First identification of canine parvovirus -2a/2b variant in unvaccinated domestic dogs with gastrointestinal signs in Türkiye.

BACKGROUND: Canine parvovirus type 2 (CPV-2) is the most common enteric virus that infects canids. CPV is the causative agent of a contagious disease defined mostly by clinical gastrointestinal signs in dogs. During the late 1970s, CPV-2 emerged as a new virus capable of infecting domestic dogs and growing across the world. The VP2 gene stands out as a key determinant in the pathogenicity, antigenicity, and host interactions of CPV-2. AIMS: The molecular characterization of the VP2 gene is crucial for understanding CPV evolution and epidemiology. MATERIALS & METHODS: Genes encoding the VP2 protein were sequenced and compared to reference strains worldwide. The maximum likelihood method was used to build a phylogenetic tree using CPV VP2 gene nucleotide sequences. RESULTS: Our phylogenetic analysis of the VP2 gene revealed that five strains were very similar and clustered together, and three strains were in the 2b clade, whereas the other two were in the 2a/2b clade. DISCUSSION: This paper reports the molecular characterization of two novel CPV-2a/2b subtypes in dogs with gastrointestinal symptoms. Genetic analysis was conducted on a CPV genomic region encompassing one of the open reading frames (ORFs) encoding the structural protein VP2. Sequence analysis indicates new and unreported sequence changes, mainly affecting the VP2 gene, which includes the mutations Ser297Ala and Leu87Met. This study represents the first evidence of a new CPV-2a/2b subtype in Türkiye. Due to VP2's crucial role in encoding the capsid protein of CPV-2 and its significant involvement in the host-virus interaction, it is critical to closely monitor its evolutionary changes and be cautious while searching for novel or pre-existing subtypes. CONCLUSION: This study highlights the significance of continuous molecular research for acquiring more insights on the circulation of novel CPV mutants.

Detection of canine parvovirus type 2c (CPV-2c) in Palestine.

INTRODUCTION: The objective of the present study was to report, for the first time, the presence of canine parvovirus type 2c (CPV-2c) in domesticated dogs with acute gastroenteritis and to characterize the antigenic variants circulating in Palestine. METHODOLOGY: A veterinary clinical-based epidemiological study was carried out between December 2022 and April 2023. Fifty fecal samples were collected from dogs with gastroenteritis and screened for CPV-2 infection by polymerase chain reaction. The distribution of positive cases according to various epidemiological factors was studied. Partial sequencing of the viral protein 2 (VP2) gene was performed for the analysis of CPV-2 variants. RESULTS: Most of the investigated samples (60%; n = 50) during the study period were found positive for CPV-2 infection. There was no difference in the distribution of positive cases of CPV-2 infection based on age group, gender, location, and vaccination status. The analysis of nucleotide and amino acid sequences from amplified products, as well as phylogenetic analysis, revealed the presence of CPV-2c clustered with Asian CPV-2c variants. CONCLUSIONS: In summary, this study represents the initial genetic analysis of CPV-2 present in Palestinian dogs with gastroenteritis and provides evidence that confirms the existence of the CPV-2c variants. To determine the prevailing CPV-2 variant associated with the infection, it is crucial to conduct further sequence analysis using large populations of both domestic and wild canines.

Molecular characterization of full-length VP2 gene of canine parvovirus type 2 strains circulating in Egypt 2019-2021.

Canine parvovirus type 2 (CPV-2) is a major cause of fatal gastroenteritis and myocarditis in puppies of domestic and wild carnivores. CPV-2 has accumulated changes over time lead to the emergence of three antigenic variants CPV-2a, CPV-2b, and CPV-2c. VP2 is the major capsid protein that determines virus antigenicity, and host range. Although the three CPV-2 variants were previously identified in Egypt, most reports covered a restricted geographic region and/or time period, and only analyzed partial fragments of VP2 gene. Therefore, this study was designed to test 100 rectal swabs collected from 7 Egyptian governorates between 2019 and 2021 for CPV-2 using PCR. A total of 65 positive samples were identified, mostly in pure dog breeds of young age. The three variants co-circulated in 2019, while CPV-2b was not detected in 2020 and 2021. The frequency of CPV-2b and CPV-2c was higher in 2019 and 2021, respectively. Analysis of CPV-2 full-length VP2 gene sequence from 19/65 positive samples has identified four common amino acid substitutions F267Y, S297A, A300G, Y324I, which are characteristic for the new CPV-2 variants currently circulating worldwide. Unique substitutions including A5G, G36R, V38E, Q370R, and G392V were recognized in certain samples, and appears to have distinct effect on receptor binding, nuclear translocation, and inter-species transmission. Phylogenetic analysis showed separation of CPV-2 strains into two clades. All strains of this study were classified in clade I with Asian strains. In conclusion, this study provides updated comprehensive molecular analysis of CPV-2 variants in Egypt.

Development and characterization of a novel specific monoclonal antibody against mink enteritis virus and its antigen epitope analysis.

This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as IgG1, the titers of the MAb is up to 1:1 × 106 and keeps stably after low-temperature storage for 9 months or 11 passages of the MAb cells. The MAb can specifically recognize MEV in the cells in IFA, but not Aleutian disease virus (ADV) or canine distemper virus (CDV). Its antigen epitope was identified as a polypeptide containing 5 key amino acids (378YAFGR382) and the homology in 20 MEV strains, 4 canine parvovirus strains, and 4 feline panleukopenia virus strains was 100%. This study supplies a biological material for developing new methods to detect MEV.

Global distribution, cross-species transmission, and receptor binding of canine parvovirus-2: Risks and implications for humans.

For canine parvovirus -2 (CPV-2), a zoonotic virus capable of cross-species transmission in animals, the amino acid changes of capsid protein VP2 are key factors when binding to other species' transferrin receptors (TfR). CPV-2 variants can spread from felines and canines, for example, to Carnivora, Artiodactyla, and Pholidota species, and CPV-2c variants are essential to spread from Carnivora to Artiodactyla and Pholidota species in particular. In our study, a CPV-2a variant maintained a relatively stable trend, and the proportion of CPV-2c gradually rose from 1980 to 2021. The VP2 amino acid sequence analysis showed that five amino acid mutations at 426E/D, 305H/D, and 297S may be necessary for the virus to bind to different host receptors. Meanwhile, receptor-binding loop regions and amino acid sites 87 L, 93 N, 232I, and 305Y were associated with CPV-2 cross-species transmission. The homology of TfRs in different hosts infected with CPV-2 ranged from 77.2 % to 99.0 %, and from pig to feline, canine, and humans was 80.7 %, 80.4 %, and 77.2 %, respectively. The amino acid residues of TfRs involved in the viral binding in those hosts are highly conserved, which suggests that CPV-2 may be capable of pig-to-human transmission. Our analysis of the origin, evolutionary trend, cross-species transmission dynamics, and genetic characteristics of CPV-2 when binding to host receptors provides a theoretical basis for further research on CPV-2's mechanism of cross-species transmission and for establishing an early warning and monitoring mechanism for the possible threat of CPV-2 to animal-human public security.

Development of a time-resolved fluorescence immunoassay kit for detecting canine coronavirus and parvovirus through double labeling.

OBJECTIVE: Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases. METHODS: A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated. RESULTS: A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than - 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%. CONCLUSION: In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.

Detection and genetic characterization of circulating canine parvovirus from stray dogs in Shanghai, China.

Canine parvovirus (CPV) is the main cause of viral diarrhea in dogs. CPV became a global disease in 1978 and was endemic all over the world. CPV-2 was the first strain to be identified, but with genetic mutations, new genotypes such as CPV-2a/2b/2c/new-2a/new-2b have emerged. In this study, 128 fecal samples of stray dogs suspected of CPV-2 infection were collected from January to March 2021 in Shanghai, China. All samples were screened by PCR and further analyzed by VP2 gene. The positive rate of CPV-2 was 9.4% (12/128), of which 6 CPV-2 isolates were successfully isolated. Phylogenetic tree analysis showed that 4 isolates were CPV-2c genotype and 2 were new-CPV-2b genotype. VP-2 is a key protein that determines the antigenic properties, host range and receptor binding of cpv-2. The results of VP2 amino acid sequence analysis in this study showed that the CPV-2c isolated strain was the same as the previous strains reported in China, including F267Y, Y324I, Q370R and A5G mutations in addition to the typical N426E mutations. Similarly, in addition to the conventional N426D, S297A, F267Y and Y324I mutations, the new CPV-2b isolate also had a new mutation of T440A. This study further confirmed the prevalence of CPV-2c and new-CPV-2b in Shanghai, and also found a new mutation site of new-CPV-2c, which provided a theoretical basis for further enriching the epidemiological data of CPV-2 in Shanghai, as well as the development of vaccines and the prevention and control of the disease.

Clinical and inflammatory response to antiviral treatments in dogs with parvoviral enteritis.

BACKGROUND: Canine parvoviral enteritis (CPE) is a fatal disease worldwide. The treatment of CPE is based mainly on supportive and symptomatic treatment. Antiviral addition to the treatment may result in a higher survival. OBJECTIVES: This study evaluated the effects of antiviral treatments with a standardized treatment (ST) on the clinical and inflammatory response of dogs with naturally occurring CPE. METHODS: Twenty-eight dogs with CPE caused by canine parvovirus type 2 were divided randomly into treatment groups. The ST group received fluid, antibiotic, antiemetic, and deworming treatments. The antiviral treatment groups received the same ST with an additional antiviral drug, recombinant feline interferon omega (rFeIFN-ω), oseltamivir (OSEL) or famciclovir (FAM). RESULTS: Compared to the healthy control, the tumor necrosis factor-α, interleukin-1ß, interferon (IFN)-α, IFN-γ, haptoglobin, and C-reactive protein values were high (p < 0.05) on day zero. At presentation, mild lymphopenia, neutropenia, and a high neutrophil to lymphocyte (LYM) ratio (NLR) were also observed. Adding rFeIFN-ω to the ST produced the best improvement in the clinical score with a decreased NLR, while leucocytes remained low and inflammatory markers stayed high on day three. The survival rates of the groups were 85.7% in ST+IFN, 71.4% in ST+OSEL, 71.4% in ST+FAM, and 57.1% in ST groups on day seven. CONCLUSIONS: Antiviral drugs may be valuable in treating CPE to improve the clinical signs and survival. In addition, the decrease in NLR in favor of LYM may be an indicator of the early prognosis before the improvement of leukocytes, cytokines, and acute phase proteins in CPE.

Epidemiological survey of infectious agents in free-ranging maned wolves (Chrysocyon brachyurus) in Northeastern Brazil.

Infectious diseases are one of the most concerning threats to maned wolves (Chrysocyon brachyurus) due to the potential impact on free-ranging populations. The species is currently classified as vulnerable according to the national list of threatened species and occurs mainly in open habitats, such as the Cerrado, a tropical savannah, which comprises its main distribution area in Brazil. In the northeastern region, it occurs in the Cerrado of Bahia, Piauí, Maranhão, and Tocantins states. Therefore, this study aimed to investigate the occurrence of infectious agents in Chrysocyon brachyurus through an epidemiological assessment of free-ranging individuals in western Bahia, specifically in the Barreiras microregion, a Cerrado area intensely fragmented and anthropized by agricultural activity. Eleven specimens were evaluated for serological titration, antigen research, and genetic material research for canine distemper virus (CDV), canine parvovirus (CPV), adenovirus-canine-type 1 (CAdV-1), canine coronavirus (CCoV), Leptospira interrogans and Toxoplasma gondii from 2020 to 2022. In addition to maned wolves, domestic dogs were also evaluated and tested. All maned wolves (100%) evaluated by the dot-ELISA technique exhibited immunoglobulin M (IgM) and seven (64%) exhibited immunoglobulin G (IgG) against CDV and CPV, while 100% exhibited IgG against CDV when using the immunochromatographic technique. Regarding CAdV-1, 90% were seropositive for IgG, while 64% exhibited IgG against T. gondii. Nine dogs from the region were also sampled, and all (100%) exhibited IgM and IgG against CDV and CPV. For IgG against T. gondii and against CAdV-1, 90% of the animals were seropositive. Molecular evaluation yielded negative results for all maned wolves and dogs assessed for CAdV-1, CDV, and T. gondii, as well as the CCoV antigen. These data indicate the occurrence of viral agents and Toxoplasma gondii in maned wolves and dogs, suggesting circulation in both populations.

Genetic characterization of the parvovirus full-length VP2 gene in domestic cats in Brazil.

Feline parvovirus (FPV) and canine parvovirus (CPV) are over 98% identical in their DNA sequences, and the new variants of CPV (2a/2b/2c) have gained the ability to infect and replicate in cats. The aim of this study was to determine the genetic diversity in the VP2 gene of parvovirus strains circulating in domestic cats in Brazil during a 10-year period (2008-2017). For parvovirus screening, specific PCR was performed, and 25 (34.7%) of 72 cats tested positive. The PCR-positive samples were further subjected to full-length VP2 sequencing (1755 bp), and eight sequences (36%) were characterized as FPV, seven (28%) as CPV-2a and (32%) nine (36%) as CPV-2b. One sequence (RJ1085/11) showing typical CPV amino acid (aa) at residues 80 R, 93 N, 103 A, 232 I, and 323 N could not be characterized at this time. The sequences in this study displayed aa changes previously described for FPV (A14T, A91S, I101T, N564S, and A568G) from cats and CPV-2a/2b (S297N and Y324L) from dogs. However, the Y324L mutation has not yet been reported in any CPV-2a/2b strains from cats. Phylogenetic analysis supported the division of these sequences into two well-defined clades, clade 1 for FPV and clade 2 for CPV2a/2b. Unusually, the sequence RJ1085/11 was grouped separately. Two recombination breakpoints were detected by Bootscan and 3Seq methods implemented in the RDP4. This study is the first report of CPV-2a/2b in cats in Brazil. The detection of FPV strains with mutations characteristic of CPV indicates that Brazilian FPV strains have undergone genetic changes.

Sensitivity of lateral flow technique for diagnosis of canine parvovirus.

In this study, we devised a nanogold lateral flow immunoassay (LFA-CPV antigen test) for detecting canine parvovirus (CPV) in living attenuated CPV vaccines. We conducted instrumental characterization of the prepared nanogold particles and the developed LFA-CPV antigen test was rigorously evaluated for its performance verification including limit of detection, sensitivity, specificity, selectivity and accuracy. The LFA-CPV antigen test demonstrated strong performance when assessed against qPCR using different batches of live attenuated CPV vaccines, indicated a sensitivity of 96.4%, specificity of 88.2%, and an overall accuracy of 95%. These results suggest that the developed LFA-CPV antigen test could serve as a viable alternative for evaluation live attenuated CPV vaccines, and provide it as a point of care test for CPV diagnosis, offering a potential substitute for traditional laboratory methods, particularly qPCR.

Canine parvovirus type 2 (CPV-2) serological and molecular patterns in dogs with viral gastroenteritis from southern Brazil.

Canine Parvovirus type 2 (CPV-2) is a highly contagious virus that can cause severe systemic disease with gastroenteric symptoms in dogs, particularly in young puppies. Originating from the feline parvovirus in the late 1970s, it swiftly propagated globally, instigating a pandemic in dogs. Despite vaccination advancements, CPV-2 remains a substantial challenge for veterinary professionals and pet owners. This study aimed to contribute knowledge about the current situation of CPV-2 among dogs in southern Brazil. In this study, the sera of 125 dogs (mostly with gastroenteritis symptoms) were screened for antibodies against CPV-2 and their faeces for the virus itself. The results showed that 40% (50/125) of dogs were infected with CPV-2. Most animals (65.5%) had previously been exposed to CPV-2 (with serotitres equal or above 1:40), and only 37.6% had protective antibody titres equal or above 1:80. The findings have also demonstrated that vaccination against CPV-2 significantly reduced the risk of infection, with positive cases decreasing from 56.9% (unvaccinated) to 2.0% (fully vaccinated). Furthermore, the prevalence of CPV-2 decreased as dogs aged, with younger dogs and those with an incomplete or non-existent vaccination history at the highest risk of infection. In conclusion, this study provides valuable insight into the prevalence and risk factors associated with CPV-2 infection in dogs in southern Brazil, thereby providing valuable knowledge for the improvement of veterinary care and pet health.

First detection and genetic characterization of canine bufavirus in domestic dogs, Thailand.

Canine bufavirus (CBuV) was reported in domestic dogs worldwide. We conducted a survey of canine bufavirus in domestic dogs in Thailand from September 2016 to October 2022. Rectal swab samples (n = 531) were collected from asymptomatic dogs and dogs with gastroenteritis signs. The samples were tested for CBuV using PCR with specific primers to the VP1/VP2 gene, and 9.42% (50/531) was CBuV positive. Our findings showed that CBuVs could be detected in both symptomatic and healthy dogs. The Thai CBuVs were found in dogs from different age groups, with a significant presence in those under 1 year (12.60%) and dogs aged 1-5 years (7.34%) (p < 0.05), suggesting a high prevalence of Thai CBuVs in dogs under 5 years of age. We performed complete genome sequencing (n = 15) and partial VP1/VP2 sequencing (n = 5) of Thai CBuVs. Genetic and phylogenetic analyses showed that whole genomes of Thai CBuVs were closely related to Chinese and Italian CBuVs, suggesting the possible origin of Thai CBuVs. The analysis of VP1 and VP2 genes in Thai CBuVs showed that 18 of them were placed in subgroup A, while only 2 belonged to subgroup B. This study is the first to report the detection and genetic characterization of CBuVs in domestic dogs in Thailand. Additionally, surveillance and genetic characterization of CBuVs in domestic animals should be further investigated on a larger scale to elucidate the dynamic, evolution, and distribution of CBuVs.

Transcriptional Differential Analysis of Nitazoxanide-Mediated Anticanine Parvovirus Effect in F81 Cells.

Canine parvovirus (CPV) is a single-stranded DNA virus that can cause typical hemorrhagic enteritis, and it is one of the common canine lethal viruses. In previous studies, we screened the Food and Drug Administration (FDA)'s drug library and identified nitazoxanide (NTZ), which has anti-CPV capabilities. To investigate the potential antiviral mechanisms, we first reconfirmed the inhibitory effect of NTZ on the CPV by inoculating with different doses and treating for different lengths of time. Then, the differences in the transcription levels between the 0.1%-DMSO-treated virus group and the NTZ-treated virus group were detected using RNA-seq, and a total of 758 differential expression genes (DEGs) were finally identified. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs revealed that these genes are involved in a variety of biological processes and/or signaling pathways, such as cell cycle, mitosis and cell proliferation and differentiation. A protein-protein interaction (PPI) analysis further identified hub genes associated with cell cycle and division among the DEGs. In addition, the expression levels of some of the enriched genes were detected, which were consistent with the high-throughput sequencing results. Moreover, when the cell cycle was regulated with cell cycle checkpoint kinase 1 (Chk1) inhibitor MK-8776 or Prexasertib HCl, both inhibitors inhibited the CPV. In summary, the transcriptome differential analysis results presented in this paper lay the foundation for further research on the molecular mechanism and potential targets of NTZ anti-CPV.

Early administration of canine parvovirus monoclonal antibody prevented mortality after experimental challenge.

OBJECTIVE: To evaluate the effectiveness of canine parvovirus monoclonal antibody (CPMA) as a treatment against canine parvovirus (CPV-2)-induced mortality and to support USDA product licensure. ANIMALS: 28 purpose-bred Beagle dogs aged 8 weeks were randomized to the treated (n = 21) or control (7) group. METHODS: Dogs were challenged intranasally with 104.2 TCID50 virulent CPV-2b on Day 0 and monitored for 14 days for fecal viral shed and clinical disease. All dogs began shedding CPV-2 on Day 4 and were treated intravenously with a single dose of either CPMA (0.2 mL/kg) or saline (equal volume). No additional treatments were given to either group. Feces and sera were collected for quantitative analysis of fecal viral shed (hemagglutination) and antibody responses (hemagglutination inhibition and dot-blot ELISA), respectively. Dogs were monitored twice daily for parameters including lymphopenia, fever, vomiting, abnormal feces, inappetence, and lethargy. Humane endpoints triggered euthanasia by a veterinarian masked to treatment groups. The primary outcome variable was prevention of mortality as compared to controls. RESULTS: Mortality was prevented in all CPMA-treated dogs compared to 57% mortality in the control group (P = .0017, Fisher exact test). Canine parvovirus monoclonal antibody-treated dogs also experienced less severe and/or shorter durations of diarrhea, fever, vomiting, CPV-2 shedding in feces, and lymphopenia. Both groups showed similar immunoglobulin M responses as measured by semiquantitative analysis. CLINICAL RELEVANCE: Intravenous administration of CPMA can effectively improve clinical outcome when administered early in CPV-2 disease. Canine parvovirus monoclonal antibody treatment after proven infection does not interfere with adaptive immunity.

A novel lateral flow immunochromatographic assay using a recombinant VP2 antigen for total antibody detection of canine parvovirus-2.

Canine parvovirus-2 (CPV-2) is a viral disease of dogs causing acute hemorrhagic gastroenteritis and myocarditis with high morbidity and mortality rates. The infection is still widespread all over the world. Vaccines developed against infection have great importance in preventing infection. However, it is difficult to recommend a practical vaccination program without knowing the antibody level of a puppy. Despite widespread vaccination, difficulties in detecting the maternal antibodies in puppies remain the main cause of vaccination failure. The hemagglutination inhibition (HAI) test is the gold standard to determine the immune status of dogs for canine parvovirus 2, but the HAI test has several disadvantages such as the need for fresh porcine blood, well-equipped laboratory, and long incubation periods. In this study, for the first time we developed a colloidal gold-based competitive lateral flow assay (cLFA) system for the rapid detection of total antibodies in canine serum using CPV-2b-VP2 derived from field isolates. The recombinantly expressed capsid protein of CPV-2 in the prokaryotic expression system was used as a labeled molecule in cLFA. We carried out studies on our cLFA system using the standard antibody solution and the clinical samples from vaccinated puppy serum. We compared the results of the LFAs with the HAI test. Competitive lateral flow assay results showed good correlation with the gold standard method, the HAI test. In the developed platform, the limit of detection of the standard antibody was determined to be 375 ng mL-1, while the cut-off level of antibodies was observed to be 1 : 40 HAI titer in clinical samples. Our reported system will be a strong alternative for CPV-2 antibody-based detection applications.

Inhibition of canine parvovirus 2 (CPV-2) replication by TAT-scFv through targeting of the viral structural protein VP2 of CPV-2.

Canine parvovirus (CPV) causes severe infectious disease with a high mortality rate in dogs. CPV is still a major health issue of dogs in the clinic. Therefore, there is an urgent need to develop effective drugs to treat the disease. In this study, we fused the transactivating transcriptional activator peptide (TAT) with scFv. TAT-scFv was identified by Western blot. CCK8 kit was used to detect the toxicity of TAT-scFv to cells. The binding activity of TAT-scFv to CPV-2-VP2 was detected by DAS ELISA. The cell uptake rate of TAT-scFv was assessed by IFA. After infection with CPV-2, F81 cells were incubated by TAT-scFv. The replication of virus was measured to determine the neutralization effect of TAT-scFv on intracellular and extracellular viruses. Protein docking was used to predict the amino acid (AA) sites of VP2 binding to TAT-scFv. TAT-scFv was expressed in Escherichia coli and purified. The DAS ELISA showed that TAT-scFv could bind with CPV-2-VP2. We demonstrated that TAT-scFv entered cells in a dose-dependent and time-dependent manner and effectively inhibited the replication of CPV-2. Using protein docking, we determined the interaction pattern and found that the N-terminal region (AA 41-49) and the C-terminal region (AA 558) of VP2 interacted with the TAT-scFv. Taken together, these results suggest that, TAT-scFv may be a potential antiviral drug for inhibiting CPV-2 replication and controlling disease caused by CPV-2.

Development of an accurate and rapid method for whole genome characterization of canine parvovirus.

Canine parvovirus is a highly contagious pathogen affecting domestic dogs and other carnivores globally. Monitoring CPV through continuous genomic surveillance is crucial for mapping variability and developing effective control measures. Here, we developed a method using multiplex-PCR-next-generation sequencing to obtain full-length CPV genomes directly from clinical samples. This approach utilizes tiling and tailed amplicons to amplify overlapping fragments of roughly 250 base pairs. This enables the creation of Illumina libraries by conducting two PCR reaction runs. We tested the assay in 10 fecal samples from dogs diagnosed with CPV and one CPV-2 vaccine strain. Furthermore, we applied it to a feline sample previously diagnosed with the feline panleukopenia virus. The assay provided 100 % genome coverage and high sequencing depth across all 12 samples. It successfully provided the sequence of the coding regions and the left and right non-translated regions, including tandem and terminal repeats. The assay effectively amplified viral variants from divergent evolutionary groups, including the antigenic variants (2a, 2b, and 2c) and the ancestral CPV-2 strain included in vaccine formulations. Moreover, it successfully amplified the entire genome of the feline panleukopenia virus found in cat feces. This method is cost-effective, time-efficient, and does not require lab expertise in Illumina library preparation. The multiplex-PCR-next-generation methodology facilitates large-scale genomic sequencing, expanding the limited number of complete genomes currently available in databases and enabling real-time genomic surveillance. Furthermore, the method helps identify and track emerging CPV viral variants, facilitating molecular epidemiology and control. Adopting this approach can enhance our understanding of the evolution and genetic diversity of Protoparvovirus carnivoran1.

Investigation of circulating infectious agents in experimental Beagle dogs of a production colony and three research facilities in China from June 2021 to May 2022.

To understand the epizootiologic characteristics of pathogens and opportunistic infections in one Beagle dog production colony and three research facilities, viruses and mycoplasma were detected in 1777 samples collected from Beagle dogs in China by polymerase chain reaction/reverse transcription polymerase chain reaction, and bacteria were isolated and identified by 16S rRNA sequence analysis. In addition, genotyping of the major circulating viruses was carried out by amplification of gene fragments and homology analysis. Canine coronavirus (CCoV), Escherichia coli, canine parvovirus (CPV), Bordetella bronchiseptica, Clostridium perfringens, Mycoplasma cynos, Klebsiella pneumoniae, Streptococcus canis, canine astrovirus (CaAstV), canine kobuvirus (CaKV), Pseudomonas aeruginosa, Proteus mirabilis, Macrococcus canis, Pasteurella canis, canine bocavirus (CBoV) and canine adenovirus (CAdV) were detected in the samples. Single, double, triple and quadruple infections accounted for 6.6%, 1.4%, 1.2% and 0.96% of samples, respectively. CCoV strains in 81 samples included three genotypes, CCoV-I, CCoV-IIa and CCoV-IIb, by analysis of S gene. The rate of single infection of CCoV-I, CCoV-IIa or CCoV-IIb was 19%, 38% or 7.4% respectively. The double and triple infection rates of CCoV were 32.8% and 2.5% respectively. All CPV strains in 36 samples belonged to CPV-2c. There were three amino acid differences in the Fiber protein of CAdV-positive sample QD2022, compared with the reference strain Toronto A26/61 and the vaccine strain YCA-18. These results suggest that CCoV and CPV are primary infectious agents, and that these two viruses were often identified in mixed infections, or coinfections alongside mycoplasma or other bacteria. These results will provide the basis for improvements in prevention and control of naturally occurring infectious diseases in Beagle dog production colonies and research facilities.

Canine bufavirus (Carnivore protoparvovirus-3) infection in dogs with respiratory disease.

Canine bufavirus (CBuV) or Carnivore protoparvovirus-3, a nonenveloped DNA virus belonging to the genus Protoparvovirus, family Parvoviridae, has been identified in dogs with respiratory and enteric diseases. Although CBuV detection has been reported in multiple countries, descriptions of pathologic findings associated with infection have not yet been provided. In this study, the authors necropsied 14 dogs (12 puppies and 2 adult dogs) from a breeding colony that died during multiple outbreaks of respiratory diseases. Postmortem investigations revealed extensive bronchointerstitial pneumonia with segmental type II pneumocyte hyperplasia in all necropsied puppies but less severe lesions in adults. With negative results of common pathogen detection by ancillary testing, CBuV DNA was identified in all investigated dogs using a polymerase chain reaction (PCR). Quantitative PCR demonstrated CBuV DNA in several tissues, and in situ hybridization (ISH) indicated CBuV tissue localization in the lung, tracheobronchial lymph node, and spinal cord, suggesting hematogenous spread. Dual CBuV ISH and cellular-specific immunohistochemistry were used to determine the cellular tropism of the virus in the lung and tracheobronchial lymph node, demonstrating viral localization in various cell types, including B-cells, macrophages, and type II pneumocytes, but not T-cells. Three complete CBuV sequences were successfully characterized and revealed that they clustered with the CBuV sequences obtained from dogs with respiratory disease in Hungary. No additional cases were identified in small numbers of healthy dogs. Although association of the bufavirus with enteric disease remains to be determined, a contributory role of CBuV in canine respiratory disease is possible.