Development and application of quadruplex real time quantitative PCR method for differentiation of Muscovy duck parvovirus, Goose parvovirus, Duck circovirus, and Duck adenovirus 3.

Introduction: Muscovy duck parvovirus (MDPV), Goose parvovirus (GPV), Duck circovirus, (DuCV) and Duck adenovirus 3 (DAdV-3) are important pathogens that cause high morbidity and mortality in ducks, causing huge economic loss for the duck industry. Methods: The present study, a quadruplex one-step real time quantitative PCR method for the detection of MDPV, GPV, DuCV, and DAdV-3 was developed. Results: The results showed that assay had no cross-reactivity with other poultry pathogens [Duck plague virus (DPV), Duck tembusu virus (DTMUV), H6 avian influenza virus (H6 AIV), New duck reovirus (NDRV), Newcastle disease virus (NDV), H4 avian influenza virus (H4 AIV), Escherichia coli (E. coli), Muscovy duck reovirus (MDRV), Egg drop syndrome virus (EDSV), Pasteurella multocida (P. multocida)]. The sensitivity result showed that the limits of detection for MDPV, GPV, DuCV, and DAdV-3 were 10, 10, 1 and 10 copies/µl, respectively; The coefficients of variation intra- and inter-method was 1-2%; The range of linear (109 to 103 copies/µL) demonstrated the R2 values for MDPV, GPV, DuCV, and DAdV-3 as 0.9975, 0.998, 0.9964, and 0.996, respectively. The quadruplex real time quantitative PCR method efficiency was 90.30%, 101.10%, 90.72%, and 90.57% for MDPV, GPV, DuCV, and DAdV-3, respectively. 396 clinical specimens collected in some duck sausages from June 2022 to July 2023 were simultaneously detected using the established quadruplex real time quantitative PCR method and the reported assays. The detection rates for MDPV, GPV, DuCV, and DAdV-3 were 8.33% (33/396), 17.93% (71/396), 33.58% (133/396), and 29.04% (115/396), respectively. The agreement between these assays was greater than 99.56%. Discussion: The developed quadruplex real-time quantitative PCR assay can accurately detect these four viruses infecting ducks, providing a rapid, sensitive, specific and accurate technique for clinical testing.

Development and application of a novel recombinase polymerase amplification-Pyrococcus furiosus argonaute system for rapid detection of goose parvovirus.

Rapid and accurate detection of goose parvovirus (GPV) is crucial for controlling outbreaks and mitigating their economic impact on the poultry industry. This study introduces recombinase polymerase amplification combined with the Pyrococcus furiosus argonaute (RPA-PfAgo) system, a novel diagnostic platform designed to address the limitations of traditional GPV detection methods. Capitalizing on the rapid DNA amplification of RPA and stringent nucleic acid cleavage by the PfAgo protein, the RPA-PfAgo system offers high specificity and sensitivity in detecting GPV. Our optimization efforts included primer and probe configurations, reaction parameters, and guided DNA selection, culminating in a detection threshold of 102 GPV DNA copies per microlitre. The specificity of the proposed method was rigorously validated against a spectrum of avian pathogens. Clinical application to lung tissues from GPV-infected geese yielded a detection concordance of 100%, surpassing that of qPCR and PCR in both rapidity and operational simplicity. The RPA-PfAgo system has emerged as a revolutionary diagnostic modality for managing this disease, as it is a promising rapid, economical, and onsite GPV detection method amenable to integration into broad-scale disease surveillance frameworks. Future explorations will extend the applicability of this method to diverse avian diseases and assess its field utility across various epidemiological landscapes.

Bone lesions and intestinal barrier disruption caused by the isolated novel goose parvovirus infection in ducks.

Short beak and dwarfism syndrome (SBDS) is attributed to Novel Goose Parvovirus (NGPV), which has inflicted significant economic losses on farming in China. Despite its significant impact, limited research has been conducted on the pathogenesis of this disease. The SD strain, a parvovirus variant isolated from ducks in Shandong province, was identified and characterized in our study. Phylogenetic analysis and sequence comparisons confirmed the classification of the SD strain as a member of NGPV. Based on this information, we established an animal model of SBDS by inoculating Cherry Valley ducks with the SD strain. Our findings indicate that infection with the SD strain leads to a reduction in body weight, beak length, width, and tibia length. Notably, significant histopathological alterations were observed in the thymus, spleen, and intestine of the infected ducks. Furthermore, the SD strain induces bone disorders and inflammatory responses. To evaluate the impact of NGPV on intestinal homeostasis, we performed 16S rDNA sequencing and gas chromatography to analyze the composition of intestinal flora and levels of short-chain fatty acids (SCFAs) in the cecal contents. Our findings revealed that SD strain infection induces dysbiosis in cecal microbial and a decrease in SCFAs production. Subsequent analysis revealed a significant correlation between bacterial genera and the clinical symptoms in NGPV SD infected ducks. Our research providing novel insights into clinical pathology of NGPV in ducks and providing a foundation for the research of NGPV treatment targeting gut microbiota.

First detection of Tetraparvovirus ungulate 1 in diseased cattle (Chinese Simmental) from Hunan province, China.

Tetraparvovirus is an emerging parvovirus infecting a variety of mammals and humans, and associated with human diseases including severe acute respiratory infection and acute encephalitis syndrome. In the present study, a Tetraparvovirus ungulate 1 (formerly known as bovine hokovirus) strain HNU-CBY-2023 was identified and characterized from diseased Chinese Simmental from Hunan province, China. The nearly complete genome of HNU-CBY-2023 is 5346 nt in size and showed genomic identities of 85-95.5% to the known Tetraparvovirus ungulate 1 strains from GenBank, indicating a rather genetic variation. Phylogenetic and genetic divergence analyses indicated that Tetraparvovirus ungulate 1 could be divided into two genotypes (I and II), and HNU-CBY-2023 was clustered into genotype II. This study, for the first time, identified Tetraparvovirus ungulate 1 from domestic cattle from mainland China, which will be helpful to understand the prevalence and genetic diversity of Tetraparvovirus ungulate 1.

Short beak and dwarfism syndrome among Pekin ducks: First detection, full genome sequencing, and immunohistochemical signals of novel goose parvovirus in tongue tissue.

Novel goose parvovirus (NGPV) is continuously threatening the global duck industry, as it causes short beak and dwarfism syndrome among different duck breeds. In this study, we investigated the viral pathogenesis in the tongue of affected ducks, as a new approach for deeper understanding of the syndrome. Seventy-three, 14- to 60-day-old commercial Pekin ducks were clinically examined. Thirty tissue pools of intestine and tongue (15 per tissue) were submitted for molecular identification. Clinical signs in the examined ducks were suggestive of parvovirus infection. All examined ducks had short beaks. Necrotic, swollen, and congested protruding tongues were recorded in adult ducks (37/73, 51%). Tongue protrusion without any marked congestion or swelling was observed in 20-day-old ducklings (13/73, 18%), and no tongue protrusion was observed in 15-day-old ducklings (23/73, 32%). Microscopically, the protruding tongues of adult ducks showed necrosis of the superficial epithelial layer with vacuolar degeneration. Glossitis was present in the nonprotruding tongues of young ducks, which was characterized by multifocal lymphoplasmacytic aggregates and edema in the propria submucosa. Immunohistochemical examination displayed parvovirus immunolabeling, mainly in the tongue propria submucosa. Based on polymerase chain reaction, goose parvovirus was detected in 9 out of 15 tongue sample pools (60%). Next-generation sequencing confirmed the presence of a variant goose parvovirus that is globally named NGPV and closely related to Chinese NGPV isolates. Novel insights are being gained from the study of NGPV pathogenesis in the tongue based on molecular and immunohistochemical identification.

Correlation between goose circovirus and goose parvovirus with gosling feather loss disease and goose broke feather disease in southern Taiwan.

BACKGROUND: Goslings in several Taiwanese farms experienced gosling feather loss disease (GFL) at 21-35 days and goose broke feather disease (GBF) at 42-60 days. The prevalence ranges from a few birds to 500 cases per field. It is estimated that about 12,000 geese have been infected, the morbidity is 70-80% and the mortality is 20-30%. OBJECTIVES: This study aims to investigate the pathogens that cause GFL and GBF. Focus on the study of the correlation between goose circovirus (GoCV) and goose parvovirus (GPV) with the goose feather loss in southern Taiwan. Furthermore, a phylogenetic tree was established to align the differences between southern and northern Taiwan and compare with virus strains from China and Europe. METHODS: Samples were collected from animal hospitals. Molecular and microscopy diagnostics were used to examine 92 geese. Specific quantitative polymerase chain reaction (Q-PCR) assays are performed to evaluate GPV and GoCV viral loads and simultaneously evaluated the feather loss conditions in geese with the scoring method. RESULTS: High prevalence of GoCV and GPV infection in geese showing signs of GFL and GBF. Inclusion body was detected in the feather follicles and Lieberkühn crypt epithelial cells. The Q-PCR showed the high correlation between feather loss and viruses during 3rd-5th week. However, the infection was not detected using the same test in 60 healthy geese. CONCLUSIONS: Thus, GFL and GBF appear to be significantly closely related to GoCV and GPV. The geese feathers showed increasing recovery after being quarantined and disinfected.

Molecular survey of selected viral pathogens in wild leopard cats (Prionailurus bengalensis) in Taiwan with an emphasis on the spatial and temporal dynamics of carnivore protoparvovirus 1.

The leopard cat (Prionailurus bengalensis) was listed as an endangered species under the Wildlife Conservation Act in Taiwan in 2009. However, no study has evaluated the possible direct or indirect effects of pathogens on the Taiwanese leopard cat population. Here, we targeted viral pathogens, including carnivore protoparvovirus 1 (genus Protoparvovirus), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), coronaviruses (CoVs), and canine distemper virus (CDV), through molecular screening. The spatial and temporal dynamics of the target pathogens were evaluated. Through sequencing and phylogenetic analysis, we clarified the phylogenetic relationship of viral pathogens isolated from leopard cats and domestic carnivores. Samples from 23 live-trapped leopard cats and 29 that were found dead were collected from 2015 to 2019 in Miaoli County in northwestern Taiwan. Protoparvoviruses and CoVs were detected in leopard cats, and their prevalence (95% confidence interval) was 63.5% (50.4%-76.6%) and 8.8% (0%-18.4%), respectively. Most of the protoparvovirus sequences amplified from Taiwanese leopard cats and domestic carnivores were identical. All of the CoV sequences amplified from leopard cats were identified as feline CoV. No spatial or temporal aggregation of protoparvovirus infection in leopard cats was found in the sampling area, indicating a wide distribution of protoparvoviruses in the leopard cat habitat. We consider sympatric domestic carnivores to be the probable primary reservoir for the identified pathogens. We strongly recommend management of protoparvoviruses and feline CoV in the leopard cat habitat, particularly vaccination programs and population control measures for free-roaming dogs and cats.

First investigation of the prevalence of parvoviruses in slaughterhouse pigs and genomic characterization of ungulate copiparvovirus 2 in Vietnam.

Ungulate protoparvovirus 1, also known as porcine parvovirus 1 (PPV1), is considered to be one of the major causes of reproductive failure in pig breeding herds. Other parvoviruses have also been identified in pigs, including ungulate tetraparvovirus 3, or PPV2, ungulate tetraparvovirus 2, or PPV3, and ungulate copiparvovirus 2, or PPV4, but their significance for pigs is unknown. In the present study, the prevalence of PPV1-4 was investigated using a total of 231 lung and serum samples collected from slaughterhouses in 13 provinces throughout Vietnam. The overall prevalence was 54.5% (126/231) for PPV1, 28.0% (65/231) for PPV2, 17.7% (41/231) for PPV3, and 7.8% (18/231) for PPV4. While PPV1 and PPV2 were found in 11 provinces, PPV4 was detected in only three provinces. Co-circulation of PPV1, PPV2 and PPV3 was frequently observed, with PPV1/PPV2 coinfection predominating, with 20.8% (48/231). All four PPVs were detected together in only one sample from Thua Thien Hue. Three nearly complete PPV4 genome sequences of 5,453 nt were determined and deposited in the GenBank database. Alignment and comparison of the three genome sequences showed 99.5-99.6% nucleotide sequence identity, and the deduced amino acid sequences of open reading frames 1-3 were 99.6-99.9% identical to each other, 98.9-99.3% identical to those of other Vietnamese strains and 99.4-99.7% identical to those of Chinese strains). Phylogenetic analysis further confirmed a close relationship between Vietnamese and Chinese PPV4 strains. These results are the first to report the prevalence of PPV1, PPV2, PPV3, and PPV4 and nearly complete genomic sequences of PPV4 in pigs from slaughterhouses in Vietnam.

Domestic dog origin of Carnivore Protoparvovirus 1 infection in a rescued free-ranging guiña (Leopardus guigna) in Chile.

Carnivore protoparvovirus 1 is one of the most important pathogens affecting both wild and domestic carnivores. Here, we reported the genetic characterization of canine parvovirus (CPV-2) strains from a rescued guiña (Leopardus guigna) and domestic dogs from Chile. Guiña strain was classified as CPV-2c, and phylogenetic analysis of the complete coding genome showed that the guiña CPV-2c strain shares a recent common ancestor with Chilean domestic dogs' strains. These viruses showed >99% identity and exhibited three changes in the NS1 protein (V596A, E661K and L582F). This is the first detection and genetic characterization of CPV-2c infection in guiña worldwide, and one of the few comparative studies that show the source of infection was domestic dogs. The current findings highlight the fact that guiña is a susceptible species to protoparvovirus infection and that domestic dogs represent an important threat to its conservation. The CPV-2 cross-species transmission between domestic dogs and guiña should be taken into account for protection programmes of this endangered species.

Isolation and characterization of a recombinant Muscovy duck parvovirus circulating in Muscovy ducks in South China.

In 2019, flocks of Muscovy ducks presented with clinical signs typical of MDPV infection. The MDPV GD201911 strain was isolated by inoculating samples from positive birds into Muscovy duck embryos. Challenge with the isolate GD201911 caused typical MDPV disease symptoms and resulted in 25%-40% mortality, depending on the challenge dose, indicating the high pathogenicity of GD201911 for Muscovy ducks. Genome sequencing and phylogenetic analysis demonstrated that GD201911 clustered with recombinant MDPV strains, indicating that recombinant MDPV is circulating in China. Epidemiological monitoring should be performed continuously to assist with decision making for disease control.

Simultaneous detection of duck circovirus and novel goose parvovirus via SYBR green I-based duplex real-time polymerase chain reaction analysis.

Beak atrophy and dwarfism syndrome (BADS) is commonly caused by co-infection with duck circovirus (DuCV) and novel goose parvovirus (NGPV). Therefore, concurrent detection of both viruses is important for monitoring and limiting BADS, although such a diagnostic test has not been reported. In this study, we developed a duplex, SYBR Green I-based real-time polymerase chain reaction (PCR) assay to enable the simultaneous detection of DuCV and NGPV. The assay readily distinguished between the two viruses, based on their different melting temperatures (Tm), where the Tm for DuCV was 80 °C and that for NGPV was 84.5 °C. Other non-target duck viruses that were tested did not show melting peaks. The detection limit of the duplex assay was 101 copies/µL for both viruses. This method exhibited high repeatability and reproducibility, and both the inter-assay and intra-assay variation coefficients were <1.6%. Thirty-one fecal samples were collected for clinical testing using real-time PCR analysis, and the results were confirmed using sequencing. The rate of co-infection was 6.5%, which was consistent with the sequencing results. This duplex real-time PCR assay offers advantages over other tests, such as rapid, sensitive, specific, and reliable detection of both viruses in a single sample, which enables the quantitative detection of DuCV and NGPV in clinical samples. Using this test may be instrumental in reducing the incidence of BADS and the associated economic losses in the duck and goose industries.

Virome of a Feline Outbreak of Diarrhea and Vomiting Includes Bocaviruses and a Novel Chapparvovirus.

An unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and PCR. We characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by 8/17 affected cats and identified three different feline bocaviruses shed by 9/17 cats. Also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. Epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease.

Purification of Recombinant Adeno-Associated Virus 2 (rAAV2) by Heparin Column Affinity Chromatography.

This protocol describes a simple single-step column purification (SSCP) of rAAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation.

Identification of a novel bovine copiparvovirus in pooled fetal bovine serum.

A novel parvovirus was identified as a cell culture contaminant by metagenomic analysis. Droplet digital PCR (ddPCR) was used to determine viral loads in the cell culture supernatant and further analysis, by ddPCR and DNA sequencing, demonstrated that fetal bovine serum (FBS) used during cell culture was the source of the parvovirus contamination. The FBS contained ~ 50,000 copies of the novel parvovirus DNA per ml of serum. The viral DNA was resistant to DNAse digestion. Near-full length sequence of the novel parvovirus was determined. Phylogenetic analysis demonstrated that virus belongs to the Copiparvovirus genus, being most closely related to bovine parvovirus 2 (BPV2) with 41% identity with the non-structural protein NS1 and 47% identity with the virus capsid protein of BPV2. A screen of individual and pooled bovine sera identified a closely related variant of the novel virus in a second serum pool. For classification purposes, the novel virus has been designated bovine copiparvovirus species 3 isolate JB9 (bocopivirus 3-JB9).

Detection and molecular characterization of novel porcine bufaviruses in Guangxi province.

Bufavirus (BuV) can infect a variety of hosts, including human, bats, rats, dog, swine and shrew species and are suggested related to diarrhea disease. Porcine bufaviruses (PoBuV) were first detected in Hungarian pig farms in 2016. To determine the prevalence and genetic diversity of PoBuV in China, we developed SYBR Green-based real-time PCR assays to detect PoBuV in Guangxi pigs. Real-time PCR detected PoBuV in 30 (29.13%, 30/103) of the samples with diarrhoeal intestinal tissues and rectal swabs. PoBuV-positive intestinal tissues and rectal swabs samples, co-infection with PEDV (15/30, 50.0%), followed by PDCoV (8/30, 26.67%), PoRV (6/30, 20.0%), PRRSV (5/30, 16.67%), and PCV2 (3/30, 10.0%) were observed. Fourteen complete genomes were cloned and sequenced. The results showed that they were 4189 bp in length and combined three open reading frames (ORFs) in the order 5'-NS1-VP1/VP2-3'. Fourteen strains shared 96.5%-99.8% identity among themselves and 92.7%-97.9% with the PoBuV reference sequences. Phylogenetic analysis based on the deduced amino acid sequence of the VP2 gene showed fourteen strains belonging to PoBuV and were grouped into the three branches. These results help to provide new insight into the molecular epidemiology of PoBuV in the world.

Research Note: Development of an ELISA to distinguish between goose parvovirus infection and vaccine immunization antibodies.

Goose parvovirus (GPV) leads to a huge loss in the poultry industry, and early diagnosis is required to prevent the disease from spreading. At present, there are a variety of detection methods for GPV infection, and the ELISA method has the advantages of simple and rapid operation. However, most ELISA methods for detecting GPV can only detect the antibody level of the sample, but cannot distinguish between the GPV infection and vaccine immunization antibodies. Therefore, this study has a wider application value by establishing the detection method based on the structure and non-structural protein of the virus. The GPV non-structural (NS1) and structure (VP3) fusion proteins were used as coating antigens to establish 2 indirect ELISA methods, and the detection conditions were optimized. A series of experiments proved that the detection method has good specificity, sensitivity, and repeatability. The test results of 120 immune sera samples and 145 natural infection serum samples showed that the positive rates of immunized serum were 9.17% (NS1) and 88.33% (VP3), and the positive rates of natural infection were 88.97% (NS1) and 86.21% (VP3), which distinguish between the GPV infection and vaccine immunization antibodies. The establishment of 2 indirect ELISA methods using NS1 and VP3 proteins as inclusion antigens provides a new method for detecting GPV infection and inactivated immune antibodies, which lays a foundation for the serological diagnosis and epidemiological monitoring of GPV.

Identification of a common conserved neutralizing linear B-cell epitope in the VP3 protein of waterfowl parvoviruses.

Waterfowl parvoviruses (WPVs) including goose parvovirus (GPV), novel GPV-related virus (NGPV) and Muscovy duck parvovirus (MDPV) cause significant economic losses and epizootic threat to the waterfowl industries, and little is known about the B-cell epitopes of WPVs. In this study, a monoclonal antibody (mAb) 5B5 against the VP3 protein of NGPV was used to identify the possible epitope in the three kinds of WPVs. The mAb 5B5 had neutralizing activities to the three viruses, and reacted with the conserved linear B-cell epitopes of 438LHNPPP443 in VP3 protein of GPV, NGPV and MDPV. To the authors' best knowledge, this is the first report on identification of the common conserved neutralizing linear B-cell epitope on VP3 protein of three different WPVs, which would facilitate the development of a novel immunodiagnostic assay for rapid detection of WPV infection.

Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR.

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/µL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.

Bocaparvovirus, Erythroparvovirus and Tetraparvovirus in New World Primates from Central America.

Parvoviruses in the genera Bocaparvovirus (HBoV), Erythroparvovirus (B19) and Tetraparvovirus (PARV4) are the only autonomous parvoviruses known to be associated with human and non-human primates based on studies and clinical cases in humans worldwide and non-human primates in Asia and Africa. Here, the presence of these agents with pathogenic potential was assessed by PCR in blood and faeces from 55 howler monkeys, 112 white-face monkeys, 3 squirrel monkeys and 127 spider monkeys in Costa Rica and El Salvador. Overall, 3.7% (11/297) of the monkeys had HboV DNA, 0.67% (2/297) had B19 DNA, and 14.1% (42/297) had PARV4 DNA, representing the first detection of these viruses in New World Primates (NWP). Sex was significantly associated with the presence of HBoV, males having greater risk up to nine times compared with females. Captivity was associated with increased prevalence for PARV4 and when all viruses were analysed together. This study provides compelling molecular evidence of parvoviruses in NWPs and underscores the importance of future research aimed at understanding how these viruses behave in natural environments of the Neotropics and what variables may favour their presence and transmission.

Lack of detection of Cutavirus DNA using PCR real time in cutaneous T-cell lymphomas.

BACKGROUND: A novel human protoparvovirus named Cutavirus has been discovered. We investigated the presence of Cutavirus in a sample of Cutaneous T-cell lymphomas by using PCR real time TaqMan® (Thermo Fisher Scientific, Waltham, MA, USA). METHODS: In total, 55 CTCL samples were analyzed using a TaqMan® Real time PCR on a 7500 ABI instrument. All of these shown internal control amplification. RESULTS: The presence of Cutavirus DNA corresponding was examined. CuV DNA sequences were not detected in any skin specimen. CONCLUSIONS: The role of Cutaviruses in cutaneous cancers remains to be investigated.