Establishment and application of multiplex PCR for rapid detection of three mink diarrhea-associated viruses.

In this report, a multiplex PCR method was developed for the detection of three diarrhea-associated viruses in mink, including circovirus (MCV), bocavirus (MBoV), and enteritis virus (MEV). Three compatible sets of primers specific for each virus were designed respectively based on their conserved sequences. After optimization of the crucial factors such as primer concentration and annealing temperature in single and multiple amplification, three specific fragments were simultaneously amplified with the highest sensitivity and specificity in one PCR reaction. The fragments amplified were 259 bp (MCV)，455 bp (MBoV) and 671 bp (MEV). The sensibility of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There were no cross-reactions with some relevant pathogens like mink coronavirus, canine distemper virus, and aleutian mink disease virus. In our study we analyzed viral DNA in mink fecal samples by multiplex PCR assay from China, which revealed the occurrence of MCV, MBoV, and MEV as 3.1 %, 5.7 %, and 9.8 %, respectively. The testing results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100 %. These results indicated that the method could provide technical support for rapid detection of the three diarrhea-associated viruses, and epidemiological investigation of mink viral diarrhea.

Chaperones Hsc70 and Hsp70 play distinct roles in the replication of bocaparvovirus minute virus of canines.

Minute virus of canines (MVC) belongs to the genus Bocaparvovirus (formerly Bocavirus) within the Parvoviridae family and causes serious respiratory and gastrointestinal symptoms in neonatal canines worldwide. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. However, little is known about the MVC-host cell interactions. In this study, we identified that two cellular proteins (Hsc70 and Hsp70) interacted with NS1 and VP2 proteins of MVC, and both two domains of Hsc70/Hsp70 were mediated for their interactions. Functional studies revealed that Hsp70 was induced by MVC infection, knockdown of Hsc70 considerably suppressed MVC replication, whereas the replication was dramatically promoted by Hsp70 knockdown. It is interesting that low amounts of overexpressed Hsp70 enhanced viral protein expression and virus production, but high amounts of Hsp70 overexpression weakened them. Upon Hsp70 overexpressing, we observed that the ubiquitination of viral proteins changed with Hsp70 overexpression, and proteasome inhibitor (MG132) restored an accumulation of viral proteins. In addition, we verified that Hsp70 family inhibitors remarkably decreased MVC replication. Overall, we identified Hsc70 and Hsp70 as interactors of MVC NS1 and VP2 proteins and were involved in MVC replication, which may provide novel targets for anti-MVC approach.

Stability and integrity of self-assembled bovine parvovirus virus­like particles (BPV­VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment.

BACKGROUND: Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences. METHODS: In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism. RESULTS: Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs. CONCLUSIONS: In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

Immune escape of bovine parvovirus by VP1 inhibiting IFN-β production through the RIG-I-like receptor pathway / Escape inmunológico del parvovirus bovino mediante la inhibición de VP1 de la producción de IFN-β a través de la vía del receptor tipo RIG-I

Objective: The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-β) production and to reveal further molecular mechanism of BPV immune escape. Method: The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-β mRNA were detected using qPCR. Results: The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-β mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-β expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. Conclusion: pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-β. Overexpression of pCMV-Myc-BPV-VP decreased IFN-β mRNA expression in HEK 293 T cells and inhibited IFN-β production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.(AU)

Structural Characterization of Canine Minute Virus, Rat and Porcine Bocavirus.

Bocaparvovirus is an expansive genus of the Parvovirinae, with a wide range of vertebrate hosts. This study investigates Canine minute virus (CnMV), Rat bocavirus (RBoV), and Porcine bocavirus 1 (PBoV1). Both CnMV and PBoV1 have been found in gastrointestinal infections in their respective hosts, with CnMV responsible for spontaneous abortions in dogs, while PBoV has been associated with encephalomyelitis in piglets. The pathogenicity of the recently identified RBoV is currently unknown. To initiate the characterization of these viruses, their capsids structures were determined by cryo-electron microscopy at resolutions ranging from 2.3 to 2.7 Å. Compared to other parvoviruses, the CnMV, PBoV1, and RBoV capsids showed conserved features, such as the channel at the fivefold symmetry axis. However, major differences were observed at the two- and threefold axes. While CnMV displays prominent threefold protrusions, the same region is more recessed in PBoV1 and RBoV. Furthermore, the typical twofold axis depression of parvoviral capsids is absent in CnMV or very small in PBoV and RBoV. These capsid structures extend the structural portfolio for the Bocaparvovirus genus and will allow future characterization of these pathogens on a molecular level. This is important, as no antivirals or vaccines exist for these viruses.

Epidemiologic and Clinical Characteristics of Human Bocavirus Infection in Children with or without Acute Gastroenteritis in Acre, Northern Brazil.

Human bocavirus (HBoV) is an emerging virus detected around the world that may be associated with cases of acute gastroenteritis (AGE). However, its contribution to AGE has not been elucidated. This study aimed to describe the frequency, clinical features, and HBoV species circulation in children up to 5 years with or without AGE symptoms in Acre, Northern Brazil. A total of 480 stool samples were collected between January and December 2012. Fecal samples were used for extraction, nested PCR amplification, and sequencing for genotyping. Statistical analysis was applied to verify the association between epidemiological and clinical characteristics. Overall, HBoV-positivity was 10% (48/480), with HBoV-positive rates of 8.4% (19/226) and 11.4% (29/254) recorded in diarrheic and non-diarrheic children, respectively. The most affected children were in the age group ranging between 7 and 24 months (50%). HBoV infection was more frequent in children who live in urban areas (85.4%), use water from public networks (56.2%), and live with adequate sewage facilities (50%). Co-detection with other enteric viruses was 16.7% (8/48) and the most prevalent coinfection was RVA+ HBoV (50%, 4/8). HBoV-1 was the most frequent species detected in diarrheic and non-diarrheic children, responsible for 43.8% (21/48) of cases, followed by HBoV-3 (29.2%, 14/48) and HBoV-2 (25%, 12/48). In this study, HBoV infection was not always associated with AGE, as most HBoV cases belonged to the non-diarrheal group. Future studies are warranted in order to determine the role of HBoV in causing acute diarrhea disease.

Low dose thymoglobulin versus basiliximab in cytomegalovirus positive kidney transplant recipients: Effectiveness of preemptive cytomegalovirus modified strategy / Timoglobulina en dosis bajas vs. basiliximab en receptores de trasplante renal positivos para citomegalovirus: efectividad de una estrategia preventiva modificada

Background: We performed a retrospective trial to determine asymptomatic CMV reactivation and CMV disease in kidney allograft recipients with positive CMV serostatus. Methods: Preemptive modified strategy under low dose thymoglobulin versus basiliximab induction was evaluated. Patients were monitored by CMV-polymerase chain reaction (PCR); if the viral load was >4000copies/μl, they received valganciclovir adjusted for their renal function. Results: 132 recipients were included in the study, 84 and 48 receiving basiliximab and thymoglobulin induction respectively, and followed up for 12 months. Asymptomatic CMV reactivation was significantly higher for thymoglobulin (77.1% vs. 16.7%, p<0.001). Treatment groups had similar rates of CMV disease (3.6% vs. 2.1%, p 0.538). The significant difference in asymptomatic CMV reactivation between two treatment groups did not have any impact on 1 year graft function (71±26ml/min vs. 74±19ml/min; p=0.475) and no histological differences in protocol biopsies were observed among patients with asymptomatic CMV reactivation vs those without CMV reactivation. Conclusions: Due to the high asymptomatic CMV reactivation incidence in patients who received thymoglobulin induction, our results suggest that valganciclovir prophylaxis may be advantageous in CMV seropositive renal transplant recipients after low dose thymoglobulin induction. A preemptive strategy appeared to significantly reduce the likelihood of CMV disease in both groups. Rejection risk and negative impact in renal function associated with asymptomatic CMV reactivation was not found in our series. (AU)

Antecedentes: Llevamos a cabo un estudio retrospectivo para determinar la reactivación y enfermedad por CMV en receptores de trasplante renal CMV seropositivos bajo diferentes esquemas de inducción. Métodos: Una estrategia preventiva modificada bajo inducción con basiliximab y timoglobulina en dosis bajas fue evaluada. Se llevó a cabo un seguimiento de la carga viral-reacción de cadena de la polimerasa-CMV; los valores mayores de 4000 copias/μl recibieron valganciclovir ajustado a la función renal. Resultados: Un total de 132 receptores de trasplante renal fueron incluidos; 84 y 48 recibieron inducción con basiliximab y timoglobulina respectivamente. Seguimiento hasta el mes 12 postrasplante. La reactivación asintomática de CMV fue significativamente mayor para timoglobulina (77,1% vs. 16,7%, p<0,001). La tasa de enfermedad por CMV fue similar en ambos grupos de tratamiento (3,6% vs. 2,1%, p=0,538). Ningún impacto en la función renal un año postrasplante fue encontrado entre los grupos a pesar de la diferencia significativa en reactivación asintomática de CMV (71±26ml/min vs. 74±19ml/min; p=0,475); igualmente, no encontramos diferencias en los hallazgos histológicos en biopsias por protocolo entre receptores con reactivación asintomática por CMV y aquellos sin reactivación. Conclusiones: La alta incidencia de reactivación asintomática por CMV en receptores seropositivos a pesar del uso de bajas dosis de timoglobulina sugiere que la profilaxis con valganciclovir es una estrategia apropiada en este grupo; sin embargo, una estrategia preventiva reduce significativamente la probabilidad de enfermedad por CMV en ambos grupos de tratamiento. El riesgo de rechazo y el impacto negativo en la función renal asociado a la reactivación asintomática por CMV no fue encontrado en nuestra experiencia.

Immune escape of bovine parvovirus by VP1 inhibiting IFN-ß production through the RIG-I-like receptor pathway.

OBJECTIVE: The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-ß) production and to reveal further molecular mechanism of BPV immune escape. METHOD: The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-ß mRNA were detected using qPCR. RESULTS: The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-ß mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-ß expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. CONCLUSION: pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-ß. Overexpression of pCMV-Myc-BPV-VP decreased IFN-ß mRNA expression in HEK 293 T cells and inhibited IFN-ß production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.

A STUDY OF THE NOVEL WU AND KI POLYOMAVIRUSES, BOCAVIRUS ADENOVIRUS IN CHILDREN WITH UPPER RESPIRATORY TRACT INFECTIONS.

OBJECTIVE: The aim: To find out the frequency of WU and KI polyomaviruses, and Human Boca and Adenoviruses infections among children with different types of acute upper respiratory tract infections and to compare the frequency of these viruses among immune-competent and immune compromised patients. PATIENTS AND METHODS: Materials and methods: A case-control study conducted in children aged 3-18 years with acute upper respiratory tract infections. The samples were taken from: Group 1: 100 immuno competent children with acute upper respiratory tract infections. Group 2: 100 immuno compromised children (Leukemic, cancer, Nephrotic syndrome, chronic renal failure and children with renal transplant) with acute upper respiratory tract infections. Group 3: 100 apparently healthy children without respiratory infections as control group. Nasal swap samples were collected from children and then viral DNA extracted from these samples. Then detection of WU, KI polyomaviruses HBoV and HAdv was done by using real time PCR. RESULTS: Results: All of 300 samples were negative for WU and KI polyomaviruses. However, human Bocavirus was detected in the three groups (immunocompromised, immunocompetent and control group) and the positivity rates were 61.61%, 37.37% and 18.18%, respectively. While human adenovirus was found only in 2% of immunocompromised patients and 1.1% of immunocompetent patients also there were cases positive for both HBoV and HAdv in 5.5% of immunocompromised patients, and 8.8% of immunocompetent patients. CONCLUSION: Conclusions: High frequency of HBoV especially in immunocompromised patients while low number of positive cases for HAdv by using nasal swab samples, WU and KI polyomaviruses could not be detected in samples.

Naturally acquired feline bocavirus type 1 and 3 infections in cats with neurologic deficits.

Feline bocaviruses (FBoVs) have been recognized as novel feline pathogens associated with gastrointestinal diseases. Although bocavirus infections in humans and animals present a broad range of clinical symptoms including neurologic diseases, the neuropathology caused by FBoV infection in cats is unknown. This study aims to investigate the presence of bocavirus in the brain samples of 78 cats showing neurologic deficits and 41 healthy cats using polymerase chain reaction (PCR) and to present the pathological findings of FBoV infection in brain tissues. Only five (6.41%, five out of 78) cats with neurological deficit were FBoV positive on PCR screening and were characterized as FBoV-1 (four out of five) and FBoV-3 (one out of five) by sequencing. Among FBoV-positive cases, viral DNA were detected by PCR in the cerebrum and brain stem of all FBoV-positive cases and rarely detected in the cerebellum of some cases. Histologically, all FBoV-positive cases revealed a variety of inflammatory responses. Among these, 80% (four out of five cases) showed multifocal neuronal vacuolation, mainly found in the cerebrum and brain stem. Eosinophilic inclusion-like materials were found within the nuclei of glial cells in the FBoV-3-positive case. In situ hybridization revealed FBoV DNA in oligodendroglia and vacuolated neurons detected using dual labelling with Olig-2 and NeuN immunohistochemistry, respectively. Transmission electron microscopy confirmed the presence of FBoV-3 virions in the nuclei of glial cells. Apart from localization in brain tissues, the FBoV DNA were also detected in multiple lymph nodes (five out of five) and some intestines (two out of five) of such positive cases, suggesting both parenteral and enteral infections. Complete genome sequence analysis revealed genetic diversity of detected FBoV-1, which were closely related to the strains found in China and Hong Kong, while the detected FBoV-3 presented distant monophyletic clade to previously detected FBoV-3 sequences. The FBoVs, together, should be considered a neurotropic virus and a possible cause for neuronal vacuolation in cats with neurologic deficits.

The enteric virome of cats with feline panleukopenia differs in abundance and diversity from healthy cats.

Feline panleukopenia (FPL) is a severe, often fatal disease caused by feline panleukopenia virus (FPV). How infection with FPV might impact the composition of the entire eukaryotic enteric virome in cats has not been characterized. We used meta-transcriptomic and viral particle enrichment metagenomic approaches to characterize the enteric viromes of 23 cats naturally infected with FPV (FPV-cases) and 36 age-matched healthy shelter cats (healthy controls). Sequencing reads from mammalian infecting viral families largely belonged to the Coronaviridae, Parvoviridae and Astroviridae. The most abundant viruses among the healthy control cats were feline coronavirus, Mamastrovirus 2 and Carnivore bocaparvovirus 3 (feline bocavirus), with frequent coinfections of all three. Feline chaphamaparvovirus was only detected in healthy controls (6 out of 36, 16.7%). Among the FPV-cases, in addition to FPV, the most abundant viruses were Mamastrovirus 2, feline coronavirus and C. bocaparvovirus 4 (feline bocaparvovirus 2). The latter and feline bocaparvovirus 3 were detected significantly more frequently in FPV-cases than in healthy controls. Feline calicivirus was present in a higher proportion of FPV-cases (11 out of 23, 47.8%) compared to healthy controls (5 out of 36, 13.9%, p = 0.0067). Feline kobuvirus infections were also common among FPV-cases (9 out of 23, 39.1%) and were not detected in any healthy controls (p < .0001). While abundant in both groups, astroviruses were more frequently present in FPV-cases (19 out of 23, 82.6%) than in healthy controls (18 out of 36, p = .0142). The differences in eukaryotic virome composition revealed here indicate that further investigations are warranted to determine associations between enteric viral co-infections on clinical disease severity in cats with FPL.

Molecular evidence of rat bocavirus among rodents in Peninsular Malaysia.

Rat bocavirus (RBoV) and rodent bocavirus (RoBoV) have previously been detected in Rattus norvegicus; however, these viruses have not been reported in rodent populations in Malaysia. We investigated the presence of RBoV and RoBoV in archived rodent specimens. DNA barcoding of the rodent cytochrome c oxidase gene identified five different species: Rattus tanezumi R3 mitotype, Rattus tiomanicus, Rattus exulans, Rattus argentiventer, and Rattus tanezumi sensu stricto. Three spleens were positive for RBoV (1.84%; 3/163), but no RoBoV was detected. Phylogenetic analyzes of the partial non-structural protein 1 gene grouped Malaysian RBoV strains with RBoV strains from China. Further studies among rats from different geographical locations are warranted for this relatively new virus.

Ungulate bocaparvovirus 4 and rodent bocavirus are different genotypes of the same species of virus.

Bocaviruses are associated with many human infectious diseases, such as respiratory tract infections, gastroenteritis, and hepatitis. Rats are known to be reservoirs of bocaviruses, including rodent bocavirus and rat bocavirus. Recently, ungulate bocaparvovirus 4, a known porcine bocavirus, has also been found in rats. Thus, investigating bocaviruses in rats is important for determining the origin of the viruses and preventing and controlling their transmission. To the best of our knowledge, no study to date has investigated bocaviruses in the livers of rats. In this report, a total of 624 rats were trapped in southern China between 2014 and 2017. Liver and serum samples from rats were tested for the prevalence of bocaviruses using PCR. Sequences related to ungulate bocaparvovirus 4 and rodent bocavirus were detected in both liver and serum samples. Interestingly, the prevalence of ungulate bocaparvovirus 4 (reference strain: KJ622366.1) was higher than that of rodent bocavirus (reference strain: KY927868.1) in both liver (2.24% and 0.64%, respectively) and serum samples (2.19% and 0.44%, respectively). The NS1 regions of ungulate bocaparvovirus 4 and rodent bocavirus related sequences displayed over 84% and 88% identity at the nucleic acid and amino acid levels, respectively. Furthermore, these sequences had similar genomic structure, genomic features, and codon usage bias, and shared a common ancestor. These viruses also displayed greater adaptability to rats than pigs. Our results suggested that ungulate bocaparvovirus 4 and rodent bocavirus may originate from rats and may be different genotypes of the same bocavirus species.

Isolation, pathogenesis, and genetic evolution of a porcine bocavirus PBoV/HB/30/2018 strain in China.

Porcine bocavirus (PBoV) was first identified in Sweden in 2009. Due to its association with healthy as well as diseased pigs, its role in clinical disease has not been reported yet. In the present study, bocavirus was identified from the intestinal content of a 30-day-old piglet and its whole genome was constructed and phylogenetic analysis was carried on. The pathogenesis of bocavirus was investigated following orogastric inoculation of the colostrum-deprived newborn piglet with bacteria free intestinal content. The bocavirus-inoculated piglets developed diarrhea, shed virus in the rectal swabs from 18 h post inoculation and developed macroscopic and microscopic lesions in small intestine with virus confirmed by conventional PCR. This study experimentally confirmed pathogenicity and characterized bocavirus as the etiological agent of diarrhea in the colostrum-deprived newborn piglets. On phylogenetic analysis, it was observed that this virus has long evolutionary history with subsequent mutation as well as better host adaptation. This study highlights the importance of identifying bocavirus as the etiological agent of viral diarrhea that could threaten livestock, public health as well as economic loss.

Molecular characterization of human bocavirus in recycled water and sewage sludge in Thailand.

The study aimed to assess the presence and molecular characterization of human bocavirus (HBoV) in recycled water and sewage sludge samples in Thailand. One hundred and two recycled water and eighty-six sewage sludge samples collected from a wastewater treatment plant were tested for the presence of HBoV using nested PCR with broad-range primer pairs targeting the capsid proteins VP1 and VP2. HBoV DNA was detected in recycled water of 9/102 (8.8%) samples and sewage sludge of 27/86 (31.4%) samples. Based on DNA sequencing and phylogenetic analysis, the HBoV DNA sequences had 98.8-100.0% nucleotide identity to the sequences from HBoV reported globally. Thirty-five HBoV-positive samples were identified to genotypes as the predominant HBoV2; 26 followed by HBoV3; 8 and the rare HBoV4; 1 sample. Concerning recycled water, HBoV2 was detected in 3 (2.9%) and HBoV3 was detected in 5 (4.9%) of all samples. The sewage sludge samples were characterized as HBoV2 in 23 (26.7%), HBoV3 in 3 (3.5%) and HBoV4 in 1 (1.2%) of all samples. The frequency of HBoV detected in recycled water and sewage sludge samples significantly differed in sample type (p-value = 0.007). The findings of three HBoV genotypes in recycled water and sewage sludge emphasized the circulation of the virus in the environment and the potential source of transmission to the community.

Detection and molecular characterization of astro and bocaviruses in dogs in Minnesota.

Canine astrovirus (CAstV) and canine bocavirus (CBoV) are involved in cases of mild, and sometimes severe, gastroenteritis in dogs. Fecal samples from two dead dogs with gastroenteritis were received at the University of Minnesota Veterinary Diagnostic Laboratory to determine the cause of death. Small round viruses of 20-35 nm diameter were observed by negative contrast electron microscopy. The samples were subjected to Illumina MiSeq sequencing. Both samples were strongly positive for CAstV; all viral reads were related to CAstV. In addition, sample number 1 had a few reads of CBoV. Two complete sequences of CAstV were identified (6625 and 6627 nt in length) with 95% nt identity. RT-PCR and PCR were used to confirm CAstV and CBoV infections in successive samples of canine gastroenteritis. Sanger sequencing was done on nucleic acids from positive samples. Of a total of ten samples, CAstV and CBoV infections were confirmed in six and eight animals, respectively. Four animals had mixed infection with both viruses. All sequences of ORF1b gene of CAstVs showed closest clusters in phylogenetic tree with 96-100% nucleotide and amino acids identity. On the other hand, identity between VP2 gene of different CBoV strains in this study ranged from 93%- 100%. All strains were located close to each other except the divergent MT078234 strain, which was arranged in a separate branch and was closer to reference strain JN648103/USA/2010. This study highlights the importance of electron microscopy and next generation sequencing for early detection and characterization of viruses associated with dog gastroenteritis.

Human bocavirus 2 detected in Rattus norvegicus feces in China.

Bocaviruses are typical zoonotic pathogens with a wide range of hosts. Here, we report the detection of human bocavirus (HBoV) in Rattus norvegicus captured in China and the results of sequencing and phylogenetic analysis based on the partial VP1 region and the entire viral genome. A total of 357 fecal samples from rats were collected in 2015-2017 and analyzed for HBoV using PCR. The detection rate of HBoV was 0.84% (3/357). Phylogenetic analysis revealed that this virus is genetically closely related to HBoV-2. R. norvegicus may be a carrier of HBoV, and its impact on public health merits attention.

First detection of feline bocaparvovirus 2 and feline chaphamaparvovirus in healthy cats in Turkey.

The pet cat's population and the number of viruses that infect them are increasing worldwide. Recently, feline chaphamaparvovirus (FeChPV, also called fechavirus) and feline bocaparvovirus (FBoV) infections, which are novel parvovirus species, have been reported in cats from different geographic regions. Here, we investigated FBoV 1-3 and FeChPVs in healthy cats in Turkey using PCR, where nuclear phosphoprotein 1 (NP1) is targeted for FBoV and NP for FeChPV. For this purpose, oropharygeal swabs were obtained from 70 healthy cats with different housing status from June 15 to December 1, 2020. After PCR screening tests, six out of 70 cats (5/47 shelter cats; 1/23 domestic cats) were found to be positive for FBOV, while two were positive for FeChPV (1/47 shelter cats; 1/23 domestic cats). No cat was found in which both viruses were detected. The nucleotide (nt) sequence comparison in the 310 base pair (bp) NP gene of the two FeChPVs identified in this study shared a high identity with each other (95.0% nt and 99% aa identities) and with previously reported FeChPVs (92.4-97.1% nt and 98.1-99.0% aa identities), including 313R/2019/ITA, 49E/2019/ITA, VRI_849, 284R/2019/ITA, and IDEXX-1. Here, the near-full length (1489 nt, 495 amino acids-aa) of the VP2 gene of the FechaV/Tur-2020/68 isolate obtained from the study was also sequenced. The nt and aa identity ratio of this isolate with other FeChPVs was 98.0-98.5%-96-96.5%, respectively. Sequences of the 465 bp NP1 gene of the six Turkish FBoV strains shared high identities with each other (99.6-100% nt and 99.3-100% aa identities) and with those of FBoV-2 strains (97.8-99.1% nt and 98.0-100% aa identities), including 16SY0701, 17CC0505-BoV2, HFXA-6, and POR1. All FBoVs detected in this study were classified as genotype 2, similar to the study conducted in Japan and Portugal. Here, the NS1 (partial), NP1, VP1 and VP2 gene of the FBoV-2/TUR/2020-14 strain obtained from the study were also sequenced and the nt and aa sequences showed high identities to the above-mentioned FBoV-2 strain/isolates (> 96%, except for the aa ratio of strain 16SY0701). In conclusion, this study shows that FBoV and FeChPV are present in healthy cats in Turkey, and these viruses can be detected from oropharyngeal swabs. Our findings contribute to further investigation of the prevalence, genotype distribution, and genetic diversity of Turkish FBoVs and FeChPVs, adding to the molecular epidemiology of FBoV and FeChPVs worldwide.

Identification and characterization of a novel bocaparvovirus in tufted deer (Elaphodus cephalophus) in China.

We used viral metagenomics and next-generation sequencing to identify a novel strain of bocaparvovirus in the intestinal tract of tufted deer (Elaphodus cephalophus), tentatively named "Elaphodus cephalophus bocaparvovirus" (ECBOV). A nearly complete genome sequence of 5,354 nucleotides was obtained, which had the typical genome organization and protein motifs of a bocaparvovirus. Sequence comparisons and phylogenetic analysis revealed that ECBOV may be a new ungulate bocaparvovirus. The identification and characterization of viruses in wildlife will facilitate our understanding of genetic evolution and cross-species transmission and thus further reduce the potential threat to human and animal health.