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Abstract Background At the end of 2021, an epidemic of reproductive abnormalities in cows occurred in Jilin Province, China, posing an urgent need for a rapid diagnosis. Results To identify the cause of the disease, a total of 172 samples were collected from 21 dead calves and 45 aborting or pregnant cows in 10 farms across the province. Routine PCR or RT-PCR detection did not find any common abortion-related agents. We then employed the viromics-based precision diagnosis method to analyze these samples, and the read-based annotation showed signals of an Akabane disease virus (AKAV) in some libraries. To further identify the virus, nested RT-PCR detection revealed that 52.3% (11/21) of dead calves and 26.6% (12/45) of cows were positive for the virus. Phylogenetic analysis of the partial fragments showed that the S segment of the virus was 100% identical to the Chinese strain TJ2016, but its M and L segments shared 94.3% and 96.5% identities with an Israeli strain. Conclusions The viromic and molecular results suggested that these animals were infected with a reassortant AKAV. Coupled with the clinical signs, the virus should be responsible for the epizootic, highlighting that molecular and serological surveys of the virus in cows during early pregnancy, as well as ecological investigation in its arthropod vectors, are necessary.

Rapid evolution of porcine reproductive and respiratory syndrome virus (PRRSV) is the bottleneck for effective prevention and control of PRRS. Thus, understanding the prevalence and genetic background of PRRSV strains in swine-producing regions is important for disease prevention and control. However, there is only limited information about the epizootiological situation of PRRS in the Xinjiang Uygur Autonomous Region, China. In this study, blood or lung tissue samples were collected from 1,411 PRRS-suspected weaned pigs from 9 pig farms in Changji, Shihezi, and Wujiaqu cities between 2020 and 2022. The samples were first tested by RT-quantitative PCR, yielding a PRRSV-2 positive rate of 53.6%. Subsequently, 36 PRRSV strains were isolated through initial adaptation in bone marrow-derived macrophages followed by propagation in grivet monkey Marc-145 cells. Furthermore, 28 PRRSV-positive samples and 20 cell-adapted viruses were selected for high-throughput sequencing (HTS) to obtain the entire PRRSV genome sequences. Phylogenetic analysis based on the nucleotide sequences of the ORF5 gene of the PRRSV strains identified in this study grouped into sub-lineages 1.8 and 8.7 the former being the dominant strain currently circulating in Xinjiang. However, the NSP2 proteins of the Xinjiang PRRSV strains shared the same deletion patterns as sub-lineage 1.8 prototype strain NADC30 with the exception of 4 strains carrying 2-3 additional amino acid deletions. Further analysis confirmed that recombination events had occurred in 27 of 37 PRRSVs obtained here with the parental strains belonging to sub-lineages 1.8 and 8.7, lineages 3 and 5, with the recombination events having occurred most frequently in the 5' and 3' termini of ORF1a and 5' terminus of ORF1b.

Abstract Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Abstract Background Ticks are medically important vectors capable of transmitting a variety of pathogens to and between host species. Although the spectrum of tick-borne RNA viruses has been frequently investigated, the diversity of tick-borne DNA viruses remains largely unknown. Methods A total of 1571 ticks were collected from forests and infested animals, and the diversity of the viruses they harbored was profiled using a DNA-specific virome method. The viromic data were phylogenetically analyzed and validated by PCR assays. Results Although diverse and abundant prokaryotic viruses were identified in the collected ticks, only eukaryotic DNA viruses with single-stranded circular genomes covering the anelloviruses and circular replication-associated (Rep) protein-encoding single-stranded (CRESS) DNA viruses were recovered from ticks. Anelloviruses were detected only in two tick pools, but CRESS DNA viruses were prevalent across these ticks except in one pool of Dermacentor spp. ticks. Phylogenetic analyses revealed that these tick-borne CRESS DNA viruses were related to viruses recovered from animal feces, tissues and even environmental samples, suggesting that their presence may be largely explained by environmental factors rather than by tick species and host blood meals. Conclusions Based on the results, tick-borne eukaryotic DNA viruses appear to be much less common than eukaryotic RNA viruses. Investigations involving a wider collection area and more diverse tick species are required to further support this speculation. Graphical Abstract

Abstract The ongoing COVID-19 pandemic has continued to affect millions of lives worldwide, leading to the urgent need for novel therapeutic strategies. G-quadruplexes (G4s) have been demonstrated to regulate life cycle of multiple viruses. Here, we identify several highly conservative and stable G4s in SARS-CoV-2 and clarify their dual-function of inhibition of the viral replication and translation processes. Furthermore, the cationic porphyrin compound 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4) targeting SARS-CoV-2 G4s shows excellent antiviral activity, while its N-methyl-2-pyridyl positional isomer TMPyP2 with low affinity for G4 has no effects on SARS-CoV-2 infection, suggesting that the antiviral activity of TMPyP4 attributes to targeting SARS-CoV-2 G4s. In the Syrian hamster and transgenic mouse models of SARS-CoV-2 infection, administration of TMPyP4 at nontoxic doses significantly suppresses SARS-CoV-2 infection, resulting in reduced viral loads and lung lesions. Worth to note, the anti-COVID-19 activity of TMPyP4 is more potent than remdesivir evidenced by both in vitro and in vivo studies. Our findings highlight SARS-CoV-2 G4s as a novel druggable target and the compelling potential of TMPyP4 for COVID-19 therapy. Different from the existing anti-SARS-CoV-2 therapeutic strategies, our work provides another alternative therapeutic tactic for SARS-CoV-2 infection focusing on targeting the secondary structures within SARS-CoV-2 genome, and would open a new avenue for design and synthesis of drug candidates with high selectivity toward the new targets.

ABSTRACT Widespread in public databases, foreign contaminant sequences pose a substantial obstacle in genomic analyses. Such contamination in viral genome databases is also notorious but more complicated and often causes questionable results in various applications, particularly in virome-based virus detection. Here, we conducted comprehensive screening and identification of the foreign sequences hidden in the largest eukaryotic viral genome collections of GenBank and UniProt using a scrutiny pipeline, which enables us to rigorously detect those problematic viral sequences (PVSs) with origins in hosts, vectors, and laboratory components. As a result, a total of 766 nucleotide PVSs and 276 amino acid PVSs with lengths up to 6,605 bp were determined, which were widely distributed in 39 families with many involving highly public health-concerning viruses, such as hepatitis C virus, Crimean-Congo hemorrhagic fever virus, and filovirus. The majority of these PVSs are genomic fragments of hosts including humans and bacteria. However, they cannot simply be regarded as foreign contaminants, since parts of them are results of natural occurrence or artificial engineering of viruses. Nevertheless, they severely disturb such sequence-based analyses as genome annotation, taxonomic assignment, and virome profiling. Therefore, we provide a clean version of the eukaryotic viral reference data set by the removal of these PVSs, which allows more accurate virome analysis with less time consumed than with other comprehensive databases. IMPORTANCE High-throughput sequencing-based viromics highly depends on reference databases, but foreign contamination is widespread in public databases and often leads to confusing and even wrong conclusions in genomic analysis and viromic profiling. To address this issue, we systematically detected and identified the contamination in the largest viral sequence collections of GenBank and UniProt based on a stringent scrutiny pipeline. We found hundreds of PVSs that are related to hosts, vectors, and laboratory components. By the removal of them, the resulting data set greatly improves the accuracy and efficiency of eukaryotic virome profiling. These results refresh our knowledge of the type and origin of PVSs and also have warning implications for viromic analysis. Viromic practitioners should be aware of these problems caused by PVSs and need to realize that a careful review of bioinformatic results is necessary for a reliable conclusion.

Porcine parvoviruses (PPVs) are a group of small non-enveloped viruses with seven species (porcine parvovirus 1–7, PPV1-7) have been identified. In this study, a novel porcine parvovirus, provisionally named porcine parvovirus 8 (PPV8), was initially identified via high-throughput sequencing (HTS) in porcine reproductive and respiratory syndrome virus-positive samples collected from swine herds in Guangdong province, 2021. The nearly full-length genome of PPV8 strain GDJM2021 is 4,380 nucleotides in length with two overlapping open ORFs encoding NS1 and VP1 respectively. Sequence analysis indicated that PPV8 shared 16.23–44.18% sequence identity at the genomic levels to PPV1-7 with the relatively highest homology to PPV1. PPV8-GDJM2021 shared 31.86–32.68% aa sequence identity of NS1 protein with those of PPV1 and porcine bufavirus (PBuV), and formed an independent branch neighboring to those formed by members of the genus Protoparvovirus. Of the 211 clinical samples collected from 1990 to 2021, 37 samples (17.5%) distributed over 12 regions in China were positive for PPV8 with time spanning 24 years (1998–2021). To our knowledge, this is the first report on the genomic characterization of the novel PPV8 and its epidemiological situations in China.

ABSTRACT Viral metagenomics is the most powerful tool to profile viromic composition for a given sample. Different viromic methods, including amplification-free ones, have been developed, but choosing them for different purposes requires comprehensive benchmarks. Here, we assessed the performance of four routinely used methods, i.e., multiple displacement amplification (MDA), direct metagenomic sequencing (MTG), sequence-independent single-primer amplification (SIA), and metatranscriptomic sequencing (MTT), using marmot rectal samples as the templates spiked with five known viruses of different genome types. The obtained clean data were differently contaminated by host and bacterial genomes, resulting in MDA having the most, with ~72.1%, but MTT had only ~7.5% data, useful for follow-up viromic analysis. MDA showed a broader spectrum with higher efficiency to profile the DNA virome, and MTT captured almost all RNA viruses with extraordinary sensitivity; hence, they are advisable in richness-based viromic studies. MTG was weak in capturing single-stranded DNA viruses, and SIA could detect both RNA and DNA viruses but with high randomness. Due to biases to certain types of viruses, the four methods caused different alterations to species abundance compared to the initial virus composition. SIA and MDA introduced greater stochastic errors to relative abundances of species, genus, and family taxa, whereas the two amplification-free methods were more tolerant toward such errors and thus are recommendable in abundance-based analyses. In addition, genus taxon is a compromising analytic level that ensures technically supported and biologically and/or ecologically meaningful viromic conclusions. IMPORTANCE Viral metagenomics can be roughly divided into species richness-based studies and species abundance-based analyses. Viromic methods with different principles have been developed, but rational selection of these techniques according to different purposes requires comprehensive understanding of their properties. By assessing the four most widely used methods using template samples, we found that multiple displacement amplification (MDA) and metatranscriptomic sequencing (MTT) are advisable for species richness-based viromic studies, as they show excellent efficiency to detect DNA and RNA viruses. Meanwhile, metagenomic sequencing (MTG) and MTT are more compatible with stochastic errors of methods introduced into relative abundance of viromic taxa and hence are rational choices in species abundance-based analyses. This study also highlights that MTG needs to tackle host genome contamination and ameliorate the capacity to detect single-stranded DNA viruses in the future, and the MTT method requires an improvement in bacterial rRNA depletion prior to library preparation.

Liver diseases seriously challenge the health of chickens raised on scaled farms and cause tremendous economic losses to farm owners. The causative agents for liver diseases are still elusive, even though various pathogens, such as the hepatitis E virus, have been reported. In the winter of 2021, a liver disease was observed on a chicken farm in Dalian, China, which increased chicken mortality by up to 18%. We conducted panvirome profiling of the livers, spleens, kidneys, and recta of 20 diseased chickens. The viromic results revealed coinfection of multiple viruses, including pathogenic ones, in these organs. The viruses were highly identical to those detected in other provinces, and the vaccine and field strains of avian encephalomyelitis virus (AEV) and chicken infectious anemia virus (CIAV) cocirculated on the farm. In particular, the liver showed higher abundance of AEV and multiple fowl adenoviruses than other organs. Furthermore, the liver also contracted avian leukemia virus and CIAV. Experimental animals with infected liver samples developed minor to medium lesions of the liver and showed a virus abundance profile for AEV across internal organs similar to that in the original samples. These results suggest that coinfection with multiple pathogenic viruses influences the occurrence and development of infectious liver disease. The results also highlight that strong farm management standards with strict biosafety measures are needed to minimize the risk of pathogenic virus introduction to the farm.

Classical swine fever virus (CSFV) is a major animal pathogen threatening the global pork industry. To date, numerous anti-CSFV monoclonal antibodies (mAbs) and their recognizing epitopes have been reported. However, few mAbs were systematically characterized for the capacity to differentiate field CSFV isolates from CSF vaccine strains, and the molecular basis associated with antigenic differences between vaccines and field isolates is still largely unknown. In the present study, recombinant CSFV structural glycoproteins E2 of both virulent and vaccine strains and Erns of vaccine strain were expressed using eukaryotic cells and murine mAbs generated against E2 and Erns. After serial screening and cloning of the hybridomas, the viral spectra of mAbs were respectively determined by indirect fluorescent antibody assay (IFA) using 108 CSFVs, followed by Western blot analysis using expressed glycoproteins of all CSFV sub-genotypes including vaccine strains. The antigenic structures recognized by these mAbs were characterized by epitope mapping using truncated, chimeric, and site-directed mutated E2 and Erns proteins. We have identified two vaccine-specific, one field isolate-specific, and two universal CSFV-specific mAbs and five novel conformational epitopes with critical amino acid (aa) motifs that are associated with these five mAbs: 213EPD215, 271RXGP274, and 37LXLNDG42 on E2 and 38CKGVP42, W81, and D100/V107 on Erns. Particularly, E213 of E2 is field isolate-specific, while N40 of E2 and D100/V107 of Erns are vaccine strain-specific. Results from our study further indicate that N40D of E2 mutation in field strains was likely produced under positive selection associated with long-term mass vaccination, leading to CSFV evasion of host immune response. Taking together, this study provides new insights into the antigenic structure of CSFV E2 and Erns and the differentiating mAbs will contribute to the development of a diagnostic strategy to differentiate C-strain vaccination from natural infection (DIVA) of CSFV in terms of elimination of CSF in China.

Although tremendous effort has been exerted to elucidate the pathogenesis of severe COVID-19 cases, the detailed mechanism of moderate cases, which accounts for 90% of all patients, remains unclear yet, partly limited by lacking the biopsy tissues. Here, we established the COVID-19 infection model in cynomolgus macaques (CMs), monitored the clinical and pathological features, and analyzed underlying pathogenic mechanisms at early infection stage by performing proteomic and metabolomic profiling of lung tissues and sera samples from COVID-19 CMs models. Our data demonstrated that innate immune response, neutrophile and platelet activation were mainly dysregulated in COVID-19 CMs. The symptom of neutrophilia, lymphopenia and massive “cytokines storm”, main features of severe COVID-19 patients, were greatly weakened in most of the challenged CMs, which are more semblable as moderate patients. Thus, COVID-19 model in CMs is rational to understand the pathogenesis of moderate COVID-19 and may be a candidate model to assess the safety and efficacy of therapeutics and vaccines against SARS-CoV-2 infection.

Eastern roe deer (Capreolus pygargus) is a small ruminant and is widespread across China. This creature plays an important role in our ecological system. Although a few studies have been conducted to investigate pathogens harbored by this species, our knowledge of the virus diversity is still very sparse. In this study, we conducted the whole virome profiling of a rescue-failed roe deer, which revealed a kobuvirus (KoV), a bocaparvovirus (BoV), and multiple circular single-stranded viruses. These viruses were mainly recovered from the rectum, but PCR detection showed systematic infection of the KoV. Particularly, the KoV and BoV exhibited closely genetic relationships with bovine and canine viruses, respectively, highly suggesting the spillover of viruses from domestic animals to wildlife. Although these viruses were unlikely to have been responsible for the death of the animal, they provide additional data to understand the virus spectrum harbored by roe deer. The transmission of viruses between domestic animals and wildlife highlights the need for extensive investigation of wildlife viruses.

As a small top predator, Amur leopard cat (Prionailurus bengalensis euptilurus) is widely distributed in northeast Asia and plays an important role in the control of small rodent populations and in the maintenance of ecological equilibrium. However, the viruses harbored by this creature have been rarely investigated. Here, we report the DNA and RNA eukaryotic virome profiling of an injured Amur leopard cat followed by PCR validation, which revealed diverse anelloviruses in multiple organs and a bocaparvovirus in the lymph, but no RNA viruses. These anelloviruses have diverse genomic structures and are classified into four phylogroups with viruses of various felines, while the bocaparvovirus is extremely similar to those recovered from diarrheal domestic cats, illustrating the transmission of the virus between domestic animals and wildlife. These data provide the first insight into the genetic diversity of Amur leopard cat viruses, highlighting the need for further investigation of wild animals.

Flavonoids have antioxidant, anti-inflammatory and immunomodulatory properties, and may alleviate food allergic reactions and intestinal inflammation induced by ω-5 gliadin, a main allergen of wheat food allergy in children. In this study, a human basophil KU812 cell degranulation model and a Caco-2 monolayer cell model were constructed in vitro to evaluate the effects of four flavonoids on the allergenicity of ω-5 gliadin peptides and ω-5 gliadin peptide-induced barrier damage in Caco-2 intestinal epithelial monolayers. The results show that baicalein, luteolin, isorhamnetin and naringenin can significantly inhibit the degranulation of KU812 cells stimulated by ω-5 gliadin-derived peptide P4 and the release of IL-6 and TNF-α. In addition, the four flavonoids significantly inhibited the ω-5 gliadin-derived peptide P4 to induce the release of IL-6, IL-8 in Caco-2 cells, inhibited the release of zonulin, and significantly increase the expression of tight junction proteins Occludin and ZO-1 in the Caco-2 cell monolayer. In conclusion, baicalein, luteolin, isorhamnetin and naringenin inhibit degranulation stimulated by wheat allergen and enhance intestinal barrier functions, which supports the potential pharmaceutical application of the four flavonoids treatment for wheat food allergy.

Mammalian orthoreoviruses (MRVs) are increasingly reported to cause various diseases in humans and other animals, with many possibly originating from bats, highlighting the urgent need to investigate the diversity of bat-borne MRVs (BtMRVs). Here, we report the detection and characterization of a reassortant MRV that was isolated from a bat colony in Xinjiang, China. The BtMRV showed a wide host and organ tropism and can efficiently propagate the cell lines of different animals. It caused mild damage in the lungs of the experimentally inoculated suckling mice and was able to replicate in multiple organs for up to three weeks post-inoculation. Complete genome analyses showed that the virus was closely related to MRVs in a wide range of animals. An intricate reassortment network was revealed between the BtMRV and MRVs of human, deer, cattle, civet and other bat species. Specifically, we found a bat-specific clade of segment M1 that provides a gene source for the reassortment of human MRVs. These data provide important insights to understand the diversity of MRVs and their natural circulation between bats, humans, and other animals. Further investigation and surveillance of MRV in bats and other animals are needed to control and prevent potential MRV-related diseases.

Classical swine fever can be controlled effectively by vaccination with C-strain vaccine. In this study, we developed a novel competitive enzyme-linked immunosorbent assay (cELISA) based on a C-strain Erns specific monoclonal antibody (mAb 1504), aiming to serologically measure immune responses to C-strain vaccine in pigs, and finally to make the C-strain become a DIVA-compatible vaccine. The cELISA system was established based on the strategy that mAb 1504 will compete with the C-strain induced antibodies in the pig serum to bind the C-strain Erns protein. The cELISA was optimized and was further evaluated by testing different categories of pig sera. It can efficiently differentiate C-strain immunized from wild-type CSFV-infected pigs and lacks cross-reaction with other common swine viruses and viruses in genus Pestivirus such as Bovine viral diarrhea virus (BVDV). The C-strain antibody can be tested in pigs 7–14 days post vaccination with this cELISA. The sensitivity and specificity of the established cELISA were 100% (95% confidence interval: 95.60 to 100%) and 100% (95% confidence interval: 98.30 to 100%), respectively. This novel cELISA is a reliable tool for specifically measuring and differentiating immune responses to C-strain vaccine in pigs. By combining with the wild-type CSFV-specific infection tests, it can make the C-strain have DIVA capability.

The article discusses the rapid diagnosis of a fox's death case using nanopore sequencing, revealing an infection with an Arctic-like rabies virus. The study highlights the importance of early diagnosis of emerging infectious diseases (EIDs) in wildlife conservation. Nanopore sequencing proved to be a portable and affordable technique for rapid diagnosis of viral EIDs, showcasing its potential in wildlife conservation efforts. The study also emphasizes the need for active surveillance of rabies in the fox population in northeastern China due to the complex circulation of rabies viruses in the region. [Extracted from the article]

The article discusses research which performed the first complete meta-transcriptomic (MTT) genomic sequence analysis of canine distemper virus (CDV) in wild boar. The study analyzed tissue samples from free-ranging wild boars and determined the CDV infection rate using rapid amplification of complementary DNA (cDNA) ends and specific reverse transcription polymerase chain reaction (RT-PCR). It investigated CDV seroprevalence in wild boars in northeast China using virus-neutralization tests.

Classical swine fever virus nonstructural protein NS5A is essential for viral genome replication, protein translation, virus assembly, and autophagy. In the present paper, the interaction of cellular PPP2R1A, PP2Ac, and DAPK3 with NS5A has been confirmed. These interactions mediate the dissociation of PP2A from Beclin 1 and its association with DAPK3. Downregulation of DAPK3 or PPP2R1A inhibits CSFV replication. NS5A induced phosphorylation of Ser88 (PK-15 cells) or Ser90 (HEK293T cells) on Beclin 1. Knockdown of Beclin 1 inhibited NS5A-induced autophagy, indicating that NS5A induces Beclin 1-dependent autophagy. Downregulation of DAPK3, PPP2R1A, or PP2Ac by siRNA reduced Beclin 1 phosphorylation and autophagy mediated by NS5A, showing a critical role of PP2A and DAPK3 in Beclin 1 phosphorylation and autophagy triggered by NS5A. In vitro analysis of Beclin 1 phosphorylation revealed PP2A as being essential for DAPK3-mediated phosphorylation of Beclin 1, indicating that PP2A may dephosphorylate DAPK3 to activate its protein kinase activity, with activated DAPK3 phosphorylated Beclin 1, and then triggering autophagy. These data show for the first time that DAPK3 can be activated through dephosphorylation by PP2A. These findings reveal a novel mechanism whereby CSFV NS5A protein activates autophagy via PP2A-DAPK3-Beclin 1 axis to favor viral replication. [ABSTRACT FROM AUTHOR]