can be used for GPVs and MDPVs differentiation [18,19]. After a bioinformatics analysis of the NS genes of GPVs (cGPVs and N-GPVs) and MDPVs specific primers and a probe were designed using Primer Premier Software version 5.0 (Premier Biosoft, Palo Alto, CA, USA) following a similar strategy that we used to develop a specific TaqMan-based real-time PCR for MDPV. Detailed information regarding the primers and probe is shown in Table 1. The amplicon was 158-bp in length. The GPV-qF (5′-TAGGGAGGAGTTAGAAGA-3′), the GPV-qR (5′-CATCCATAGAATTGTCATAAGTA-3′), and the GPV-qP (FAM-5′-ACCTGGTAATTGTTCYTGCTT CTCT-3′-Eclipse) were synthesized by a commercial company (TaKaRa, Dalian, China). DNA and cDNA were quantified using a NANODROP 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and stored at − 80°C until use.

Plasmid construction
The partial NS gene of cGPV (strain G7, GenBank accession number KR029617) [3] was amplified by PCR with the primer sets forward primer (GNSF) 5′-ATA-CATATTGCACTACCTGATAC-3′ and reverse primer (GNSR) 5′-TTATTGTTCATTTTCAGCATCATC-3′. The amplified PCR products were then analysed with electrophoresis on 1.0% agarose gels. The expected PCR amplicons were T-A cloned using the pMD18-T Vector Cloning Kit (TaKaRa, Dalian, China). The recombinant plasmids were then sequenced in both directions using the Sanger method by a commercial company (Sangon, Shanghai, China). The selected plasmid, pT-G, was quantified using a NANODROP 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The number of plasmid pT-G copies was calculated using the following formula [24]. Ten-fold dilutions of the plasmid pT-G, ranging from 5.02 × 10 7 to 5.02 × 10 0 copies/μl, were prepared with EASY Dilution (TaKaRa, Dalian, China). Each diluted plasmid, with different aliquots, was stored at − 80°C until use.

Real-time PCR protocol optimization
The TaqMan qPCR assay was developed and validated with a Mastercycler ep realplex (Eppendorf, Germany). Different concentrations of the primers and probe were   prepared into reaction tubes to optimize the assay by evaluating the highest fluorescence and lowest threshold cycle (Ct). The reaction concentrations were determined as follows: 12.5 μl of Premix Ex Taq (Probe qPCR, TaKaRa, Dalian, China), 0.6 μl of each primer (GPV-qF and GPV-qR, 10 μmol/l each), 1.2 μl of probe (GPV-qP, 10 μmol/l), 1 μl of DNA template, and Nuclease-free water in an amount to adjust the total reaction volume to 25 μl. The following thermoprofile was set: 1 cycle of 95°C for 30 s, 40 cycles of 95°C for 5 s, 58°C for 10 s, and 72°C for 15 s.
To determine the reproducibility of the real-time PCR, plasmid pT-G at concentrations of 5.02 × 10 5 , 5.02 × 10 3 , and 5.02 × 10 1 copies/μl were used to evaluate the coefficient of variation (CV). These plasmids were repeatedly amplified three different times daily to assess intra-assay variability and three different times weekly to assess inter-assay variability. The CVs were calculated according to the formula using the geometric mean Ct value deviation.

Clinical samples application
A total of 100 individual dead suspected cases of infected waterfowls (geese, Muscovy ducks, Cherry Valley ducks, and Mule ducks, 25 birds in each group on the basis of species) ( Table 3) were used to validate the Taq-Man qPCR assay. These birds were collected from privately owned animals via participating veterinary hospital (namely as Poultry Disease Treatment Centre, a department of our institute). Each liver tissue was centrifuged at 4000 rpm at 4°C for 30 min after mechanical grinding. Viral DNA was extracted with EasyPure Viral DNA/RNA Kit (TransGen Biotech, Beijing, China). Conventional PCR (cPCR) was also performed simultaneously to detect infections in the above samples [25]. Based on the host specificity of cGPV and N-GPV, positive signals from geese and Muscovy ducks were considered to be cGPV-positive, whereas positive signals from Cherry Valley ducks and Mule ducks were considered to be N-GPV-positive.

Vertical transmission application
Previous studies confirmed that cGPV could spread through vertical transmission to susceptible young goslings via eggs. The same phenomenon can also be found with N-GPV in Cherry Valley ducks and Pekin ducks. To test the hypothesis that N-GPV could be vertically transmitted in Mule ducks, 25 Mule duck embryos (18day post fertilization) and 25 newly hatched Mule ducklings (1-day-old), were collected from farms where N-GPV infections has previously occurred. The liver of each embryo and newly hatched duckling was designated as one sample. Viral DNA was extracted with EasyPure Viral DNA/RNA Kit (TransGen Biotech, Beijing, China). These samples were also simultaneously assayed using the cPCR method.