Circovirus Hepatitis Infection in Heart-Lung Transplant Patient, France.

In March 2022, a 61-year-old woman in France who had received a heart-lung transplant sought treatment with chronic hepatitis mainly characterized by increased liver enzymes. After ruling out common etiologies, we used metagenomic next-generation sequencing to analyze a liver biopsy sample and identified an unknown species of circovirus, tentatively named human circovirus 1 (HCirV-1). We found no other viral or bacterial sequences. HCirV-1 shared 70% amino acid identity with the closest known viral sequences. The viral genome was undetectable in blood samples from 2017-2019, then became detectable at low levels in September 2020 and peaked at very high titers (1010 genome copies/mL) in January 2022. In March 2022, we found >108 genome copies/g or mL in the liver and blood, concomitant with hepatic cytolysis. We detected HCirV-1 transcripts in 2% of hepatocytes, demonstrating viral replication and supporting the role of HCirV-1 in liver damage.

Rapid detection of porcine circovirus type 2 by a red latex microsphere immunochromatographic strip.

To establish a rapid and specific antigen detection method for porcine circovirus type 2 (PCV2), monoclonal antibodies (mAbs) were produced against the PCV2 epidemic strains and a red latex microsphere immunochromatographic strip was established. A total of eight anti-PCV2b and four anti-PCV2d mAbs were produced, and seven mAbs were confirmed to react with PCV2a, PCV2b, and PCV2d strains using an immunoperoxidase monolayer assay. The results of micro-neutralization tests showed that the mAbs 2C8, 9H4, 10G7, 7B9, and 7C7 had good neutralizing activity, whereas the neutralizing activity of the mAbs 4B3, 4C9, 6H9, and 7E2 was lower than 50%. Three mAbs, 4B3, 7C7, and 9H4, and PCV2 pAb were selected for the establishment of a red latex microsphere immunochromatographic strip, and the combination of mAb 7C7 labeled with red latex microspheres and mAb 9H4 exhibited the greatest detection ability. The immunochromatographic strip had minimum detection limits of 102.5 TCID50/0.1 ml, 100.7 TCID50/0.1 ml, and 101.5 TCID50/0.1 ml for PCV2a/CL, PCV2b/MDJ, and PCV2d/LNHC, respectively. Furthermore, no cross-reactivity was found for African swine fever virus, classical swine fever virus, porcine respiratory and reproductive syndrome virus, porcine parvovirus, porcine pseudorabies virus, porcine circovirus type 1, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine rotavirus, or porcine deltacoronavirus using the immunochromatographic strip. Using PCR as a reference standard, the detection sensitivity, specificity, and overall coincidence rate of the immunochromatographic strip were 81.13%, 100%, and 90.00%. Additionally, the detection ability of the immunochromatographic strip was correlated with that of virus titration. The immunochromatographic strip was used to detect 183 clinical disease samples, and the average positive detection rate was 22.95%. In summary, this method has good sensitivity and specificity and is simple, convenient, and quick to operate. It has high application value for on-site diagnosis of PCV2 and virus quantification. KEY POINTS: • A red latex microsphere immunochromatographic strip for PCV2 detection was developed. • The method was not only simple to operate, but also takes less time. • The method had good sensitivity and specificity.

Metagenomic analysis of endemic viruses in oral secretions from Chinese pigs.

BACKGROUND: Pigs are unique reservoirs for virus ecology. Despite the increased use of improved biosecurity measures, pig viruses readily circulate in Chinese swine farms. OBJECTIVES: The main objective of this study was to examine archived swine oral secretion samples with a panel of pan-species viral assays such that we might better describe the viral ecology of swine endemic viruses in Chinese farms. METHODOLOGY: Two hundred (n = 200) swine oral secretion samples, collected during 2015 and 2016 from healthy pigs on six swine farms in two provinces in China, were screened with molecular pan-species assays for coronaviruses (CoVs), adenoviruses (AdVs), enteroviruses (EVs), and paramyxoviruses (PMV). Samples were also screened for porcine circovirus (PCV) 3, porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV). RESULTS: Among 200 swine oral secretion samples, 152 (76.0%) were found to have at least one viral detection. Thirty-four samples (17%) were positive for more than one virus, including 24 (70.5%) with dual detection and 10 (29.5%) with triple detection. Seventy-eight (39.0%) samples were positive for porcine AdVs, 22 (11.0%) were positive for porcine CoVs, 21 (10.5%) were positive for IAVs, 13 (6.5%) were positive for PCV, 7 (3.5%) were positive for PMV, six (3.0%) were positive for PRRSV and five (2.5%) were positive for porcine EV. CONCLUSION: Our findings underscore the high prevalence of numerous viruses among production pigs in China and highlight the need for routine, periodic surveillance for novel virus emergence with the goal of protecting pigs.

Advances in Crosstalk between Porcine Circoviruses and Host.

Porcine circoviruses (PCVs), including PCV1 to PCV4, are non-enveloped DNA viruses with a diameter of about 20 nm, belonging to the genus Circovirus in the family Circoviridae. PCV2 is an important causative agent of porcine circovirus disease or porcine circovirus-associated disease (PCVD/PCVAD), which is highly prevalent in pigs and seriously affects the swine industry globally. Furthermore, PCV2 mainly causes subclinical symptoms and immunosuppression, and PCV3 and PCV4 were detected in healthy pigs, sick pigs, and other animals. Although the pathogenicity of PCV3 and PCV4 in the field is still controversial, the infection rates of PCV3 and PCV4 in pigs are increasing. Moreover, PCV3 and PCV4 rescued from infected clones were pathogenic in vivo. It is worth noting that the interaction between virus and host is crucial to the infection and pathogenicity of the virus. This review discusses the latest research progress on the molecular mechanism of PCVs-host interaction, which may provide a scientific basis for disease prevention and control.

Frequency of porcine circovirus 3 detection and histologic lesions in clinical samples from swine in the United States.

Porcine circovirus 3 (PCV3) is widespread in pigs worldwide. Diverse clinical signs and lesions have been associated with PCV3, but the role of PCV3 as a cause of disease in swine remains unclear. We investigated the association of PCV3 with clinical signs and histologic lesions in 730 diagnostic swine cases between February 2016 and January 2018. The cases contained 2,177 samples submitted from 474 sites located in 21 states in the United States. PCR assay results were positive for PCV3 for 577 of 2,177 (27%) samples, 255 of 730 (35%) cases, 181 of 474 (38%) sites, and 17 of 21 (81%) states. We detected PCV3 in 19 of 28 specimen types and in pigs of all ages and clinical presentations, including healthy pigs, with the highest detection rate in adult pigs. PCV3 detection was not associated with respiratory, gastrointestinal, or CNS signs, weight loss, or sudden death. Of 58 types of histologic lesions evaluated, PCV3 detection was associated with myocarditis, cardiac vasculitis, and interstitial pneumonia in growing pigs. A high PCV3 detection rate was observed in aborted fetuses.

Immunoglobin G Sero-Dynamics Aided Host Specific Linear Epitope Identification and Differentiation of Infected from Vaccinated Hosts.

Differentiation of infected from vaccinated hosts (DIVH) is a critical step in virus eradication programs. DIVH-compatible vaccines, however, take years to develop, and are therefore unavailable for fighting the sudden outbreaks that typically drive pandemics. Here, we establish a protocol for the swift and efficient development of DIVH assays, and show that this approach is compatible with any type of vaccines. Using porcine circovirus 2 (PCV2) as the experimental model, the first step is to use Immunoglobin G (IgG) sero-dynamics (IsD) curves to aid epitope discovery (IsDAED): PCV2 Cap peptides were categorized into three types: null interaction, nonspecific interaction (NSI), and specific interaction (SI). We subsequently compared IsDAED approach and traditional approach, and demonstrated identifying SI peptides and excluding NSI peptides supports efficient diagnostic kit development, specifically using a protein-peptide hybrid microarray (PPHM). IsDAED directed the design of a DIVH protocol for three types of PCV2 vaccines (while using a single PPHM). Finally, the DIVH protocol successfully differentiated infected pigs from vaccinated pigs at five farms. This IsDAED approach is almost certainly extendable to other viruses and host species. IMPORTANCE Sudden outbreaks of pandemics caused by virus, such as SARS-CoV-2, has been determined as a public health emergency of international concern. However, the development of a DIVH-compatible vaccine is time-consuming and full of uncertainty, which is unsuitable for an emergent situation like the ongoing COVID-19 pandemic. Along with the development and public health implementation of new vaccines to prevent human diseases, e.g., human papillomavirus vaccines for cervical cancer; enterovirus 71 vaccines for hand, foot, and mouth disease; and most recently SARS-CoV-2, there is an increasing demand for DIVH. Here, we use the IsDAED approach to confirm SI peptides and to exclude NSI peptides, finally to direct the design of a DIVH protocol. It is plausible that our IsDAED approach is applicable for other infectious disease.

Characterization of a dynamic self-replicating mammalian expression vector based on the circular ssDNA genome of beak and feather disease virus.

In vivo nucleic expression technologies using DNA or mRNA offer several advantages for recombinant gene expression. Their inherent ability to generate natively expressed recombinant proteins and antigens allows these technologies to mimic foreign gene expression without infection. Furthermore, foreign nucleic acid fragments have an inherent ability to act as natural immune adjuvants and stimulate innate pathogen- and DNA damage-associated receptors that are responsible for activating pathogen-associated molecular pattern (PAMP) and DNA damage-associated molecular pattern (DAMP) signalling pathways. This makes nucleic-acid-based expression technologies attractive for a wide range of vaccine and oncolytic immunotherapeutic uses. Recently, RNA vaccines have demonstrated their efficacy in generating strong humoral and cellular immune responses for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). DNA vaccines, which are more stable and easier to manufacture, generate similar immune responses to RNA, but typically exhibit lower immunogenicity. Here we report on a novel method of constructing self-amplifying DNA expression vectors that have the potential to amplify and enhance gene/antigen expression at a cellular level by increasing per cell gene copy numbers, boost genomic adjuvating effects and mitigate through replication many of the problems faced by non-replicating vectors such as degradation, methylation and gene silencing. These vectors employ a viral origin rolling circle replication cycle in mammalian host cells that amplifies the vector and gene of interest (GOI) copy number, maintaining themselves as nuclear episomes. We show that these vectors maintain persistently elevated GOI expression levels at the cellular level and induce morphological cellular alterations synonymous with increased cellular stress.

Molecular detection and phylogenetic analysis of pigeon circovirus from racing pigeons in Northern China.

BACKGROUND: Pigeon circovirus (PiCV) infections in pigeons (Columba livia) have been reported worldwide. Currently, pigeon racing is becoming increasingly popular and considered to be a national sport in China, and even, the greatest competitions of racing pigeons are taking place in China. However, there are still no epidemiologic data regarding PiCV infections among racing pigeons in China. The purpose of our study was to provide information of prevalence, genetic variation and evolution of PiCV from racing pigeons in China. RESULTS: To trace the prevalence, genetic variation and evolution of PiCV in sick and healthy racing pigeons, 622 samples were collected from 11 provinces or municipalities in China from 2016 to 2019. The results showed that the positive rate of PiCV was 19.3% (120/622) at the sample level and 59.0% (23/39) at the club level, thus suggesting that the virus was prevalent in Chinese racing pigeons. A sequence analysis revealed that the cap genes of the PiCV strains identified in our study displayed a high genetic diversity and shared nucleotide homologies of 71.9%-100% and amino acid homologies of 71.7%-100%. 28 and 36 unique amino acid substitutions were observed in the Cap and Rep proteins derived from our PiCV strains, respectively. A cladogram representation of PiCV strains phylogeny based on 90 cap gene sequences showed that the strains in this study could be further divided into seven clades (A, B, C, E, G, H, and I) and some of them were closely related to worldwide strains from different types of pigeons. A large number of recombination events (31 events) were also detected in the PiCV genomes from Chinese racing pigeons. CONCLUSIONS: These findings indicate that PiCV strains circulating in China exhibit a high genetic diversity and also contribute to information of prevalence, genetic variation and evolution of PiCV from racing pigeons in China.

Identification of novel circovirus and anelloviruses from wolverines using a non-invasive faecal sampling approach.

Viruses in the families Circoviridae and Anelloviridae have circular single-stranded DNA genomes and have been identified in various animal species. Some members of the Circoviridae family such as beak and feather disease and porcine circovirus have been found to cause disease in their host animals. Anelloviruses on the other hand have not been identified to cause disease in their hosts but are highly prevalent in mammalian species. Using a non-invasive sampling approach, we identified novel circovirus and anelloviruses from faecal samples of wolverines dwelling in Montana, USA. Wolverines are forest carnivores that feed on a wide variety of carrion and other prey species, and they occupy diverse habitats across northern Europe to North America. Little is known about viruses associated with wild wolverines. Our investigation of the faecal samples resulted in the identification of a novel circovirus from three out of four wolverine samples, two collected in 2018 and one in 2019. Comparison with other circoviruses shows it is most closely related to a porcine circovirus 3, sharing ~69% identity. Additionally, three anellovirus genomes were recovered from two wolverine faecal samples which share 68--69% ORF1 nucleotide similarity with an anellovirus from another mustelid species, pine martens. Here we identify novel single-stranded DNA viruses associated with wolverine and open up new avenues for research.

Genetic diversity and prevalence of porcine circovirus type 3 (PCV3) and type 2 (PCV2) in the Midwest of the USA during 2016-2018.

In recent years, reports indicated that PCV3 may be involved in porcine dermatitis and nephropathy syndrome (PDNS)-like disease similar to that linked to PCV2. A total of 2,125 porcine samples from 910 cases were collected during 2016-2018 and tested for presence of PCV3 and PCV2 by real-time PCR assays. Results showed high prevalence of PCV3 and PCV2: 28.4% samples from 41.2% cases were PCV3 positive and 16.4% samples from 16.7% cases were PCV2 positive. The overall coinfection rate was 5.4% and 8.4% at the sample and case level, respectively. Temporal analysis indicated that PCV3 positive case rate increased from 31.6% in 2016, 40.9% in 2017, to 55.6% in 2018. Although its prevalence was lower, PCV2-positive case rate in 2018 (28.8%) doubled that in 2017 (14.4%). The coinfection case rate also increased from 3.4% in 2016, 8.0% in 2017 to 16.1% in 2018. The high positive rate of PCV3 (56.9%) and PCV2 (33.8%) in oral fluids, PCV3 in foetuses (57.1%) and PCV2 in tonsils (54.8%) implied viral transmission route and tissue tropism. In phylogenetic analysis, two small PCV3 clusters (1 and 2) were separated but others were clustered with low bootstrapping values indicating overall low genetic diversity. Genotypes, PCV2a-h, were confirmed by analysing 2,944 strains, with a new genotype proposed as PCV2i. In this study, 61 PCV3 unique whole genomes were sequenced; 12 belonged to a separate cluster that were characterized by five consistent amino acid changes in the capsid protein (24V, 27K, 56D, 98R and 168K) and may be associated with potential differences in immunogenicity. Among the 43 unique PCV2 whole genomes sequenced, 31 belonged to PCV2d, 7 to PCV2a and 5 to PCV2b. Thus, our study demonstrates that PCV2d is the predominant genotype and PCV3 is widely circulating in the Midwest of the USA.

Genomic characterization of a novel bat-associated Circovirus detected in European Miniopterus schreibersii bats.

Circoviruses are small circular DNA viruses causing severe pig and poultry disease, recently identified in various bat species worldwide. We report the detection and full-genome molecular characterization of a novel bat-associated Circovirus identified in faecal samples of Miniopterus schreibersii bats (Schreiber's bent-winged bats) from Sardinia, Italy. Full-genomic sequencing revealed a new putative member of Circoviridae family, with a genome size of 2063 nt. Sequencing allowed the characterization of the two major ORFs, inversely arranged, encoding replicase and capsid proteins, as well as the finding of a polythymidine tract within the genome, and highlighted phylogenetic relationships of the novel virus. This is the first report of circovirus in European bats. Giving the high level of genetic diversity of bat circoviruses, it is paramount to further investigate the relationships between these viruses and bats.