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.

Growth characteristics of experimental live influenza vaccine strains with modified NP and NS genes

BACKGROUND: Vaccination is the most effective means of fighting influenza epidemics, but the immunogenicity of licensed influenza vaccines is not always satisfactory. One of the ways to increase the immunogenicity of an attenuated live influenza vaccine is to shorten the open reading frame of the NS1 protein, a modulator of innate antiviral immunity. In addition, the T-cell response to vaccination can be optimized by including the NP gene from the epidemic parental virus into the genome of vaccine strains. MATERIALS AND METHODS: The open reading frame of the NS1 protein of the master donor virus A/Leningrad/134/17/57 was truncated to 126 amino acids by site-directed mutagenesis. The HA, NA, and NP genes of the model virus A/Anhui/1/2013 (H7N9) were cloned into the pCIPolISapIT vector. The rescue of recombinant influenza viruses was performed by transfection of Vero cells with a desired set of plasmids. The growth properties of the recombinant viruses were determined in embryonated chicken eggs incubated at different temperatures, as well as in the tissues of the respiratory tract of mice (nasal turbinates, lungs). RESULTS: Experimental live influenza vaccine strains of subtype H7N9 with genome compositions 6:2 and 5:3 and carrying a full-length or truncated NS1 gene were actively replicated in eggs under optimal conditions, while maintaining the temperature-sensitive and cold-adapted phenotypes characteristic of classical live influenza vaccine strains. All viruses lacked the ability to grow in the lungs of C57BL/6J mice, which confirms the attenuated phenotype of the viruses. In the nasal passages of mice, only viruses with the full-length NS1 gene replicated, while viruses expressing the truncated NS1 protein were not detected in the respiratory tract of animals. CONCLUSIONS: The results indicate that modification of the NS1 gene of the vaccine virus and the inclusion of wild-type NP gene in its genome does not affect its growth characteristics in eggs. A decrease in the activity of viral replication in the upper respiratory tract of mice with a shortening of the NS1 open reading frame indicates an increase in the attenuating properties of modified vaccines, which opens up prospects for the use of new vaccines in children under three years of age.

Dengue Virus Non-structural (NS) 1 Gene as A Molecular Marker for Early Detection of in vitro Dengue Virus Infection

Serology-based dengue assays at times produce inaccurate results especially in the early phase of disease onset. A more precise diagnostic approach detecting dengue infections in the early phase enables better management of the disease. This helps reduce dengue-associated morbidity and mortality. Besides, an early diagnosis of dengue is also very beneficial in a dengue outbreak and in endemic regions. In this light, this study aimed to determine the potential of the dengue virus (DENV) non-structural 1 (NS1) gene as an early detection biomarker. The cytopathic effects (CPE) were monitored and the cell death of DENV serotype-2 (DENV2)-infected Vero cells was evaluated for fourteen consecutive days. Only Lemos and in-house NS1-specific primer pairs showed positive amplifications in the preliminary primer validation. Thus, both of the primer pairs were then used to amplify the NS1 gene from the infected cells. The NS1 gene was detected as early as day-2 post-infection using the in-house primers. There was no amplicon produced using the Lemos primers. This is speculated to be attributable to the relatively lower complementarity of the primer sequences with that of the template and low amount of viral mRNA in the DENV2-infected cells. Conclusively, the DENV NS1 gene is a potential early detection marker, however, the NS1-specific primers should be pre-validated to ensure a reliable dengue diagnosis.

First Clinical Case of Equine Parvovirus-Hepatitis-Related Theiler’s Disease in Asia

Equine parvovirus-hepatitis (EqPV-H) is a newly identified etiologic agent of Theiler’s disease (TD). We present a case of EqPV-H-related fulminant hepatitis in a 14-year-old thoroughbred mare in Korea. The mare had acute hepatopathy and gastrointestinal symptoms, with abnormal liver-related blood parameters. The horse was born in the USA and imported to Korea in 2017, with no history of administration of equine biological products after entry into Korea. The horse was diagnosed with EqPV-H-associated hepatitis after abdominal ultrasonography, laparotomy, and nested polymerase chain reaction (PCR) and in situ hybridization (ISH) assays. The serum, nasal swab, oral swab, and liver biopsy were positive for EqPV-H according to the PCR assay. Genetic analysis of the partial NS1 gene of EqPV-H showed a unique nucleotide substitution, distinct from that in previously deposited strains. EqPV-H DNA was found not only in hepatocytes but also in bile duct epithelium and Kupffer cells, particularly via ISH. To the best of our knowledge, this is the first case of EqPV-H-associated TD in Asia, providing the first clinical evidence for viral shedding from the mouth and nose, and identification of EqPV-H in the liver. This study contributes to a better understanding of the pathological features of EqPV-H-associated TD.

Identification of porcine circovirus type 2 (PCV2), type 3 (PCV3) and porcine parvovirus (PPV) in swine by multiplex PCR test

This study aimed to simultaneously detect three important viruses reported to be involved in the reproductive problems of sows. A multiplex PCR (mPCR) test was developed to provide rapid diagnosis of porcine circovirus type 2 and 3 (PCV2, PCV3) and to illustrate parvovirus (PPV) prevalence in sow herds. Three pairs of specific primers were designed to target PCV2 Cap gene, PCV3 Cap gene and PPV NS1 gene, with predicted mPCR products of 702 bp, 267 bp and 380 bp, respectively. The detection limit of mPCR was 100 copies/reaction per target gene. The mPCR was run against a panel of 94 swine serum samples whose infection status had been pre-determined by commercial real-time PCR kits. Sequencing of mPCR products performed with clinical serum samples accurately confirmed the results. Overall, the results indicated that the mPCR functioned accurately and specifically and matched 100% with the single-target real-time PCRs. The mPCR was developed successfully and can be used in routine diagnosis of PCV2, PCV3 and PPV.

Parvovirus-induced encephalitis in a juvenile raccoon

A juvenile raccoon was euthanized because of severe neurologic signs. At postmortem examination, no significant gross lesions were present. Histologic evaluation demonstrated nonsuppurative encephalitis in thalamus, brainstem, and hippocampus, cerebellar Purkinje cell loss, as well as poliomyelitis and demyelination of the spinal cord. Parvovirus antigen–specific immunohistochemistry revealed immunopositive neurons in the brainstem, cerebral cortex, and hippocampus. A few Purkinje cells were also immunopositive. DNA extracted from formalin-fixed, paraffin-embedded brain tissue (thalamus, hippocampus, cerebral cortex) yielded a positive signal using PCR targeting both feline and canine parvovirus. Sequencing analyses from a fragment of the NS1 gene and a portion of the VP2 gene confirmed the presence of DNA of a recent canine parvovirus variant (CPV-2a–like virus) in the cerebellum. Our case provides evidence that a recent canine parvovirus (CPV) strain ( Carnivore protoparvovirus 1) can infect cerebral and diencephalic neurons and cause encephalitis in an otherwise healthy raccoon. Parvovirus-induced encephalitis is a differential diagnosis of rabies and canine distemper in raccoons with neurologic signs.

Identification of Porcine circovirus type (PCV2) and type 3 (PCV3), Porcine 2 parvovirus (PPV) in swine by multiplex PCR test

Background : Aiming to simultaneously detect three important viruses known to be involved in reproductive problems of sows, a multiplex PCR (mPCR) test was developed to provide rapid diagnosis of porcine circovirus type 2 and 3 (PCV2, PCV3) and to illustrate parvovirus (PPV) prevalence in sow herds. Methods : Three pairs of specific primers were designed to target PCV2 Cap gene, PCV3 Cap gene and PPV NS1 gene, with predicted mPCR products of 702 bp, 267 bp and 380 bp, respectively. Results : The detection limit of mPCR was 100 copies/ reaction per target gene. Sequencing of mPCR products performed with clinical serum samples accurately confirmed results. The mPCR was run against a panel of 94 swine serum samples whose infection status had been pre-determined by commercial real-time PCR kits. Overall, the mPCR results matched 100% with the real-time PCRs. Conclusions : The developed mPCR test functions successfully and can be used in routine rapid diagnosis of PCV2, PCV3 and PPV.

Cerebellar hypoplasia and dysplasia in a juvenile raccoon with parvoviral infection

A juvenile raccoon ( Procyon lotor) was submitted dead to the Minnesota Veterinary Diagnostic Laboratory for rabies testing without history. The animal had marked hypoplasia of the cerebellum. Histology demonstrated that most folia lacked granule cells and had randomly misplaced Purkinje cells. Immunohistochemistry revealed the presence of parvoviral antigen in a few neurons and cell processes. PCR targeting feline and canine parvovirus yielded a positive signal. Sequencing analyses from a fragment of the nonstructural protein 1 ( NS1) gene and a portion of the viral capsid protein 2 ( VP2) gene confirmed the presence of DNA of a recent canine parvovirus variant (CPV-2a–like virus) in the cerebellum. Our study provides evidence that (canine) parvovirus may be associated with cerebellar hypoplasia and dysplasia in raccoons, similar to the disease that occurs naturally and has been reproduced experimentally by feline parvoviral infection of pregnant cats, with subsequent intrauterine or neonatal infections of the offspring.

Association of Human Parvovirus B19 Infection with Development and Clinical Course of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome

Our aim was to estimate the presence of B19V infection markers, the level of cytokines and time period since the appearance of infection in association with ME/CFS clinical symptoms. In 200 ME/CFS patients and 104 control group individuals the presence of B19V-specific IgG/IgM class antibodies, B19V NS1 gene sequence, mRNA expression, viral load and level of cytokines were determined. B19V-specific IgG-antibodies were found in 70% of ME/CFS patients and 67.4% of controls, IgM-antibodies in 8% of patients and in none of controls, B19V genomic sequences in 29% of patients and 3.8% of controls. 58.6% of positive patients had active and 41.4% had latent/persistent B19V infection. B19V NS1 gene expression was detected in 43% of patients. B19V load varied from < 0.2 copies to median 38.2 copies/µg of DNA. According to the antibody pattern, 36% of patients had a recent, and 43% had sustained B19V infection. Patients with the B19V genomic sequence and NS1 specific antibodies significantly more often had lymphadenopathy and multi-joint pain. Onset of the symptoms corresponded to time of appearance of B19V infection. IL-10 and TNF-levels were higher in patients with elevated B19V load. B19V genome 1 was identified in Latvian ME/CFS patients. The results indicated that at least in some cases B19V infection plays an important role in ME/CFS development