Transcriptome analysis of infected Crandell Rees Feline Kidney (CRFK) cells by canine parvovirus type 2c Laotian isolates.

The advent of RNA sequencing technology provides insight into the dynamic nature of tremendous transcripts within Crandell-Reese feline kidney (CRFK) cells in response to canine parvovirus (CPV-2c) infection. A total of 1,603 genes displayed differentially expressed genes (DEGs), including 789 up-regulated genes and 814 downregulated genes in the infected cells. Gene expression profiles have shown a subtle pattern of defense mechanism and immune response to CPV through significant DEGs when extensively examined via Gene Ontology (GO) and pathway analysis. Prospective GO analysis was performed and identified several enriched GO biological process terms with significant participating roles in the immune system process and defense response to virus pathway. A Gene network was constructed using the 22 most significantly enriched genes of particular interests in defense response to virus pathways to illustrate the key pathways. Eleven genes (C1QBP, CD40, HYAL2, IFNB1, IFNG, IL12B, IL6, IRF3, LSM14A, MAVS, NLRC5) were identified, which are directly related to the defense response to the virus. Results of transcriptome profiling permit us to understand the heterogeneity of DEGs during in vitro experimental study of CPV infection, reflecting a unique transcriptome signature for the CPV virus. Our findings also demonstrate a distinct scenario of enhanced CPV responses in CRFK cells for viral clearance that involved multistep and perplexity of biological processes. Collectively, our data have given a fundamental role in anti-viral immunity as our highlights of this study, thus providing outlooks on future research priorities to be important in studying CPV.

T598 and T601 phosphorylation sites of canine parvovirus NS1 are crucial for viral replication and pathogenicity.

Canine parvovirus-2 (CPV-2) is an important pathogen causing severe diseases in dogs and other wild carnivores. Phosphorylation of NS1 may be related to CPV-2 pathogenicity, but the exact mechanism is unclear. Here, we conducted parvovirus disease surveillance in Shaanxi Province of China and 51 fecal swabs were detected to be infected with CPV-2. The 7 CPV-2 strains were identified, all of which belonged to CPV-2c. The complete genome sequence of one of the strains (CPV-2c XY) was cloned into pKQLL plasmid to construct a full-length infectious clone plasmid pX-CPV-2c, which carried a genetic marker. The plasmid pX-CPV-2c was transfected into F81 cells for virus rescue. And the rescued virus, which was designed as X-CPV-2c, showed the similar biological property to parental CPV-2c XY in vitro and in vivo. We further constructed four NS1 phosphorylation site mutant strains (X-CPV-2cT584A, X-CPV-2cS592A, X-CPV-2cT598A/T601A and X-CPV-2cT617A) on the basis of X-CPV-2c. After the analysis and comparison of biological characteristics, the low pathogenic strain X-CPV-2cT598A/T601A was further screened out, which emphasized the importance of phosphorylation sites 598 T/601 T for the pathogenicity of CPV-2. Overall, our data indicated that T598 and T601, the C-terminal phosphorylation site of CPV-2 NS1, play important roles in viral pathogenicity and laid the foundation for the development of new attenuated live vaccine vectors.

Comparison of Eight Commercially Available Faecal Point-of-Care Tests for Detection of Canine Parvovirus Antigen.

A real-time polymerase chain reaction (qPCR) is considered the gold standard for the laboratory diagnosis of canine parvovirus (CPV) infection but can only be performed in specialized laboratories. Several point-of-care tests (POCT), detecting CPV antigens in faeces within minutes, are commercially available. The aim of this study was to evaluate eight POCT in comparison with qPCR. Faecal samples of 150 dogs from three groups (H: 50 client-owned, healthy dogs, not vaccinated within the last four weeks; S: 50 shelter dogs, healthy, not vaccinated within the last four weeks; p = 50 dogs with clinical signs of CPV infection) were tested with eight POCT and qPCR. Practicability, sensitivity, specificity, positive (PPV) and negative predictive values (NPV), as well as overall accuracy were determined. To assess the differences between and agreement among POCT, McNemar's test and Cohen's Kappa statistic were performed. Specificity and PPV were 100.0% in all POCT. Sensitivity varied from 22.9-34.3% overall and from 32.7-49.0% in group P. VetexpertRapidTestCPVAg® had the highest sensitivity (34.3% overall, 49.0% group P) and differed significantly from the 3 POCT with the lowest sensitivities (Fassisi®Parvo (27.7% overall, 36.7% group P), Primagnost®ParvoH+K (24.3% overall, 34.7% group P), FASTest®PARVOCard (22.9% overall, 32.7% group P)). The agreement among all POCT was at least substantial (kappa >0.80). A positive POCT result confirmed the infection with CPV in unvaccinated dogs, whereas a negative POCT result did not definitely exclude CPV infection due to the low sensitivity of all POCT.

Molecular Investigation of Canine Parvovirus-2 (CPV-2) Outbreak in Nevis Island: Analysis of the Nearly Complete Genomes of CPV-2 Strains from the Caribbean Region.

To date, there is a dearth of information on canine parvovirus-2 (CPV-2) from the Caribbean region. During August-October 2020, the veterinary clinic on the Caribbean island of Nevis reported 64 household dogs with CPV-2-like clinical signs (hemorrhagic/non-hemorrhagic diarrhea and vomiting), of which 27 animals died. Rectal swabs/fecal samples were obtained from 43 dogs. A total of 39 of the 43 dogs tested positive for CPV-2 antigen and/or DNA, while 4 samples, negative for CPV-2 antigen, were not available for PCR. Among the 21 untested dogs, 15 had CPV-2 positive littermates. Analysis of the complete VP2 sequences of 32 strains identified new CPV-2a (CPV-2a with Ser297Ala in VP2) as the predominant CPV-2 on Nevis Island. Two nonsynonymous mutations, one rare (Asp373Asn) and the other uncommon (Ala262Thr), were observed in a few VP2 sequences. It was intriguing that new CPV-2a was associated with an outbreak of gastroenteritis on Nevis while found at low frequencies in sporadic cases of diarrhea on the neighboring island of St. Kitts. The nearly complete CPV-2 genomes (4 CPV-2 strains from St. Kitts and Nevis (SKN)) were reported for the first time from the Caribbean region. Eleven substitutions were found among the SKN genomes, which included nine synonymous substitutions, five of which have been rarely reported, and the two nonsynonymous substitutions. Phylogenetically, the SKN CPV-2 sequences formed a distinct cluster, with CPV-2b/USA/1998 strains constituting the nearest cluster. Our findings suggested that new CPV-2a is endemic in the region, with the potential to cause severe outbreaks, warranting further studies across the Caribbean Islands. Analysis of the SKN CPV-2 genomes corroborated the hypothesis that recurrent parallel evolution and reversion might play important roles in the evolution of CPV-2.

First detection of canine parvovirus 2b DNA in a crab-eating fox pup (Cerdocyon thous, Linnaeus, 1766).

The crab-eating fox (Cerdocyon thous) is a small wild mammal present in all Brazilian biomes and in some countries of South America. This study aimed to verify the involvement of viral infectious agents in the death of a wild crab-eating fox pup (Cerdocyon thous) in Brazil. The Center for Medicine and Research of Wild Animals of the Universidade Estadual Paulista received a free-living crab-eating fox aged approximately 21 days and apparently healthy. After 13 days, the animal presented anorexia, diarrhea, fever, prostration, and neurological signs progressing to death with an inconclusive diagnosis. In a retrospective study, tissue fragments stored at - 80 °C were used to identify nucleic acids from major canine viruses, such as canine parvovirus-2 (CPV-2), canine adenovirus A types 1 and 2, canid alphaherpesvirus 1, and canine distemper virus. The amplified product with the expected length for CPV-2 was obtained from the heart fragment. After performing nucleotide (nt) sequencing of the amplicon, it was possible to demonstrate that the crab-eating fox strain exhibited high (99.8%) nt identity with the CPV-2b prototype (CPV-39 strain). Additionally, deduced amino acid (aa) sequence analysis showed the GAT codon for the aa Asp (D) at position 426 of the CPV-2 viral protein VP2, which characterizes the subtype 2b. To the best of the authors' knowledge, this report describes the first detection of CPV-2b DNA in tissue fragments from a crab-eating fox.

Changes in Mass Treatment of the Canine Parvovirus ICU Population in Relation to Public Policy Changes during the COVID-19 Pandemic.

Previous work has indicated that canine parvovirus (CPV) prevalence in the Central Texas region may follow yearly, periodic patterns. The peak in CPV infection rates occurs during the summer months of May and June, marking a distinct "CPV season". We hypothesized that human activity contributes to these seasonal changes in CPV infections. The COVID-19 pandemic resulted in drastic changes in human behavior which happened to synchronize with the CPV season in Central Texas, providing a unique opportunity with which to assess whether these society-level behavioral changes result in appreciable changes in CPV patient populations in the largest CPV treatment facility in Texas. In this work, we examine the population of CPV-infected patients at a large, dedicated CPV treatment clinic in Texas (having treated more than 5000 CPV-positive dogs in the last decade) and demonstrate that societal-behavioral changes due to COVID-19 were associated with a drastic reduction in CPV infections. This reduction occurred precisely when CPV season would typically begin, during the period immediately following state-wide "reopening" of business and facilities, resulting in a change in the typical CPV season when compared with previous years. These results provide evidence that changes in human activity may, in some way, contribute to changes in rates of CPV infection in the Central Texas region.

Canine parvovirus induces G1/S cell cycle arrest that involves EGFR Tyr1086 phosphorylation.

Canine parvovirus (CPV) has been used in cancer control as a drug delivery vehicle or anti-tumor reagent due to its multiple natural advantages. However, potential host cell cycle arrest induced by virus infection may impose a big challenge to CPV associated cancer control as it could prevent host cancer cells from undergoing cell lysis and foster them regain viability once the virotherapy was ceased. To explore CPV-induced cell cycle arrest and the underlying mechanism toward improved virotherapeutic design, we focus on epidermal growth factor receptor (EGFR), a cellular receptor interacting with TfR that mediates CPV-host interactions, and alterations on its tyrosine phosphorylation sites in response to CPV infection. We found that CPV could trigger host G1/S cell cycle arrest via the EGFR (Y1086)/p27 and EGFR (Y1068)/STAT3/cyclin D1 axes, and EGFR inhibitor could not reverse this process. Our results contribute to our understandings on the mechanism of CPV-induced host cellular response and can be used in the onco-therapeutic design utilizing CPV by preventing host cancer cells from entering cell cycle arrest.

Phylogenetic and Geospatial Evidence of Canine Parvovirus Transmission between Wild Dogs and Domestic Dogs at the Urban Fringe in Australia.

Canine parvovirus (CPV) is an important cause of disease in domestic dogs. Sporadic cases and outbreaks occur across Australia and worldwide and are associated with high morbidity and mortality. Whether transmission of CPV occurs between owned dogs and populations of wild dogs, including Canis familiaris, Canis lupus dingo and hybrids, is not known. To investigate the role of wild dogs in CPV epidemiology in Australia, PCR was used to detect CPV DNA in tissue from wild dogs culled in the peri-urban regions of two Australian states, between August 2012 and May 2015. CPV DNA was detected in 4.7% (8/170). There was a strong geospatial association between wild-dog CPV infections and domestic-dog CPV cases reported to a national disease surveillance system between 2009 and 2015. Postcodes in which wild dogs tested positive for CPV were 8.63 times more likely to also have domestic-dog cases reported than postcodes in which wild dogs tested negative (p = 0.0332). Phylogenetic analysis of CPV VP2 sequences from wild dogs showed they were all CPV-2a variants characterized by a novel amino acid mutation (21-Ala) recently identified in CPV isolates from owned dogs in Australia with parvoviral enteritis. Wild-dog CPV VP2 sequences were compared to those from owned domestic dogs in Australia. For one domestic-dog case located approximately 10 km from a wild-dog capture location, and reported 3.5 years after the nearest wild dog was sampled, the virus was demonstrated to have a closely related common ancestor. This study provides phylogenetic and geospatial evidence of CPV transmission between wild and domestic dogs in Australia.

Canine papillomavirus type 16 associated to squamous cell carcinoma in a dog: virological and pathological findings.

Papillomaviruses (PVs) are circular double-stranded DNA virus belonging to Papillomaviridae family. During the infection cycle, PVs translate proteins that can influence cell growth and differentiation, leading to epidermal hyperplasia and papillomas (warts) or malignant neoplasms. Canis familiaris papillomaviruses (CPVs) have been associated with different lesions, such as oral and cutaneous papillomatosis, pigmented plaques, and squamous cell carcinomas (SCCs). Here, we report a clinical case of a mixed bred female dog with pigmented plaques induced by CPV16 (Chipapillomavirus 2) that progressed to in situ and invasive SCCs. Gross and histological findings were characterized, and the lesions were mainly observed in ventral abdominal region and medial face of the limbs. In situ hybridization (ISH) revealed strong nuclear hybridization signals in the neoplastic epithelial cells, as well as in the keratinocytes and koilocytes of the pigmented viral plaques. The full genome of the CPV16 recovered directly from the lesions was characterized, and the phylogenetic relationships were determined. The identification of oncoprotein genes (E5, E6, and E7) by high throughput sequencing (HTS) and their expected domains are suggestive of the malignant transformation by CPV16.

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana.

BACKGROUND: Domestic dogs (Canis familiaris) have the potential to act as disease reservoirs for wildlife and are important sentinels for common circulating pathogens. Therefore, the infectious disease seroprevalence among domestic dogs in northern Botswana may be indicative of pathogen exposure of various wildlife species. The objective of this study was to assess the seroprevalence of Ehrlichia spp., Borrelia burgdorferi, Anaplasma spp., Dirofilaria immitis, canine adenovirus, canine parvovirus, and canine distemper virus in domestic dogs as proxies of disease prevalence in the local wildlife in the Okavango Delta region of Botswana. Statistical analysis assessed crude and factor-specific seroprevalence proportions in relation to age, sex, and geographical location as predictors of seropositivity. Logistic regression was used to identify adjusted predictors of seropositivity for each of the pathogens of interest. RESULTS: Samples from 233 dogs in a total of seven locations in Maun, Botswana, and surrounding villages were collected and serologically analyzed. No dogs were seropositive for B. burgdorferi, while low seroprevalence proportions were observed for Anaplasma spp. (2.2%) and D. immitis (0.9%). Higher seroprevalence proportions were observed for the tick-borne pathogen Ehrlichia spp. (21.0%), and 19.7% were seropositive for canine adenovirus (hepatitis). The highest seroprevalence proportions were for canine parvovirus (70.0%) and canine distemper virus (44.8%). The predictors of seropositivity revealed that adults were more likely to be seropositive for canine adenovirus, canine distemper virus, and canine parvovirus than juveniles, and location was a risk factor for canine adenovirus, canine distemper virus, canine parvovirus, and Ehrlichia spp. CONCLUSIONS: Results indicate that increasing tick control and vaccination campaigns for domestic dogs may improve the health of domestic animals, and potentially wildlife and humans in the Okavango Delta since viral and vector-borne bacterial pathogens can be transmitted between them.

Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine Parvovirus Host Adaptation and Spread.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep-sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and microscale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ∼99% identical in nucleotide sequence, with only a limited number (<40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intrahost level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few subconsensus single nucleotide variants (SNVs) above ∼0.5%, and experimental passages demonstrate that substantial preexisting genetic variation is not necessarily required for rapid host receptor-driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intrahost diversity are not necessarily required for virus host adaptation.IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and interhost diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here, we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intrahost evolutionary processes of an ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained transmission does not necessarily depend on a high level of intrahost diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

Canine parvovirus (CPV) phylogeny is associated with disease severity.

After its first identification in 1978, canine parvovirus (CPV) has been recognized all around the world as a major threat for canine population health. This ssDNA virus is characterized by a high substitution rate and several genetic and phenotypic variants emerged over time. Overall, the definition of 3 main antigenic variants was established based on specific amino acid markers located in a precise capsid position. However, the detection of several minor variants and incongruence observed between the antigenic classification and phylogeny have posed doubts on the reliability of this scheme. At the same time, CPV heterogeneity has favored the hypothesis of a differential virulence among variants, although no robust and consistent demonstration has been provided yet. The present study rejects the antigenic variant concept and attempts to evaluate the association between CPV strain phylogeny, reconstructed using the whole information contained in the VP2 coding gene, and several clinical and hemato-biochemical parameters, assessed from 34 CPV infected dogs at admission. By using different statistical approaches, the results of the present study show an association between viral phylogeny and host parameters ascribable to immune system, coagulation profile, acute phase response and, more generally, to the overall picture of the animal response. Particularly, a strong and significant phylogenetic signal was proven for neutrophil count and WBC. Therefore, despite the limited sample size, a relation between viral phylogeny and disease severity has been observed for the first time, suggesting that CPV virulence is an inherited trait. The likely existence of clades with different virulence highlights once more the relevance of intensive epidemiological monitoring and research on CPV evolution to better understand the virulence determinants, their epidemiology and develop adequate countermeasures.

Emergence of novel canine parvovirus type 2 and its pathogenesis in raccoon dogs.

Three parvoviruses were isolated from the raccoon dogs experiencing severe enteritis, named RDPV-DP1, RDPV-DP2 and RDPV-DP3, respectively. The VP2 genes of the 3 isolates showed 99.9% identity at the nucleotide level, and shared 99.1%-99.5% identity with the reference CPVs. The RDPVs resembled original CPV-2, but with four mutations. The RDPVs displayed S297A of VP2 protein as CPV-2a or CPV-2b prevalent throughout most of the world. Residue N375D was found in the 3 isolates, resembling CPV-2a/2b/2c. And the 3 isolates had a natural mutation of VP2 residue V562L, which is adjacent to residue 564 and 568 and might be involved in host range. Interestingly, VP2 S27T was firstly found in the isolates. Phylogenetic analysis of VP2 genes revealed that the RDPVs were clustered into one small evolutionary branch and shared the identical branch with 7 CPV-2 isolates from raccoon dogs and one CPV-2 isolate from fox, not with CPV vaccine viruses. Phylogenetic analysis of NS1 genes demonstrated that the RDPVs shared the identical branch with the reference CPV-2a/2b/2c. Experimental infection showed that RDPV infection caused a high morbidity in raccoon dogs. It implied that the RDPV was virulent to raccoon dogs and continued to evolve in China.

Substitutions at residues 300 and 389 of the VP2 capsid protein serve as the minimal determinant of attenuation for canine parvovirus vaccine strain 9985-46.

Identifying molecular determinants of virulence attenuation in live attenuated canine parvovirus (CPV) vaccines is important for assuring their safety. To this end, we identified mutations in the attenuated CPV 9985-46 vaccine strain that arose during serial passage in Crandell-Rees feline kidney cells by comparison with the wild-type counterpart, as well as minimal determinants of the loss of virulence. Four amino acid substitutions (N93K, G300V, T389N and V562L) in VP2 of strain 9985-46 significantly restricted infection in canine A72 cells. Using an infectious molecular clone system, we constructed isogenic CPVs of the parental virulent 9985 strain carrying single or double mutations. We observed that only a single amino acid substitution in VP2, G300V or T389N, attenuated the virulent parental virus. Combinations of these mutations further attenuated CPV to a level comparable to that of 9985-46. Strains with G300V/T389N substitutions did not induce clinical symptoms in experimentally infected pups, and their ability to infect canine cells was highly restricted. We found that another G300V/V562L double mutation decreased affinity of the virus for canine cells, although its pathogenicity to dogs was maintained. These results indicate that mutation of residue 300, which plays a critical role in host tropism, is not sufficient for viral attenuation in vivo, and that attenuation of 9985-46 strain is defined by at least two mutations in residues 300 and 389 of the VP2 capsid protein. This finding is relevant for quality control of the vaccine and provides insight into the rational design of second-generation live attenuated vaccine candidates.

Serum Derived Transfer Factor Stimulates the Innate Immune System to Improve Survival Traits in High Risk Pathogen Scenarios.

Preclinical Research Transfer Factors (TFs) are low molecular weight (<5,000 daltons) biological response mediators. In the present study, a serum derived TF improved the ability of the recipient animal to survive high-risk infectious challenges (salmonellosis and canine parvoviral enteritis (CPV)) by altering the host's cytokine response profile. Mice mortally challenged with 5,000 colony-forming units of Salmonella experienced a group mortality of 73% while mice treated with a single 5 mg dose of the TF demonstrated a significant decrease in morbidity (7%, p ≤ 0.01). The splenic bacterial load in untreated mice was over 10,000 times higher than that in the TF treated mice. Twenty-four hours post-administration, the treated murine population expressed a rapid temporal increase in serum IL-6 (26-fold) and INF-γ (77-fold) concentrations. IL-6 can act as a critical signal regulating action against bacterial pathogens. A comparative double-blind study performed using dogs confirmed to be undergoing a canine parvovirus challenge showed that when conventional supportive therapy was supplemented with a single 5 mg TF dose there was a reduction (p ≤ 0.01) in group mortality (68% of the TF treated group survived versus 32% of the placebo group), an observation consistent with the observed increase in INF-γ, a cytokine associated with promoting antiviral activity. Drug Dev Res 78 : 189-195, 2017. © 2017 Wiley Periodicals, Inc.

MT-PCR panel detection of canine parvovirus (CPV-2): Vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples.

Canine parvovirus (CPV-2) remains an important cause of devastating enteritis in young dogs. It can be successfully prevented with live attenuated CPV-2 vaccines when given at the appropriate age and in the absence of maternal antibody interference. Rapid diagnosis of parvoviral enteritis in young dogs is essential to ensuring suitable barrier nursing protocols within veterinary hospitals. The current diagnostic trend is to use multiplexed PCR panels to detect an array of pathogens commonly responsible for diarrhea in dogs. The multiplexed PCR assays do not distinguish wild from vaccine CPV-2. They are highly sensitive and detect even a low level of virus shedding, such as those caused by the CPV-2 vaccine. The aim of this study was to identify the CPV-2 subtypes detected in diagnostic specimens and rule out occult shedding of CPV-2 vaccine strains. For a total of 21 samples that tested positive for CPV-2 in a small animal fecal pathogens diagnostic multiplexed tandem PCR (MT-PCR) panel during 2014-2016 we partially characterized the VP2 gene of CPV-2. Vaccine CPV-2 strain, wild type CPV-2a subtypes and vaccine-like CPV-2b subtypes were detected. High copy number was indicative of wild-type CPV-2a presence, but presence of vaccine-like CPV-2b had a variable copy number in fecal samples. A yardstick approach to a copy number or Ct-value to discriminate vaccine strain from a wild type virus of CPV-2 can be, in some cases, potentially misleading. Therefore, discriminating vaccine strain from a wild type subtype of CPV-2 remains ambitious.

Canine parvovirus NS1 induced apoptosis involves mitochondria, accumulation of reactive oxygen species and activation of caspases.

The non-structural protein (NS1) of parvoviruses plays an important role in viral replication and is thought to be responsible for inducing cell death. However, the detailed mechanism and the pathways involved in canine parvovirus type 2 NS1 (CPV2.NS1) induced apoptosis are not yet known. In the present study, we report that expression of CPV2.NS1 in HeLa cells arrests cells in G1 phase of the cell cycle and the apoptosis is mitochondria mediated as indicated by mitochondrial depolarization, release of cytochrome-c and activation of caspase 9. Treatment of cells with caspase 9 inhibitor Z-LEHD-FMK reduced the induction of apoptosis significantly. We also report that expression of CPV2.NS1 causes accumulation of reactive oxygen species (ROS) and treatment with an antioxidant reduces the ROS levels and the extent of apoptosis. Our results provide an insight into the mechanism of CPV2.NS1 induced apoptosis, which might prove valuable in developing NS1 protein as an oncolytic agent.

Influence of chemotherapy for lymphoma in canine parvovirus DNA distribution and specific humoral immunity.

In man, the combination of cancer and its treatment increases patients' susceptibility to opportunistic infections, due to immune system impairment. In veterinary medicine little information is available concerning this issue. In order to evaluate if a similar dysfunction is induced in small animals undergoing chemotherapy, we assessed the complete blood count, leukocytic, plasma and fecal canine parvovirus (CPV) viral load, and anti-CPV protective antibody titers, in dogs with lymphoma treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) protocol, before and during chemotherapy. There was no evidence of decreased immune response, either at admission or after two chemotherapy cycles, indicating that the previously established immunity against CPV was not significantly impaired, supporting the idea that immunosuppression as a result of hematopoietic neoplasms and their treatment in dogs requires further investigation and conclusions cannot be extrapolated from human literature.

Soluble form of canine transferrin receptor inhibits canine parvovirus infection in vitro and in vivo.

Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

Apoptosis induced by NS1 gene of Canine Parvovirus-2 is caspase dependent and p53 independent.

Apoptosis is programmed cell death that normally occurs during development and aging in multicellular animals. Apoptosis also occurs as a defense mechanism against disease or harmful external agents. It can be initiated by a variety of stimuli including viruses and viral proteins. Canine parvovirus type 2 (CPV-2) that causes acute disease in dogs has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. Though non structural protein 1 (NS1) of many parvoviruses has been found to be apoptotic, no report on the apoptotic potential of NS1 of CPV-2 (CPV-2.NS1) exists. In this study, we evaluated the apoptotic potential of CPV-2.NS1 in HeLa cells. CPV-2.NS1 has been found to induce apoptosis which was evident through characteristic DNA fragmentation, increase in hypodiploid cell count, phosphatidyl serine translocation and activation of caspase-3. Increase in caspase-3 activity and no change in p53 activity with time in CPV-2.NS1 expressing HeLa cells showed the induction of apoptosis to be caspase dependent and p53 independent.