Genetic diversity of canine parvovirus variants circulating in Nigeria

Canine parvovirus (CPV) is a fast-evolving single-stranded DNA virus that causes severe and fatal gastrointestinal disease in dogs. Lately, several mutations affecting viral protein (VP) capsid resulting in highly pathogenic variants with distinctive immunological and clinicopathological characteristics abound. This study involved screening stools of 44 randomly selected clinical cases of canine gastroenteritis from 4 cities (Ibadan, Jos, Makurdi, and Zaria) in Nigeria for CPV antigen using an on-the-spot immunoassay test kit, as well as, molecular detection of viral nucleic acid by polymerase chain reaction. Subsequently, nucleic acid sequencing of 1195-bp amplicons encompassing the VP2 encoding region was done. The resultant 40 high-quality amino acid sequences obtained were analysed for the identification and grouping of the viruses into their discrete variants - CPV-2a, CPV-2b, or CPV-2c, using key amino acids substitutions - Asn, Asp, or Glu respectively at position 426 of the VP2 gene. One-third (11/40; 27.5%) of the analysed sequences were identified as CPV-2a and two-third (29/40; 72.5%) as CPV-2c. The original CPV and CPV-2b were not detected. Also, the “new CPV-2a variant” with mutation S297A identified had two additional mutations (Y324I and T440A) associated with selective pressure and vaccination failure in their sequences. Similarly, unique CPV-2c mutants carrying genetic markers (S297A, Y324I, and Q370R) that are highly related to CPVs of Asian origin were observed. These findings revealed a high level of divergence of existing CPVs in circulation; suggesting that CPV is rapidly evolving in Nigeria lately.     

Molecular epidemiology of canine parvovirus in Morocco

Since it first emergence in the mid-1970's, canine parvovirus 2 (CPV-2) has evolved giving rise to new antigenic variants termed CPV-2a, CPV-2b and CPV-2c, which have completely replaced the original strain and had been variously distributed worldwide. In Africa limited data are available on epidemiological prevalence of these new types. Hence, the aim of the present study was to determine circulating variants in Morocco. Through TaqMan-based real-time PCR assay, 91 samples, collected from symptomatic dogs originating from various cities between 2011 and 2015, were diagnosed. Positive specimens were characterised by means of minor groove binder (MGB) probe PCR. The results showed that all samples but one (98.9%) were CPV positive, of which 1 (1.1%) was characterised as CPV-2a, 43 (47.7%) as CPV-2b and 39 (43.3%) as CPV-2c. Interestingly, a co-infection with CPV-2b and CPV-2c was detected in 4 (4.4%) samples and 3 (3.3%) samples were not characterised. Sequencing of the full VP2 gene revealed these 3 uncharacterised strains as CPV-2c, displaying a change G4068A responsible for the replacement of aspartic acid with asparagine at residue 427, impacting the MGB probe binding. In this work we provide a better understanding of the current status of prevailing CPV strains in northern Africa.     

Feline host range of canine parvovirus: recent emergence of new antigenic types in cats

Since the emergence of Canine parvovirus (CPV-2) in the late 1970s, CPV-2 has evolved consecutively new antigenic types, CPV-2a and 2b. Although CPV-2 did not have a feline host range, CPV-2a and 2b appear to have gained the ability to replicate in cats. Recent investigations demonstrate the prevalence of CPV-2a and 2b infection in a wide range of cat populations. We illustrate the pathogenic potential of CPV in cats and assess the risk caused by CPV variants.     

Continuing evolution of canine parvovirus in China: Isolation of novel variants with an Ala5Gly mutation in the VP2 protein

Canine parvovirus (CPV) type 2c is a new antigenic variant of CPV-2. Since the year 2000 it has spread to several countries, causing severe hemorrhagic enteritis in dogs. In 2014 and 2015, 58 fecal samples were collected from dogs in Beijing with suspected CPV infection. Regardless of the vaccination status of the dogs, 43 samples were found positive for CPV according to PCR results; i.e., 18, 7, and 18 respectively belonged to antigenic types new CPV-2a, new CPV-2b, and CPV-2c. A phylogenetic tree based on their VP2 gene sequences indicated that the Chinese CPV-2c strains form a separate cluster. In addition to synonymous mutations, the CPV-2c strains also contain a unique coding mutation in VP2 that leads to glycine at residue 5, instead of the highly conserved alanine at this position in all other CPV-2c strains sequenced to date. Using F81 cells, several novel isolates of CPV-2c, each with the Ala5Gly mutation, were obtained. One of these was used to infect experimentally beagle dogs, which subsequently developed the typical clinical symptoms of CPV infection. Hence, it appears that CPV-2c is still evolving in China, a finding that warrants continuous surveying and the eventual adaptation of current vaccines.     

Molecular typing of canine parvovirus strains circulating from 2008 to 2012 in an organized kennel in India reveals the possibility of vaccination failure

Canine parvovirus-2 (CPV-2), which emerged in 1978, is considered as the major viral enteric pathogen of the canine population. With the emergence of new antigenic variants and incidences of vaccine failure, CPV has become one of the dreaded diseases of the canines worldwide. The present study was undertaken in an organized kennel from North India to ascertain the molecular basis of the CPV outbreaks in the vaccinated dogs. 415 samples were collected over a 5 year period (2008–2012). The outbreak of the disease was more severe in 2012 with high incidence of mortality in pups with pronounced clinical symptoms. Molecular typing based on the VP2 gene was carried out with the 11 isolates from different years and compared with the CPV prototype and the vaccine strains. All the isolates in the study were either new CPV-2a (2012 isolates) or new CPV-2b (2008 and 2011 isolates). There were amino acid mutations at the Tyr324Ile and at the Thr440Ala position in five isolates from 2012 indicating new CPV mutants spreading in India. The CPV vaccines used in the present study failed to generate protective antibody titer against heterogeneous CPV antigenic types. The findings were confirmed when the affected pups were treated with hyper-immune heterogeneous purified immunoglobulin’s against CPV in dogs of different antigenic types.     

Selective regimen shift and demographic growth increase associated with the emergence of high-fitness variants of canine parvovirus.

The natural evolution of Canine parvovirus (CPV) is characterized by a variety of mutations, mainly in the VP1/VP2 gene. Although positive selection has been previously reported in CPV, little is known about its overall contribution to viral adaptation in the canine population. Herein, the influences of the evolutive constraints on CPV during a period of viral adaptation into a previously uninfected population are more clearly investigated. To do this, 31 sequences of VP1/VP2 gene obtained from symptomatic domestic dogs in Brazil were used, sampled from 1980 to 2000. Marked evolutionary changes in CPV associated with a process of fine-tuning adaptation were observed. Specifically, sequences from the 1980s revealed two distinct antigenic types (i.e. 2a and 2b) cocirculating in Brazil. Moreover, analysis of the selective regimen showed that 90% of the VP2 sites were conserved (d(N)/d(S)=0). In contrast, sequences from the 1990s were composed solely of CPV-2a with 96% of VP2 sites under purifying selection (d(N)/d(S)<1) and site 297 under strong positive selection (omega=4.9). Important features regarding the demographic history of CPV in Brazil were also observed. The viral population size passed through a short period of explosive growth that declined and then stabilized into a constant rate of spread. Remarkably, the explosive growth coincided with the appearance of CPV variants that presented a unique repertoire of mutations never before seen in other worldwide strains. The analysis also showed that the estimated nucleotide substitution was similar to those commonly observed in fast evolving RNA viruses. The present results demonstrated the adaptive potential of CPV to acquire, in short interval of 10 years, key mutations in the VP1/VP 2 gene that increased viral fitness and enabled the virus to disseminate even in vaccinated dogs.     

Molecular characterisation of canine parvoviruses from clinical samples and vaccines in Nigeria

Canine parvovirus (CPV) the causative agent of canine parvovirus enteritis is an intractable pathogen of dogs characterised by mutations, evolutionary changes and eventual vaccine failure. The disease is a serious problem in dogs with limited studies conducted in Nigeria. Therefore, this study was designed to characterise the subtypes of CPV isolates in six commonly used vaccines and 157 clinical samples collected from seven states in Nigeria from June 2016 to March 2018. Faecal samples collected from the clinical cases were subjected to in-clinic immunoassay to detect viral antigens. Polymerase chain reaction (PCR) was used to amplify viral VP2 gene in the samples and commonly used vaccines in Nigeria. Thereafter, PCR products were sequenced and analysed. The result showed that 93.0% of the dogs tested positive for CPV in both assays; 72.8% were puppies less than six months old, with 58.3% of them vaccinated. Partial VP2 gene sequence and phylogenetic analysis of 11 random clinical samples showed that CPV-2c 7(63.6%) and CPV-2a 4(36.4%) were the predominant subtypes in Nigeria; with genetic signatures that are 98.7% to 99.9% closely related to Asian and European strains, respectively. No CPV-2b was detected. Amino acid mutation analysis divulged some imperative transmutation sites: D305Y, Y324I, Q370R, N375D, T440A, Y444S, I447M and Y451C in the isolates. The viruses in the vaccines were characterised as the wild-type CPV. The genetic variability, viral population heterogeneity and phylogenetic linkage with isolates from other countries probably suggest transboundary migrations and local differentiations are contributing to continuous CPV evolution and vaccine failure in Nigeria.     

Isolation,molecular characterization and phylogenetic analysis of canine parvovirus

Canine parvovirus type 2 (CPV-2) causes acute haemorrhagic enteritis in dogs. Canine parvovirus is prone to genetic evolution and has undergone several mutations that produced different strains like CPV-2a, CPV-2b, New CPV-2a, New CPV-2b and CPV-2c in the past three decades. Mutations affecting the VP2 gene of CPV have been responsible for evolution of different antigenic variants. Sequence analysis of VP2 gene of the virus and subsequent characterization is important for molecular epidemiology. The present study was conducted to isolate and to characterize the virus by amplifying partial VP2 gene and further sequence analysis and also to estimate phylogenetic relationship of field virus with the reference strains. Out of 77 samples, 51 samples were found to be positive by PCR and all the 51 samples were subjected for virus isolation in CRFK cell line. Sixteen viruses could be isolated and 10 randomly selected isolates were subjected to sequence analysis along with four random clinical samples. All the 10 isolates and 4 clinical samples were characterized as New CPV-2a (CPV2a with 297-Ser → Ala). One of the field isolates was found to be phylogenetically closely related to New CPV-2a strains of Japan and India; another field isolates was found to share ancestral origins with New CPV-2a strains of Korea, USA, Italy, Brazil, Germany, Taiwan and Vietnam; rest other sequences had distinct lineage but shared molecular relationship with New CPV-2a reference strains.     

Molecular epidemiology and genetic evolution of canine parvovirus in East China,during 2018‐2020

Canine parvovirus type 2 (CPV-2) emerged in the late 1970s, which caused high rates of morbidity and mortality in dogs. In last decade, five genetic variants (CPV-2a, CPV-2b, CPV-2c, New CPV-2a, and New CPV-2b) were frequently reported in the dog population, and replaced the original CPV-2, rising widespread concerns. However, little is known about their recent genetic diversity and evolution. The aim of this study was to analyze the characteristics of the CPV-2 strains collected in East China from 2018 to 2020. The 57 CPV-2 strains were isolated from rectal swab samples (n=140). They belong to three different genotypes, based on VP2 protein amino acid sequence. The results revealed a high prevalence of CPV-2c (77.19%) compared to the New CPV-2a (5.26%) and New CPV-2b (17.54%) strains. Further analysis showed that nucleotide homology of the VP2 gene among the 57 CPV strains was 98.9%~100%, and the homology with 24 reference strains from different countries and regions was 98.1%~100%. The phylogenetic tree of VP2 gene sequence showed that 44 CPV-2c strains were distantly related to CPV-2, CPV-2a, CPV-2b, New CPV-2a, New CPV-2b and European/American CPV-2c strains, and were closely related to Asian CPV-2c strains. The results showed that these Asian CPV-2c strains had become the dominant strain, which renewed the knowledge of CPV-2 molecular epidemiology in East China.     

Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus

Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV.     

Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus

A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.     

Enhanced antibody response to pneumococcal polysaccharide vaccine after prior immunization with conjugate pneumococcal vaccine in HIV-infected adults

The antibody production by HIV-infected adults after two vaccinations with conjugated pneumococcal vaccine (CPV) and consecutive vaccination with polysaccharide pneumococcal vaccine (PPV) was studied. Thirty days after the second CPV, the geometric mean antibody concentrations (GMC) against pneumococcal polysaccharide serotypes (PPS) 6B, 14 and 19F were significantly lower in the group HIV-infected individuals with <200x10(6)/l CD4(+) T lymphocytes (group 1) than in the group with >/=200x10(6)/l CD4(+) T lymphocytes (group 2) and healthy controls. Thirty days after PPV vaccination the GMC against PPS 6B, 14, 19F and 23F in group 1, and against 6B and 19F in group 2, were significantly lower compared with healthy controls. Both in HIV-infected and in healthy individuals who received CPV and PPV the postvaccination GMC against PPS 14, 19F and 23F were higher compared with historical controls who were not previously immunized with CPV but only received PPV. We conclude that the antibody response to CPV is impaired in HIV-infected individuals. Higher antibody concentrations were achieved in HIV-infected and healthy individuals after sequential vaccination with CPV and PPV compared with PPV vaccination alone.     

Virus neutralizing antibody response in mice and dogs with a bicistronic DNA vaccine encoding rabies virus glycoprotein and canine parvovirus VP2

A bicistronic DNA vaccine against rabies and parvovirus infection of dogs was developed by subcloning rabies glycoprotein and canine parvovirus (CPV) VP2 genes into a bicistronic vector. After characterizing the expression of both the proteins in vitro, the bicistronic DNA vaccine was injected in mice and induced immune response was compared with monocistronic DNA vaccines. There was no significant difference in ELISA and virus neutralizing (VN) antibody responses against rabies and CPV in mice immunized with either bicistronic or monocistronic DNA vaccine. Further, there was significantly similar protection in mice immunized with either bicistronic or monocistronic rabies DNA vaccine on rabies virus challenge. Similarly, dogs immunized with monocistronic and bicistronic DNA vaccines developed comparable VN antibodies against rabies and CPV. This study indicated that bicistronic DNA vaccine can be used in dogs to induce virus neutralizing immune responses against both rabies and CPV.     

Occurrence of severe gastroenteritis in pups after canine parvovirus vaccine administration: a clinical and laboratory diagnostic dilemma

A total of 29 faecal samples collected from dogs with diarrhoea following canine parvovirus (CPV) vaccination were tested by minor groove binder (MGB) probe assays for discrimination between CPV vaccine and field strains and by diagnostic tests for detection of other canine pathogens. Fifteen samples tested positive only for CPV field strains; however, both vaccine and field strains were detected in three samples. Eleven samples were found to contain only the vaccine strain, although eight of them tested positive for other pathogens of dogs. Only three samples were found to contain the vaccine strain without evidence of canine pathogens. The present study confirms that most cases of parvovirus-like disease occurring shortly after vaccination are related to infection with field strains of canine parvovirus type 2 (CPV-2) rather than to reversion to virulence of the modified live virus contained in the vaccine.     

B cell mediated priming following pneumococcal colonization

The primary reservoir for Streptococcus pneumoniae is the human nasopharynx, and colonization is often the initial step in pathogenesis. Recently we have demonstrated that pneumococcal colonization primes the immune response to subsequent vaccination with the pneumococcal conjugate vaccine (CPV). In this study we wished to determine if colonization stimulates the production of B cell memory that is activated following vaccination with CPV. To test this hypothesis, we colonized mice with S. pneumoniae serotype 14, adoptively transferred their B cells and CD4+ T cells into na?ve recipients, and vaccinated the recipients with CPV. Our results indicate that pneumococcal colonization stimulates the production of memory B cells which are responsible for enhancing the immune response to CPV vaccination.     

Molecular epidemiology of Canine parvovirus shows CPV-2a genotype circulating in dogs from western India

Canine parvovirus (CPV) has emerged as an acute pathogen of young canine causing haemorrhagic enteritis and myocarditis. It is widely distributed and underreported in India. Therefore the study was conducted to type the CPV circulating in western Maharashtra. The faecal samples (n = 150) from clinically ill dogs showing diarrhoea and vomition were collected and subjected to haemagglutination (HA) with porcine RBC's. The DNA was extracted from the samples showing HA titres above 64 and subjected for amplification of VP₂ gene fragment by PCR. The amplicons were subjected for restriction fragment length polymorphism (RFLP), sequencing and BEAST phylogenetic analysis. The results revealed 6% positivity by PCR. The RFLP results indicated single cleavage site for ApaLI and HinfI with an exception of two sites for HinfI. The nucleotide sequences showed nonfunctional nucleotide changes at different locations. The sequence analysis indicated that the nucleotide divergence within isolates under study was 0.00–0.42%, while the nucleotide homology was 99.58–100%. The most recent common ancestor was determined by molecular clock analysis using Bayesian methods. The sequence and phylogenetic analysis suggested the isolates as CPV-2a and KATN1 (KU866391, 2014) isolate from Tamilnadu, India as time to most recent common ancestor (TMRCA). The results revealed the circulating CPV in canines from western India as CPV2a genotype.     

Long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination

Canine parvovirus (CPV) modified live virus vaccines are able to infect vaccinated dogs replicating in the bloodstream and enteric mucosa. However, the exact duration and extent of CPV vaccine-induced viremia and fecal shedding are not known. With the aim to fill this gap, 26 dogs were administered two commercial vaccines containing a CPV-2 or CPV-2b strain and monitored for 28 days after vaccination. By using real-time PCR, vaccine-induced viremia and shedding were found to be long lasting for both vaccinal strains. Vaccinal CPV-2b shedding was detected for a shorter period than CPV-2 (12 against 19 mean days) but with greater viral loads, whereas viremia occurred for a longer period (22 against 19 mean days) and with higher titers for CPV-2b. Seroconversion appeared as early as 7 and 14 days post-vaccination for CPV-2b and CPV-2 vaccines, respectively. With no vaccine there was any diagnostic interference using in-clinic or hemagglutination test, since positive results were obtained only by fecal real-time PCR testing. The present study adds new insights into the CPV vaccine persistence in the organism and possible interference with diagnostic tests.     

A goat poxvirus-vectored peste-des-petits-ruminants vaccine induces long-lasting neutralization antibody to high levels in goats and sheep

Recombinant capripoxvirus (CPV) is a promising candidate differentiating infected from vaccinated animals (DIVA) vaccine against peste-des-petits-ruminants (PPR). In order for recombinant CPV to be successfully used in the field, there should exist dependable indicators for quality control of vaccine products, surveillance and vaccination evaluation. Viral neutralization antibody (VNA) is correlated to protection against PPR and is a technically feasible indicator for this purpose. The immunogenicity of this vectored vaccine in goats and sheep, however, has not been fully evaluated. In this study, we generated two recombinant CPV viruses, rCPV-PPRVH and rCPV-PPRVF, that express PPR virus (PPRV) glycoproteins H and F, respectively. Vaccination studies with different dosages of recombinant viruses showed that rCPV-PPRVH was a more potent inducer of PPRV VNA than rCPV-PPRVF. One dose of rCPV-PPRVH was enough to seroconvert 80% of immunized sheep. A second dose induced significantly higher PPRV VNA titers. There was no significant difference in PPRV VNA responses between goats and sheep. Subcutaneous inoculation also induced a significant PPRV VNA response. PPRV VNA could be detected for over 6 months in more than 80% of vaccinated goats and sheep. Boost vaccination at 6-month intervals induced significant re-boost efficacy of PPRV VNA in goats and sheep. More over, two doses of rCPV-PPRVH could completely overcome the interference caused by pre-existing immunity to the CPV vaccine backbone in animals. Vaccination with rCPV-PPRVH also protected goats from virulent CPV challenge. Our results demonstrate that VNA can serve as a dependent indicator for effective vaccination and immune protection of animals in the field. The recombinant CPV vaccine used in our studies could be a practical and useful candidate DIVA vaccine in countries where PPR newly emerges or where stamp-out plans are yet to be implemented.     

32例三阴性乳腺癌患者易感基因胚系突变分析

目的:应用全外显子测序技术初步探讨三阴性乳腺癌(TNBC)患者易感基因突变情况。方法:收集本院就诊的32例TNBC患者,均经临床手术病理确诊。采集患者外周血提取基因组DNA进行全外显子组测序,通过生物信息学分析筛选与乳腺肿瘤相关的易感基因变异。结果:32例TNBC患者中14例检测到BRCA1/2罕见变异,明确致病性或可疑致病变异6例,突变携带频率为18.8%。其中BRCA1:c.5468-1_5474del和c.4749_4750del是较常见的突变;BRCA2:c.6027A>C为新的变异;BRCA2:c.3794G>T、c.7901T>A,BRCA1:c.4616T>C首次在中国人群中发现。除了BRCA1/2变异外,还检测到83个乳腺肿瘤易感基因变异,每个患者携带2.6个变异。2个以上患者携带的乳腺癌易感基因包括ALK、APC、CDH1、PTCH2、RB1CC1、RAD51D、RAD54L、TSC1等。结论:BRCA1/2是TNBC患者最重要的易感基因,其他与DNA损伤修复相关的基因突变可能与TNBC患者的表型有一定的相关性。     

Efficacy of a bivalent inactivated non-adjuvanted feline calicivirus vaccine: relation between in vitro cross-neutralization and heterologous protection in vivo

Feline calicivirus (FCV) is a major pathogen of the cat characterized by a strong genomic, antigenic and clinical diversity. Despite vaccination, FCV infection is highly prevalent, and for a few years, outbreaks of virulent systemic disease (VSD) have been reported in North America and Europe. An inactivated non-adjuvanted bivalent vaccine was recently developed by combining antigens derived from two broadly cross-reactive FCV strains. The antigenic relatedness between the vaccine strains and other antigenic variants was demonstrated by cross-neutralization studies in vitro. This study showed that vaccine-induced protection against heterologous challenges was correlated to in vitro cross-neutralization, and it validated the use of cross-neutralization tests to select vaccine FCV strains. This correlation applies also for the highly virulent strains causing VSD (VS-FCV).