Whole genome sequence analysis of CPV-2 isolates from 1998 to 2020.

Canine parvovirus-2 (CPV-2) is a virus with worldwide spread causing canine gastroenteritis. New strains of this virus have unique characteristics and are resistant to some vaccine strains. Therefore, understanding the root causes of resistance has proven to be of increasing concern to many scientists. This study collected 126 whole genome sequences of CPV-2 subtypes with specific collection dates from the NCBI data bank. The whole genome sequences of CPV-2 collected from different countries were analyzed to detect the new substitutions and update these mutations. The result indicated 12, 7, and 10 mutations in NS1, VP1, and VP2, in that respective order. Moreover, the A5G and Q370R mutations of VP2 are the most common changes in the recent isolates of the CPV-2C subtype, and the new N93K residue of VP2 is speculated to be the cause of vaccine failure. To summarize, the observed mutations, which are increasing over time, causes several changes in viral characteristic. A comprehensive understanding of these mutations can lead us to control potential future epidemics associated with this virus more efficiently.

Spillover of Canine Parvovirus Type 2 to Pigs, South Dakota, USA, 2020.

In 1978, canine parvovirus type 2 originated from spillover of a feline panleukopenia-like virus, causing a worldwide pandemic of enteritis and myocarditis among canids. In 2020, the virus was identified in pigs in South Dakota, USA, by PCR, sequencing, in situ hybridization, and serology. Genetic analysis suggests spillover from wildlife.

Evaluating the surrogacy of multiple vaccine-induced immune response biomarkers in HIV vaccine trials.

Identifying biomarkers as surrogates for clinical endpoints in randomized vaccine trials is useful for reducing study duration and costs, relieving participants of unnecessary discomfort, and understanding vaccine-effect mechanism. In this article, we use risk models with multiple vaccine-induced immune response biomarkers to measure the causal association between a vaccine's effects on these biomarkers and that on the clinical endpoint. In this setup, our main objective is to combine and select markers with high surrogacy from a list of many candidate markers, allowing us to get a more parsimonious model which can potentially increase the predictive quality of the true markers. To address the missing "potential" biomarker value if a subject receives placebo, we utilize the baseline immunogenicity predictor design augmented with a "closeout placebo vaccination" group. We then impute the missing potential marker values and conduct marker selection through a stepwise resampling and imputation method called stability selection. We test our proposed strategy under relevant simulation settings and on (partially simulated) biomarker data from a HIV vaccine trial (RV144).

Molecular characterization and antiviral effects of canine interferon regulatory factor 1 (CaIRF1).

BACKGROUND: Interferon regulatory factor 1 (IRF1) is an important transcription factor that activates the type I interferon (IFN-I) response and plays a vital role in the antiviral immune response. Although IRF1 has been identified in several mammals, little information related to its function in canines has been described. RESULTS: In this study, canine IRF1 (CaIRF1) was cloned. After a series of bioinformatics analyses, we found that the CaIRF1 protein structure was similar to that of other animal IRF1 proteins, including a conserved DNA-binding domain (DBD), an IRF-association domain 2 (IAD2) domain and two nuclear localization signals (NLSs). An indirect immunofluorescence assay (IFA) revealed that CaIRF1 was mainly distributed in the nucleus. Overexpression of CaIRF1 in Madin-Darby canine kidney cells (MDCK) induced high levels of interferon ß (IFNß) and IFN-stimulated response element (ISRE) promoter activation and induced interferon-stimulated gene (ISG) expression. Subsequently, we assayed the antiviral activity of CaIRF1 against vesicular stomatitis virus (VSV) and canine parvovirus type-2 (CPV-2) in MDCK cells. Overexpression of CaIRF1 effectively inhibited the viral yields of VSV and CPV-2, while knocking down of CaIRF1 expression mildly increased viral gene copies. CONCLUSIONS: CaIRF1 is involved in the cellular IFN-I signaling pathway and plays an important role in the antiviral response.

Molecular characteristics and genetic evolutionary analyses of circulating parvoviruses derived from cats in Beijing.

BACKGROUND: Feline parvovirus (FPV) is a member of the family Parvoviridae, which is a major enteric pathogen of cats worldwide. This study aimed to investigate the prevalence of feline parvovirus in Beijing of China and analyze the genetic features of detected viruses. RESULTS: In this study, a total of 60 (8.5%) parvovirus-positive samples were detected from 702 cat fecal samples using parvovirus-specific PCR. The complete VP2 genes were amplified from all these samples. Among them, 55 (91.7%) sequences were characterized as FPV, and the other five (8.3%) were typed as canine parvovirus type 2 (CPV-2) variants, comprised of four CPV-2c and a new CPV-2b strain. In order to investigate the origin of CPV-2 variants in cats, we amplified full-length VP2 genes from seven fecal samples of dogs infected with CPV-2, which were further classified as CPV-2c. The sequences of new CPV-2b/MT270586 and CPV-2c/MT270587 detected from feline samples shared 100% identity with previous canine isolates KT156833 and MF467242 respectively, suggesting the CPV-2 variants circulating in cats might be derived from dogs. Sequence analysis indicated new mutations, Ala91Ser and Ser192Phe, in the FPV sequences, while obtained CPV-2c carried mutations reported in Asian CPV variants, showing they share a common evolutionary pattern with the Asian 2c strains. Interestingly, the FPV sequence (MT270571), displaying four CPV-specific residues, was found to be a putative recombinant sequence between CPV-2c and FPV. Phylogenetic analysis of the VP2 gene showed that amino acid and nucleotide mutations promoted the evolution of FPV and CPV lineages. CONCLUSIONS: Our findings will be helpful to further understand the circulation and evolution of feline and canine parvovirus in Beijing.

Transferrin receptor binds virus capsid with dynamic motion.

Canine parvovirus (CPV) is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Cross-species transmission of CPV occurs as a result of mutations on the viral capsid surface that alter the species-specific binding to the host receptor, transferrin receptor type-1 (TfR). The interaction between CPV and TfR has been extensively studied, and previous analyses have suggested that the CPV-TfR complex is asymmetric. To enhance the understanding of the underlying molecular mechanisms, we determined the CPV-TfR interaction using cryo-electron microscopy to solve the icosahedral (3.0-Å resolution) and asymmetric (5.0-Å resolution) complex structures. Structural analyses revealed conformational variations of the TfR molecules relative to the binding site, which translated into dynamic molecular interactions between CPV and TfR. The precise footprint of the receptor on the virus capsid was identified, along with the identity of the amino acid residues in the virus-receptor interface. Our "rock-and-roll" model provides an explanation for previous findings and gives insights into species jumping and the variation in host ranges associated with new pandemics in dogs.

Identification and Molecular Characterization of a Divergent Asian-like Canine Parvovirus Type 2b (CPV-2b) Strain in Southern Italy.

Canine parvovirus type 2 (CPV-2) is an infectious agent relevant to domestic and wild carnivorans. Recent studies documented the introduction and spread of CPV-2c strains of Asian origin in the Italian canine population. We investigated tissue samples from a puppy collected during necropsy for the presence of viral enteropathogens and all samples tested positive only for CPV-2. The full coding sequence of a CPV-2b (VP2 426Asp) strain was obtained. This virus was related to CPV-2c strains of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (NS1: 60Val, 545Val, 630Pro; VP2: 5Gly, 267Tyr, 324Ile) and mutations rarely reported in Asian CPV-2b strains (NS1: 588N; VP2: 370Arg). Phylogenetic analyses placed this strain in well-supported clades, including Asian CPV-2c-like strains, but always as a basal, single-sequence long branch. No recombination was observed for this strain, and we speculate that it could have originated from an Asian CPV-2c-like strain that acquired the 426Asp mutation. This study reports the first evidence of an Asian-like CPV-2b strain in Italy, confirming the occurrence of continuous changes in the global CPV-2 spread. Since positive convergent mutations complicate data interpretation, a combination of phylogenetic and mutation pattern analyses is crucial in studying the origin and evolution of CPV-2 strains.

Criminological profile of minors who have committed child-to-parent violence.

The rise of CPV cases in the last decade has become a matter of concern among researchers, who have investigated prevalence rates and factors related to this type of behavior. This study aims to analyze the criminological profile of the minors who have committed CPV compared to minors who have committed other type of crimes. The participants were 341 juveniles with a disciplinary record in the Juvenile Court of a Spanish province, whose ages ranged from 14 to 17 years old (M = 15.86, SD = 1.02). The results showed that the CPV group represented a moderate level of recidivism and the comparison group had a low risk of recidivism. The CPV group had mostly committed CPV, while the comparison group had tended to commit property crimes. The CPV group had generally served probation or confinement sentences, while the comparison group had mostly been acquitted or served probation.

Generation and evaluation of IgY-scFv based mimetics against canine parvovirus.

Antibody mimetics may be used for various biomedical applications, especially those for which conventional antibodies are ineffective. In this study, we developed a smaller molecular chicken IgY mimetic peptide (IgY-peptide) based on the complementarity-determining regions (CDRs) of the anti-canine parvovirus (CPV) IgY-scFv prepared previously. The mimetic peptide showed no cross-reactivity with canine distemper virus (CDV) and canine coronavirus (CCV) and showed excellent protective properties for Crandell-Rees Feline Kidney (CRFK) cells against CPV. This study is the first attempt to develop a mimetic IgY-peptide and demonstrates the ease and feasibility in generating such a novel antibody-like functional molecule for biomedical purposes.

A novel one-step amplification refractory mutation system PCR (ARMS-PCR) for differentiation of canine parvovirus-2 variants.

Enteritis caused by CPV-2 antigenic variants (CPV-2a, 2b, and 2c) is frequently reported in dogs worldwide leading to significant morbidity and mortality. Here, we describe about a simple, single-step, ARMS-PCR strategy targeting the mutant 426 amino acid of VP2 to differentiate CPV-2 antigenic types. A total of 150 fecal samples were subjected to ARMS-PCR of which 18 were typed as CPV-2a, 79 were typed as CPV-2b, and 6 were typed as CPV-2c. The ARMS-PCR results were validated by randomly sequencing partial VP2 gene of 14 samples. Phylogenetic analysis of partial VP2 gene sequencing of each of the CPV-2 variants revealed that CPV-2a and CPV-2b isolates formed a separate clade of Indian lineage, while CPV-2c shared common evolutionary origin with Asian lineage. The developed technique is first of its kind, one-step, rapid, sequencing independent method for typing of CPV-2 antigenic variants.

Identification and characterization of DNA aptamers specific to VP2 protein of canine parvovirus.

Canine parvovirus-2 (CPV-2) is ubiquitously distributed in dog population worldwide causing a severe and often fatal gastroenteritis. Owing to its highly contagious nature, rapid detection of CPV is crucial in effective control of the disease. Aptamers have emerged as potential alternative to antibodies as affinity reagents in diagnostic field. Present study was aimed to select and validate ssDNA aptamers specific to CPV. Systematic evolution of ligands through exponential enrichment (SELEX) method was employed for selection of CPV structural protein (VP2) specific DNA aptamers. SELEX was performed using a pool of ssDNA library comprising 30 random nucleotide region. A total of seven rounds of SELEX were performed using VP2 protein as target antigen which yielded ten aptamers (1A-10A) with distinct sequences. Target binding of all ten aptamers was assessed by dot blot and enzyme-linked oligonucleotide assay (ELONA) which revealed that 5A, 6A, 9A, and 10A were superior binders. In silico analysis of the aptamers revealed the existence of binding site on VP2 protein, and binding pattern was similar to in vitro findings. The affinity (KD) of all these four binders against CPV was evaluated by ELONA indicating relatively higher affinity of 6A and 10A than remaining two DNA sequences. Out of which, aptamer 6A displayed cross-reactivity with canine distemper virus and canine corona virus. Hence, aptamer 10A was considered as better binding sequence having high specificity and affinity for CPV. The study confirms the future utility of selected aptamers in development of a reliable diagnostic for rapid detection of CPV. KEY POINTS: • Canine parvovirus-specific ssDNA aptamers were identified with nanomolar affinity (100-150 nM). • Three aptamers displayed negligible cross-reactivity with other related viruses. • Aptamer 10A displayed high binding affinity and specificity to CPV.

The detection of canine parvovirus type 2c of Asian origin in dogs in Romania evidenced its progressive worldwide diffusion.

BACKGROUND: Canine parvovirus (CPV) is one of the most important pathogens of dogs. Despite vaccination, CPV infections are still ubiquitous in dogs, and the three antigenic variants 2a, 2b and 2c are variously distributed in the canine population worldwide. To date, no information is available on CPV variants circulating in some European countries. The aim of this study was to genetically characterise the CPV detected in ten dogs with clinical signs of acute gastroenteritis in Romania. The presence of Carnivore protoparvovirus 1 DNA was investigated in faecal samples using an end-point PCR targeting the complete VP2 gene and positive amplicons were sequenced and analysed. RESULTS: All ten dogs with acute gastroenteritis tested positive to Carnivore protoparvovirus 1 DNA in faecal samples. The identified viruses belonged to CPV-2c type, showed identical sequences of the VP2 gene and were characterised by distinctive amino acid residues in the deduced VP2 protein: 5-glicine (5Gly), 267-tirosine (267Tyr), 324-isoleucine (324Ile) and 370-arginine (370Arg). These distinctive amino acid residues have already been reported in CPV-2c widespread in Asia and occasionally detected in Italy and Nigeria. CONCLUSIONS: Since CPV-2c with VP2 amino acid residues 5Gly, 267Tyr, 324Ile and 370Arg were never reported before 2013, it can be assumed that this virus is progressively expanding its spread in the world dog population. This study adds new data about the presence of this new virus in Europe and underline worrying questions about its potential impact on the health of the canine population.

Assessment of certain biomarkers for predicting survival in response to treatment in dogs naturally infected with canine parvovirus.

Canine parvovirus (CPV) enteritis is an important cause of morbidity and mortality in puppies despite aggressive treatment. Identification of reliable biomarkers for CPV enteritis is essential to determine the severity, duration of hospitalization, and predict the clinical outcome. Meanwhile, the biomarkers will assist in decision-making with clients about the further course of treatment or euthanasia. The present study was conducted to evaluate the changes of total leukocyte count (TLC), neutrophil count, and serum concentrations of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), intestinal fatty acid binding protein-2 (IFABP-2), albumin, ceruloplasmin (Cp), cortisol, free triiodothyronine (FT3) and free thyroxine (FT4) in survivors and non-survivors as a predictor of the clinical outcome. Marked leukopenia, neutropenia, hypoalbuminemia, elevated levels of CK-MB, IFABP-2, Cp, and cortisol were noticed in CPV-infected dogs than healthy dogs but, LDH, FT3 and FT4 concentrations did not differ significantly. The CPV-infected non-survivors had persistent leukopenia, neutropenia and elevated CK-MB, IFABP-2, Cp and cortisol concentrations at 72 h of commencement of treatment. In CPV-infected survivors, TLC and neutrophil count were significantly increased, and CK-MB, IFABP-2, Cp and cortisol concentrations were significantly decreased at 72 h of commencement of treatment. The positive predictive values (PPVs) for survival using cut-off value of TLC (>3.2 × 103/µL), neutrophil count (>1.65 × 103/µL), CK-MB (≤234.50 U/L), IFABP-2 (≤7.61 ng/mL), Cp (≤0.605 g/L) and cortisol (≤16.90 ng/mL) were determined as 89.47%, 88.88%, 94.73%, 93.33%, 94.44% and 89.47%, respectively with better area under receiver operating characteristic (ROC) curve as well as sensitivity. The magnitude of decrease in TLC, neutrophil count, and increase in CK-MB, IFABP-2, Cp and cortisol concentrations at 72 h of initiation of treatment in dogs with parvoviral enteritis could be useful indicators for the prognosis of the disease. Based on sensitivity (%) and specificity (%) from ROC curve analysis and PPV (%), it is concluded that serum CK-MB concentration will serve as the most useful biomarker followed by Cp and absolute neutrophil count.

A systematic literature review and meta-analysis of characterization of canine parvoviruses 2 prevalent in mainland China.

BACKGROUND: Canine parvovirus 2 (CPV-2) is a pathogenic virus that infects dogs, causing a highly infectious disease. Monitoring CPV-2 spread is an important part of prevention; however, the prevalence and epidemiological characteristics of CPV-2 have not been systematically evaluated and analyzed in mainland China. Therefore, a systematic review and meta-analysis were performed to assess prevalence and epidemiological characteristics of CPV-2 in domestic dogs in mainland China. METHODS: In this study, Chinese and English literature on CPV-2 epidemiology published between January 2006 and December 2019 was evaluated. Regarding meta-analysis, the random-effect model was employed by forest plot with 95% of confidence interval. The number of CPV-2 infections was identified and the pooled prevalence of infection, as well as the epidemiological characteristics, was calculated using meta-analysis. RESULTS: A total of 39 studies (data from 137,844 dogs) met the evaluation criteria and were used in our study. The pooled prevalence of CPV-2 infection in mainland China was 36%. CPV-2 infection were associated with age, breed, sampling season and immunization status, but not with gender, publication time and diagnostic methods. CONCLUSIONS: Our results indicated that CPV-2 is prevalent among dogs in China. It is therefore necessary to carry out continuous surveillance and epidemiological studies of CPV-2. In addition, accordingly, effective measures should be taken to prevent the transmission and spread of CPV-2 among the Chinese dog population.

CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2.

Canine parvovirus type 2 (CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide. In today's world, dogs are an integral part of our communities as well as dogs breeding and rearing has become a lucrative business. Therefore, a fast, accurate, portable, and cost-effective CPV-2 detection method with the ability for on-site detection is highly desired. In this study, we for the first time proposed a nanosystem for CPV-2 DNA detection with RNA-guided RNA endonuclease Cas13a, which upon activation results in collateral RNA degradation. We expressed LwCas13a in prokaryotic expression system and purified it through nickel column. Activity of Cas13a was verified by RNA-bound fluorescent group while using a quenched fluorescent probe as signals. Further Cas13a was combined with Recombinase polymerase amplification (RPA) and T7 transcription to establish molecular detection system termed specific high-sensitivity enzymatic reporter un-locking (SHERLOCK) for sensitive detection of CPV-2 DNA. This nanosystem can detect 100 amol/L CPV-2 DNA within 30 min. The proposed nanosystem exhibited high specificity when tested for CPV-2 and other dog viruses. This CRISPR-Cas13a mediated sensitive detection approach can be of formidable advantage during CPV-2 outbreaks because it is time-efficient, less laborious and does not involve the use of sophisticated instruments.

Bacterial diversity in the feces of dogs with CPV infection.

Canine parvovirus (CPV) is a contagious disease in dogs that has high morbidity and mortality. In cases of infection, the pups tend to have a higher mortality and more severe clinical symptoms than the adult dogs because the dehydration is difficult for pups to bear. Following the natural infection, there is a rapid antibody response neutralizing the extracellular virus. As a result, virus titers in tissue and feces become markedly reduced. Hence, it is important to have an effective symptomatic therapy of supporting animals to survive in the early stages of CPV infection. Furthermore, the co-infection with bacteria could increase the severity of lesions and clinical signs as well. In this paper, we obtained the bacterial diversity in feces of CPV infected dogs with the enrichment of five bacteria genera (Shigella, Peptoclostridium, Peptostreptococcus, Streptococcus, Fusobacterium). These microorganisms may partly result in the intestinal pathology of the infection. In summary, the discussion of the bacterial biodiversity in feces of CPV infected dogs provides further insights into the pathology of CPV disease and the targets of developing more effective treatment strategies.

Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2.

A visible and equipment-free recombinase polymerase amplification assay combined with a lateral flow strip (LFS RPA) was developed to detect canine parvovirus type 2 (CPV-2), which is the etiological agent of canine parvovirus disease. The CPV-2 LFS RPA assay was developed based on the VP2 gene and is performed in a closed fist using body heat for 15 min; the products are visible to the naked eye on the LFS within 5 min. The assay could detect CPV-2a, CPV-2b and CPV-2c, and there was no cross-reaction with the other viruses tested. Using the standard CPV-2 DNA as a template, the analytical sensitivity was 1.0 × 102 copies per reaction, which was the same result as that of a real-time PCR. The assay performance was further evaluated by testing 60 canine fecal samples, and CPV-2 DNA was detected in 46 samples (76.7%, 46/60) by LFS RPA, which was the same result as that of the real-time PCR assay and higher than that of the SNAP method (48.3%, 29/60). The novel CPV-2 LFS RPA assay is an attractive and promising tool for rapid and convenient diagnosis of CPV disease, especially cage side and in underequipped laboratories.

In vitro virucidal activity of sodium hypochlorite against canine parvovirus type 2.

Canine parvovirosis is a very contagious, severe and often lethal infectious disease of dogs caused by canine parvovirus type 2 (CPV-2). Parvoviruses are very resistant to several disinfectants while are sensitive to halogens such as sodium hypochlorite which is often used for decontamination of veterinary clinics and animal housing facilities due to its broad spectrum of activity. If compliance with vaccination programmes and with proper disinfection plans is ensured, there should be no continuous, nor frequent, CPV-2 outbreaks in kennels and veterinary clinics. However, a continuous spread of CPV-2 infections is observed, even in kennels where an appropriate vaccination programme is applied, and this imposes a re-evaluation of disinfection protocols using sodium hypochlorite. The aim of the present study was to determine the effect of concentration, contact time and presence of organic matter on the virucidal activity of sodium hypochlorite against several CPV-2 strains. A sensitive in vitro assay capable of measuring the infectivity of CPV-2 was employed to determine the efficacy of three different concentrations of sodium hypochlorite. The data indicate that using a 0.75% sodium hypochlorite solution for a short contact time (1 min) can reduce significantly the CPV-2 titres and that even lower concentrations, i.e. 0.37%, can efficiently inactivate the viruses provided that the contact time is extended to 15 min. Results also confirm the importance of cleaning before disinfection since the presence of organic matter totally abrogated the virucidal activity of sodium hypochlorite solutions against the three CPV-2 strains.

Evaluation of the humoral immune response induced by vaccination for canine distemper and parvovirus: a pilot study.

BACKGROUND: Canine Distemper Virus (CDV) and Canine Parvovirus (CPV) lead to infections with high mortality rates in dogs. These viruses affect unvaccinated dogs or dogs with incomplete vaccination protocols. Vaccination plays an important role in reducing death rates, preventing clinical cases and controlling the spread of virus However, the efficacy of vaccination might be affected by different factors including vaccine scheduling and the neutralization of the vaccine targets by maternal antibodies. In face of these factors, the main goals of this study are (i) to investigate the antibody responses of puppies undergoing different primary vaccination protocols against CPV and CDV and (ii) to estimate the time until seroreversion in adult dogs unvaccinated for at least 3 years. RESULTS: Antibody protection against CDV and CPV was evaluated in a total of 20 dogs: 5 puppies that initiated immunization at 6 weeks after birth (group A), 8 animals that started vaccination between 8 and 12 weeks of age (group B), and 7 adult dogs that have not been vaccinated for at least 3 years (group C). Blood samples were collected from each animal, with 3 to 4 weeks apart. Antibody responses were measured using indirect ELISA. In the second immunization point, no significant differences were found between the seroconversion of groups A and B for each viral infection (p = 0.81 and 0.20 for CDV and CPV, respectively). In the third immunization, there was evidence for a shorter time to achieve a protective titer against CPV in group B when compared to group A (p = 0.015). Similar evidence was not found for CDV (p-value = 0.41). In Group C, the average time until seroveversion was estimated at 2.86 years and 7.63 years for CDV and CPV, respectively. CONCLUSION: Vaccine response to CDV and CPV is specific in each individual. Effective immune protection in primary vaccination depends mainly on the initial titer of maternal antibodies acquired by the neonate. Other factors such as environmental exposure, immunization schedules and immune system activity influence the duration of immunity in adult dogs. The variability found reinforces the need to determine individual humoral immunity levels in order to assess vaccine efficacy.

Malignant Transformation of a Canine Papillomavirus Type 1-Induced Persistent Oral Papilloma in a 3-Year-Old Dog.

This case report describes a rare case of a persistent canine papillomavirus type 1 (CPV-1)-induced oral papilloma that underwent malignant transformation into an oral squamous cell carcinoma (OSCC) in a 3-year-old Labrador retriever cross. Initially, the patient had multiple and multifocal verrucous lesions populating the oral cavity exclusively. The papillomas persisted despite multiple surgical ablations, azithromycin, interferon α-2b, alternative medicines, and off-label drug use of an immunostimulant. After 1 year and 6 months, an aggressive lesion developed at the level of the left mandibular first molar (309) and progressed to a well-differentiated invasive OSCC. The presence of CPV-1 DNA in the OSCC, and the known oncogenic abilities of CPV-1, suggests that this virus might have played a significant role in the emergence of the OSCC that ultimately led to the patient's euthanasia due to poor quality of life. This is the first well-documented case where OSCC has developed from an oral papilloma caused by CPV-1 in which the presence of coinfection by another papillomavirus was excluded by multiple polymerase chain reaction tests using various primers.