Design of primers in the molecular detection of Feline Panleukopenia Virus

Feline Panleukopenia is a disease characterized by a reduction in the number of circulating leukocytes and enteritis with degeneration of the intestinal villi. The etiologic agent, called Feline Panleukopenia virus (FPV), belongs to the Parvoviridae family, is highly contagious and has high mortality and morbidity. Although vaccination of healthy cats is the most effective way to prevent the disease, once the symptoms appear, the treatment is supportive, presenting high mortality in the first days of the disease. FPV positive cats should be hospitalized and isolated for at least 2 weeks to avoid viral transmission. Early detection is usually done with the enzyme-linked immunoadsorption assay (ELISA) test that detects viral antigens in stool samples. As a complementary diagnostic method, in this work it was proposed to implement the Polymerase Chain Reaction (PCR) aimed at the diagnosis of FPV initially in positive samples from two feline vaccines and one canine vaccine. As an approximation to real samples, the commercial vaccines were mixed with feces and blood from a healthy cat. The results showed that the In Silico design was successful strategy based on the VP2 gene sequences of FPV available in GenBank® in conjunction with the sharp visualization of positive controls of the expected size and without amplification. nonspecific or negative controls. The fragments obtained has a nucleotide identity percentage greater than 97% with respect to the information available in Genbank® and corroborates the detection of a VP2 fragment. Thus, the future option of applying the same protocol in a diagnostic way is opened, using samples obtained from suspected patients who are infected with FPV.

Isolation and Identification of Three Parvovirus Strains in Raccoon Dogs From Liaoning Province, China

To understand the epidemiological status of parvovirus (RDPV) in raccoon dogs, intestinal tissues of raccoon dogs in Liaoning Province of China were collected and evaluated. Three strains of raccoon dog parvovirus were successfully isolated from 12 intestinal tissues. Nine samples were positive for RDPV, with a positive rate of 75%. The VP2 and NS1 genes of the viruses were cloned and subjected to sequencing for analysis. The nucleotide sequences of the VP2 gene showed 99.94% similarity to the CPV-2a/Racoon dog/QHD/2/19(MT183665) strain, and the nucleotide sequences of the NS1 gene showed 99.75% similarity to RDPV-DP1 NS1(MF996335) strain. The three isolates belonging to the CPV-2a cluster were further confirmed by amino acid sequence alignment and phylogenetic analysis. Our study enriched the epidemiological data of parvovirus in raccoon dogs in the investigating region, and the results will be helpful for future investigation of the variations and transmission of raccoon dog parvoviruses.

Single-Dose Vaccination of Recombinant Chimeric Newcastle Disease Virus (NDV) LaSota Vaccine Strain Expressing Infectious Bursal Disease Virus (IBDV) VP2 Gene Provides Full Protection against Genotype VII NDV and IBDV Challenge

Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) are the two most important and widespread viruses causing huge economic losses in the global poultry industry. Outbreaks of genotype VII NDV and IBDV variants in vaccinated poultry flocks call for genetically matched vaccines. In the present study, a genetic matched chimeric NDV LaSota vaccine strain expressing VP2 gene of IBDV variant, rLaS-VIIF/HN-VP2 was generated for the first time, in which both the F and HN genes of LaSota were replaced with those of the genotype VII NDV strain FJSW. The cleavage site of the FJSW strain F protein in the rLaS-VIIF/HN-VP2 genome was mutated to the avirulent motif found in LaSota. Expression of IBDV VP2 protein was confirmed by western blot. The rLaS-VIIF/HN-VP2 maintained the efficient replication ability in embryonated eggs, low pathogenicity and genetic stability comparable to that of parental LaSota virus. One dose oculonasal vaccination of one-week-old SPF chickens with rLaS-VIIF/HN-VP2 induced full protection against genotype VII NDV and IBDV lethal challenge. These results indicate that the rLaS-VIIF/HN-VP2 is a promising bivalent vaccine to prevent infections of IBDV and genotype VII NDV.

Genotyping and Molecular Characterization of Infectious Bursal Disease Virus Identified in Important Poultry-Raising Areas of China During 2019 and 2020

Infectious bursal disease (IBD) is an acute and highly contagious immunosuppressive disease caused by the infectious bursal disease virus (IBDV), which seriously threatens the healthy development of the poultry industry. Since its spread to China in the early 1990s, the very virulent IBDV (vvIBDV) characterized by high lethality, has been the focus of prevention and control. However, the novel variant IBDV (nVarIBDV), which has been widely prevalent in China since 2017, has brought a new threat to the poultry industry. In this study, the prevalence of IBDV in the important poultry-raising areas of China from 2019 to 2020 was detected. Of these, 45.1% (101/224) of the samples and 61.9% (26/42) of the chicken flocks were shown to be positive for IBDV. For 50 IBDVs, the sequences of the hypervariable region of the VP2 gene in segment A and of the B-marker of the VP1 gene in segment B were analyzed. The results revealed the coexistence of a number of different IBDV genotypes, including A2dB1 (nVar, 26/50, 52.0%), A3B3 (HLJ0504-like, 15/50, 30.0%), A1B1 (classical, 1/50, 2.0%), and A8B1 (attenuated, 1/50, 2.0%). This indicated that the newly emerging nVarIBDV of A2dB1 and the persistently circulating HLJ0504-like vvIBDV of A3B3 are the two important epidemic strains. Furthermore, we established that segment reassortment has occurred among these circulating strains. This study is the first to reveal the novel epidemic characteristics of IBDV since the report of the emerging nVarIBDV of A2dB1 in China.

Prevalence and Genetic Characteristics of Human Bocaviruses Detected in Patients with Acute Respiratory Infections in Bulgaria

Нuman bocaviruses (hBoVs) are often associated with acute respiratory infections (ARIs). Information on the distribution and molecular epidemiology of hBoVs in Bulgaria is currently limited. The objectives of this study were to investigate the prevalence and genetic characteristics of hBoVs detected in patients with ARIs in Bulgaria. From October 2016 to September 2019, nasopharyngeal/oropharyngeal swabs were prospectively collected from 1842 patients of all ages and tested for 12 common respiratory viruses using a real-time RT-PCR. Phylogenetic and amino acid analyses of the hBoV VP1/VP2 gene/protein were performed. HBoV was identified in 98 (5.3%) patients and was the 6th most prevalent virus after respiratory-syncytial virus (20.4%), influenza A(H1N1)pdm09 (11.1%), A(H3N2) (10.5%), rhinoviruses (9.9%), and adenoviruses (6.8%). Coinfections with other respiratory viruses were detected in 51% of the hBoV-positive patients. Significant differences in the prevalence of hBoVs were found during the different study periods and in patients of different age groups. The detection rate of hBoV was the highest in patients aged 0–4 years (6.9%). In this age group, hBoV was the only identified virus in 9.7%, 5.8%, and 1.1% of the children diagnosed with laryngotracheitis, bronchiolitis, and pneumonia, respectively. Among patients aged ≥5 years, hBoV was detected as a single agent in 2.2% of cases of pneumonia. Phylogenetic analysis showed that all Bulgarian hBoV strains belonged to the hBoV1 genotype. A few amino acid substitutions were identified compared to the St1 prototype strain. This first study amongst an all-age population in Bulgaria showed a significant rate of hBoV detection in some serious respiratory illnesses in early childhood, year-to-year changes in the hBoV prevalence, and low genetic variability in the circulating strains.

Sequence diversity and evolution of infectious bursal disease virus in Iraq

Background: Infectious Bursal Disease (IBD) is a highly infectious disease which causes huge economic losses to the poultry industry due to the direct impact of the illness and indirect consequences such as decreasing the general immunity of the flock, leaving it naive to other diseases. In Iraq, IBD is highly prevalent despite vaccination programs, yet studies on sequence diversity of the causative virus are still rare. Methods: A sample from Bursa of Fabricius from an IBD outbreak in a flock in the city of Najaf in Iraq was smeared on an FTA card. Amplicons of targeted regions in VP1 and VP2 genes were generated and sequenced. Sequences were then compared with other local and global sequences downloaded from GenBank repositories. Sequence alignment and DNA sequence analyses were achieved using MUSCLE, UGENE and MEGAx software. The molecular clock and sequence evolutionary analyses were applied using MEGAx tools. Results: The strain sequenced in this study belongs to a very virulent Infectious Bursal Disease Virus (vvIBDV) as the DNA and phylogenetic analysis of VP1 and VP2 gene sequences showed a mutual clustering with similar sequences belonging to vvIBDV genogroup 3. Analyses of the hyper variable region of VP2 gene (hvVP2) of IBDV isolates from Iraq indicates a presence of sequence diversity. Interestingly, the two vaccine strains Ventri IBDV Plus and ABIC MB71 that showed the highest sequence similarity to the local isolates in the hvVP2 region are not used in vaccination routine against IBDV in Iraq. Conclusion: Sequences of vvIBDV in Iraq are diverse. Remarkably, some of the available vaccine strains show high sequence similarity with local strains in Iraq; however, they are not included in the routine vaccination programs. Analysis of more samples involving more geographical regions is needed to draw a detailed map of antigenic diversity of IBDV in Iraq.

Genetic analysis of CPV vaccine strain NL-35-D and evaluation of induced neutralizing antibody against virulent wild-type CPV and FPV Shanghai isolates

Background Canine parvovirus (CPV) emerged in 1970s as a new causative disease of dogs worldwide. The virus was shown to be closely related, genetically and antigenically, to feline parvovirus (FPV) and to FPV-like parvoviruses from which were presumably originated by host species shift and subsequent rapid adaptation. CPV is the most important viral cause of enteritis and death in puppies, and vaccination is the most important and effective measure to prevent CPV infection. The live attenuated vaccine strain NL-35-D is widely used as a commercial vaccine in China and other countries, but its genome sequence has not been reported so far. Results We reported a near full length sequence of NL-35-D vaccine strain (Genbank: MW650831), and identified a CPV-SH2001 (Genbank: MW650830) and a FPV-SH2001 (Genbank: MW650831) isolates from the fecal samples of stray dogs and cats in Shanghai, respectively. Immunofluorescence assay revealed that the CPV and FPV isolates could be proliferated by F81 cells. we sequenced the full length genome of NL-35-D virus strain, phylogenetic analysis of full-length genome and VP2 gene in comparison with the CPV and FPV strains isolated worldwide. According to the genetic analysis of VP2 gene, the CPV-SH2001 and FPV-SH2001 isolates were clustering far from the NL-35-D. However, the results demonstrated that NL-35-D vaccine strain still shares part of neutralizing epitopes with CPV and FPV isolates. The neutralizing antibody serum test also suggested vaccine strain NL-35-D is still effective against the present field isolated CPV and FPV strains. Therefore, these shared antigenic epitopes may be critical to the immune efficacy of the vaccine and deserve further testing and validation. Conclusions Our results revealed that pups inoculated with CPV vaccine had significantly high neutralizing antibody titers against CPV and FPV virus strains. Pups vaccinated with CPV possess antibodies against heterologous viruses, the antibodies probably at levels still can provide complete or partial immunity in young pups.

Molecular Analysis of Full-Length VP2 of Canine Parvovirus Reveals Antigenic Drift in CPV-2b and CPV-2c Variants in Central Chile

Canine parvovirus (CPV) is a major pathogen in canines, with a high mortality rate in unvaccinated puppies. CPV is traditionally classified into three antigenic variants (CPV-2a, CPV-2b and CPV-2c) based on the amino acid sequence of the VP2 protein. Currently, various mutations are described in the receptor-binding area or in the regions of greatest antigenicity of the VP2 protein, giving rise to new viral variants that are capable of immunological escape, affecting the protective immunity of traditional vaccines. In the present study, a molecular characterization of the VP2 gene was performed, which included phylogenetic analysis, amino acid characterization and determination of selection pressures. Blood samples were initially collected from canine patients with clinical signs of gastrointestinal infection, of which 69 were positive for CPV as measured by means of PCR and 18 samples were selected for the amplification of the complete VP2 gene. The analysis revealed a higher rate of CPV-2c-positive patients compared to CPV-2b. Furthermore, the amino acid characterization of VP2 indicated mutations in the regions of highest antigenicity previously described in the literature (CPV-2b: 297 and 324; CPV-2c: 440), as well as others not previously documented (CPV-2b: 514; CPV-2c: 188, 322, 379, 427 and 463). Our analysis of selection pressure showed that the VP2 gene is under negative selection. However, positive selection point sites were identified, both in CPV-2c (324, 426 and 440) and CPV-2b (297 and 324), at sites that have been associated with evasion of the immune response via antigenic drift, which possibly has implications for the protective immunity generated by traditional vaccines.

Integrated Use of Molecular Techniques to Detect and Genetically Characterise DNA Viruses in Italian Wolves (Canis lupus italicus)

In this study, internal organs (tongue, intestine, and spleen) of 23 free-ranging Italian wolves (Canis lupus italicus) found dead between 2017 and 2019 were tested for Carnivore protoparvovirus 1, Canine adenovirus (CAdV), and Canine circovirus (CanineCV) using real-time PCR assays. Genetic characterisation of the identified viruses was carried out by amplification, sequencing, and analysis of the complete viral genome or informative viral genes. All the wolves tested positive for at least one of the DNA viruses screened, and 11/23 were coinfected. Carnivore protoparvoviruses were the most frequently detected viruses (21/23), followed by CanineCV (11/23) and CAdV (4/23). From the analysis of the partial VP2 gene of 13 carnivore protoparvoviruses, 12 were canine parvovirus type 2b, closely related to the strains detected in dogs and wild carnivores from Italy, and one was a feline panleukopenia-like virus. Of the four CAdV identified, two were CAdV-1 and two were CAdV-2. The complete genome of seven CanineCVs was sequenced and related to the CanineCV identified in dogs, wolves, and foxes worldwide. Close correlations emerged between the viruses identified in wolves and those circulating in domestic dogs. Further studies are needed to investigate if these pathogens may be potentially cross-transmitted between the two species.