circRNA_8521 promotes Senecavirus A infection by sponging miRNA-324 to regulate LC3A.

Senecavirus A (SVA) causes outbreaks of vesicular disease in pigs, which imposes a considerable economic burden on the pork industry. As current SVA prevention measures are ineffective, new strategies for controlling SVA are urgently needed. Circular (circ)RNA is a newly characterized class of widely expressed, endogenous regulatory RNAs, which have been implicated in viral infection; however, whether circRNAs regulate SVA infection remains unknown. To investigate the influence of circRNAs on SVA infection in porcine kidney 15 (PK-15) cells, RNA sequencing technology was used to analyze the circRNA expression profiles of SVA-infected and uninfected PK-15 cells, the interactions between circRNAs, miRNAs, and mRNAs potentially implicated in SVA infection were predicted using bioinformatics tools. The prediction accuracy was verified using quantitative real-time (qRT)-PCR, Western blotting, as well as dual-luciferase reporter and RNA pull-down assays. The results showed that 67 circRNAs were differentially expressed as a result of SVA infection. We found that circ_8521 was significantly upregulated in SVA-infected PK-15 cells and promoted SVA infection. circ_8521 interacted with miR-324. miR-324 bound to LC3A mRNA which inhibited the expression of LC3A. Knockdown of LC3A inhibited SVA infection. However, circ_8521 promoted the expression of LC3A by binding to miR-324, thereby promoting SVA infection. We demonstrated that circ_8521 functioned as an endogenous miR-324 sponge to sequester miR-324, which promoted LC3A expression and ultimately SVA infection.

Phylodynamic analysis of foot-and-mouth disease virus evolution in Mar Chiquita, Argentina.

Foot-and-mouth disease is a highly contagious disease affecting cloven-hoofed animals, resulting in considerable economic losses. Its causal agent is foot-and-mouth disease virus (FMDV), a picornavirus. Due to its error-prone replication and rapid evolution, the transmission and evolutionary dynamics of FMDV can be studied using genomic epidemiological approaches. To analyze FMDV evolution and identify possible transmission routes in an Argentinean region, field samples that tested positive for FMDV by PCR were obtained from 21 farms located in the Mar Chiquita district. Whole FMDV genome sequences were obtained by PCR amplification in seven fragments and sequencing using the Sanger technique. The genome sequences obtained from these samples were then analyzed using phylogenetic, phylogeographic, and evolutionary approaches. Three local transmission clusters were detected among the sampled viruses. The dataset was analyzed using Bayesian phylodynamic methods with appropriate coalescent and relaxed molecular clock models. The estimated mean viral evolutionary rate was 1.17 × 10- 2 substitutions/site/year. No significant differences in the rate of viral evolution were observed between farms with vaccinated animals and those with unvaccinated animals. The most recent common ancestor of the sampled sequences was dated to approximately one month before the first reported case in the outbreak. Virus transmission started in the south of the district and later dispersed to the west, and finally arrived in the east. Different transmission routes among the studied herds, such as non-replicating vectors and close contact contagion (i.e., aerosols), may be responsible for viral spread.

Multiple functions of the nonstructural protein 3D in picornavirus infection.

3D polymerase, also known as RNA-dependent RNA polymerase, is encoded by all known picornaviruses, and their structures are highly conserved. In the process of picornavirus replication, 3D polymerase facilitates the assembly of replication complexes and directly catalyzes the synthesis of viral RNA. The nuclear localization signal carried by picornavirus 3D polymerase, combined with its ability to interact with other viral proteins, viral RNA and cellular proteins, indicate that its noncatalytic role is equally important in viral infections. Recent studies have shown that 3D polymerase has multiple effects on host cell biological functions, including inducing cell cycle arrest, regulating host cell translation, inducing autophagy, evading immune responses, and triggering inflammasome formation. Thus, 3D polymerase would be a very valuable target for the development of antiviral therapies. This review summarizes current studies on the structure of 3D polymerase and its regulation of host cell responses, thereby improving the understanding of picornavirus-mediated pathogenesis caused by 3D polymerase.

IFITM1 and IFITM2 inhibit the replication of senecavirus A by positive feedback with RIG-I signaling pathway.

The role of host factors in the replication of emerging senecavirus A (SVA) which induced porcine idiopathic vesicular disease (PIVD) distributed worldwide remains obscure. Here, interferon-induced transmembrane (IFITM) protein 1 and 2 inhibit SVA replication by positive feedback with RIG-I signaling pathway was reported. The expression levels of IFITM1 and IFITM2 increased significantly in SVA infected 3D4/21 cells. Infection experiments of cells with over and interference expression of IFITM1 and IFITM2 showed that these two proteins inhibit SVA replication by regulating the expression of interferon beta (IFN-ß), IFN-stimulated gene 15 (ISG-15), interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IFN regulatory factor-3 (IRF3), and IRF7. Further results showed that antiviral responses of IFITM1 and IFITM2 were achieved by activating retinoic acid-inducible gene I (RIG-I) signaling pathway which in turn enhanced the expression of IFITM1 and IFITM2. It is noteworthy that conserved domains of these two proteins also paly the similar role. These findings provide new data on the role of host factors in infection and replication of SVA and help to develop new agents against the virus.

Co-infecting viruses of species Bovine rhinitis B virus (Picornaviridae) and Bovine nidovirus 1 (Tobaniviridae) identified for the first time from a post-mortem respiratory sample of a sheep (Ovis aries) in Hungary.

In this study, a picornavirus and a nidovirus were identified from a single available nasopharyngeal swab (NPS) sample of a freshly deceased sheep, as the only vertebrate viruses found with viral metagenomics and next-generation sequencing methods. The sample was originated from a mixed feedlot farm in Hungary where sheep and cattle were held together but in separate stalls. Most of the sheep had respiratory signs (coughing and increased respiratory effort) at the time of sampling. Other NPS were not, but additional enteric samples were collected from sheep (n = 27) and cattle (n = 11) of the same farm at that time. The complete/nearly complete genomes of the identified viruses were determined using RT-PCR and Nanopore (MinION-Flonge) / Dye-terminator sequencing techniques. The results of detailed genomic and phylogenetic analyses indicate that the identified picornavirus most likely belongs to a type 4 genotype of species Bovine rhinitis B virus (BRBV-4, OR885914) of genus Aphthovirus, family Picornaviridae while the ovine nidovirus (OvNV, OR885915) - as a novel variant - could belong to the recently created Bovine nidovirus 1 (BoNV) species of genus Bostovirus, family Tobaniviridae. None of the identified viruses were detectable in the enteric samples using RT-PCR and generic screening primer pairs. Both viruses are well-known respiratory pathogens of cattle, but their presence was not demonstrated before in other animals, like sheep. Furthermore, neither BRBV-4 nor BoNVs were investigated in European cattle and/or sheep flocks, therefore it cannot be determined whether the presence of these viruses in sheep was a result of a single host species switch/spillover event or these viruses are circulating in not just cattle but sheep populations as well. Further studies required to investigate the spread of these viruses in Hungarian and European sheep and cattle populations and to identify their pathogenic potential in sheep.

Biochemical analysis of the host factor activity of ZCCHC14 in hepatitis A virus replication.

Relatively little is known of the mechanisms underlying hepatitis A virus (HAV) genome replication. Unlike other well-studied picornaviruses, HAV RNA replication requires the zinc finger protein ZCCHC14 and non-canonical TENT4 poly(A) polymerases with which it forms a complex. The ZCCHC14-TENT4 complex binds to a stem-loop located within the internal ribosome entry site (IRES) in the 5' untranslated RNA (5'UTR) and is essential for viral RNA synthesis, but the underlying mechanism is unknown. Here, we describe how different ZCCHC14 domains contribute to its RNA-binding, TENT4-binding, and HAV host factor activities. We show that the RNA-binding activity of ZCCHC14 requires both a sterile alpha motif (SAM) and a downstream unstructured domain (D4) and that ZCCHC14 contains two TENT4-binding sites: one at the N-terminus and the other around D4. Both RNA-binding and TENT4-binding are required for HAV host factor activity of ZCCHC14. We also demonstrate that the location of the ZCCHC14-binding site within the 5'UTR is critical for its function. Our study provides a novel insight into the function of ZCCHC14 and helps elucidate the mechanism of the ZCCHC14-TENT4 complex in HAV replication.IMPORTANCEThe zinc finger protein ZCCHC14 is an essential host factor for both hepatitis A virus (HAV) and hepatitis B virus (HBV). It recruits the non-canonical TENT4 poly(A) polymerases to viral RNAs and most likely also a subset of cellular mRNAs. Little is known about the details of these interactions. We show here the functional domains of ZCCHC14 that are involved in binding to HAV RNA and interactions with TENT4 and describe previously unrecognized peptide sequences that are critical for the HAV host factor activity of ZCCHC14. Our study advances the understanding of the ZCCHC14-TENT4 complex and how it functions in regulating viral and cellular RNAs.

Human Rhinovirus: Molecular and Clinical Overview / Rinovirus humano: descripción molecular y clínica

Human rhinoviruses (HRVs) are associated with a wide spectrum of clinical manifestations, ranging from mild cold symptoms to more severe respiratory illnesses, significantly burdening global healthcare systems. At the molecular level, HRVs belong to the Picornaviridae family and are classified into three species: HRV-A, HRV-B, and HRV-C. Advances in genomic sequencing and phylogenetic analysis have revealed a remarkable genetic diversity within HRV species, with over 160 serotypes identified. This genetic variability contributes to the ability of HRVs to evade host immune responses and facilitates their continuous circulation in the population. This review provides an overview of the molecular and clinical aspects of HRV infections.(AU)

Adjuvant screening of the Senecavirus A inactivated vaccine in mice and evaluation of its immunogenicity in pigs.

BACKGROUND: Senecavirus A (SVA) causes an emerging vesicular disease (VD) with clinical symptoms indistinguishable from other vesicular diseases, including vesicular stomatitis (VS), foot-and-mouth disease (FMD), and swine vesicular disease (SVD). Currently, SVA outbreaks have been reported in Canada, the U.S.A, Brazil, Thailand, Vietnam, Colombia, and China. Based on the experience of prevention and control of FMDV, vaccines are the best means to prevent SVA transmission. RESULTS: After preparing an SVA inactivated vaccine (CH-GX-01-2019), we evaluated the immunogenicity of the SVA inactivated vaccine mixed with Imject® Alum (SVA + AL) or Montanide ISA 201 (SVA + 201) adjuvant in mice, as well as the immunogenicity of the SVA inactivated vaccine combined with Montanide ISA 201 adjuvant in post-weaned pigs. The results of the mouse experiment showed that the immune effects in the SVA + 201 group were superior to that in the SVA + AL group. Results from pigs immunized with SVA inactivated vaccine combined with Montanide ISA 201 showed that the immune effects were largely consistent between the SVA-H group (200 µg) and SVA-L group (50 µg); the viral load in tissues and blood was significantly reduced and no clinical symptoms occurred in the vaccinated pigs. CONCLUSIONS: Montanide ISA 201 is a better adjuvant choice than the Imject® Alum adjuvant in the SVA inactivated vaccine preparation, and the CH-GX-01-2019 SVA inactivated vaccine can provide effective protection for pigs.

Picornavirus 2C proteins: structure-function relationships and interactions with host factors.

Picornaviruses, which are positive-stranded, non-enveloped RNA viruses, are known to infect people and animals with a broad spectrum of diseases. Among the nonstructural proteins in picornaviruses, 2C proteins are highly conserved and exhibit multiple structural domains, including amphipathic α-helices, an ATPase structural domain, and a zinc finger structural domain. This review offers a comprehensive overview of the functional structures of picornaviruses' 2C protein. We summarize the mechanisms by which the 2C protein enhances viral replication. 2C protein interacts with various host factors to form the replication complex, ultimately promoting viral replication. We review the mechanisms through which picornaviruses' 2C proteins interact with the NF-κB, RIG-I, MDA5, NOD2, and IFN pathways, contributing to the evasion of the antiviral innate immune response. Additionally, we provide an overview of broad-spectrum antiviral drugs for treating various enterovirus infections, such as guanidine hydrochloride, fluoxetine, and dibucaine derivatives. These drugs may exert their inhibitory effects on viral infections by targeting interactions with 2C proteins. The review underscores the need for further research to elucidate the precise mechanisms of action of 2C proteins and to identify additional host factors for potential therapeutic intervention. Overall, this review contributes to a deeper understanding of picornaviruses and offers insights into the antiviral strategies against these significant viral pathogens.

Modulation of nucleotide metabolism by picornaviruses.

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.

Identification and Genomic Characterization of Bovine Boosepivirus A in the United States and Canada.

Boosepivirus is a new genus in the Picornaviridae family. Boosepiviruses (BooVs) are genetically classified into three species: A, B, and C. Initially, Boosepivirus A and B were identified in cattle, whereas Boosepivirus C was detected in sheep. Recent evidence showed that Boosepivirus B was detected in sheep and Boosepivirus C was identified in goats, suggesting that Boosepvirus might cross the species barrier to infect different hosts. Different from BooV B, BooV A is less studied. In the present study, we reported identification of two North American BooV A strains from cattle. Genomic characterization revealed that US IL33712 (GenBank accession #PP035161) and Canada 1087562 (GenBank accession #PP035162) BooV A strains are distantly related to each other, and US IL33712 is more closely correlated to two Asian BooV A strains. US-strain-specific insertions, NorthAmerican-strain-specific insertions, and species A-specific insertions are observed and could contribute to viral pathogenicity and host adaptation. Our findings highlight the importance of continued surveillance of BooV A in animals.

The construction and immunogenicity analyses of a recombinant pseudorabies virus with Senecavirus A VP3 protein co-expression.

Senecavirus A (SVA)-associated porcine idiopathic vesicular disease (PIVD) and Pseudorabies (PR) are highly contagious swine disease that pose a significant threat to the global pig industry. In the absence of an effective commercial vaccine, outbreaks caused by SVA have occurred in many parts of the world. In this study, the PRV variant strain PRV-XJ was used as the parental strain to construct a recombinant PRV strain with the TK/gE/gI proteins deletion and the VP3 protein co-expression, named rPRV-XJ-ΔTK/gE/gI-VP3. The results revealed that PRV is a suitable viral live vector for VP3 protein expressing. As a vaccine, rPRV-XJ-ΔTK/gE/gI-VP3 is safe for mice, vaccination with it did not cause any clinical symptoms of PRV. Intranasal immunization with rPRV-XJ-ΔTK/gE/gI-VP3 induced strong cellular immune response and high levels of specific antibody against VP3 and gB and neutralizing antibodies against both PRV and SVA in mice. It provided 100% protection to mice against the challenge of virulent strain PRV-XJ, and alleviated the pathological lesion of heart and liver tissue in SVA infected mice. rPRV-XJ-ΔTK/gE/gI-VP3 appears to be a promising vaccine candidate against PRV and SVA for the control of the PRV variant and SVA.

Rapid detection of porcine sapelovirus by reverse transcription recombinase polymerase amplification assay.

BACKGROUND: Porcine Sapelovirus (PSV) infection has been confirmed in pigs worldwide, mostly asymptomatic, but in some cases, it can lead to significant issues in the gastrointestinal, respiratory, neurological, or reproductive systems. PSV is considered an emerging pathogen of porcine species. Recombinase polymerase amplification (RPA) is a simple and fast isothermal technique that uses three enzymes for amplification without the use of any sophisticated equipment. METHODS AND RESULTS: The reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and optimized for field based detection of PSV. The assay was developed by targeting 5´UTR region of PSV genome and optimized for reaction time, temperature, primer and MgOAc concentration. The analytical sensitivity and specificity of assay was determined. The assay was evaluated on 85 porcine faecal samples collected from field. In addition to conventional format, this assay was also optimized for visual dye-based detection format and lateral flow strips based detection (in combination with probe). The developed assay works at constant temperature of 35 °C for 20 min with forward primer concentration 20pm, reverse primer concentration 10pm and MgOAc concentration of 14mM. This assay is highly sensitive and detects up to 28 copies of viral nucleic acid both in the conventional as well as in fluorescent dye based detection format. Using the newly developed assay 21 samples out of 85 samples were found positive, showing positivity rate of 24.7%. The positivity rate of RT-RPA assay corroborated with the gold standard RT-PCR test. CONCLUSIONS: This study presented the development of an RT-RPA isothermal assay for rapid and accurate detection of PSV. The assay is highly sensitive, specific, works at a low and constant temperature, does not require any high-end instrument and can be a potential diagnostics tool for pen-side testing of PSV in the field conditions. The newly developed RT-RPA assay could successfully detect PSV circulating in swine population of Haryana, India. This is a first report of this kind from the region.

Construction and immunogenicity of Senecavirus A virus-like particle vaccine with adjuvant.

Senecavirus A (SVA) is constantly associated with vesicular disease in pigs, and the clinical symptoms of pig infection with SVA are indistinguishable from other porcine vesicular diseases. Vaccine is one of the best methods to eliminate and control the spread of SVA. Virus-like particles (VLPs) can play important roles in prevention for infectious diseases. Here, the SVA VLPs was assembled by the baculovirus expression vector system, and the immunogenicity of the SVA VLPs mixed with different adjuvants were evaluated in mice and pigs. Two recombinant baculoviruses (rPFBD-VP1-VP3 and rPFBD-VP2-VP4) were constructed, which co-infected with Sf9 suspension cells to assemble SVA VLPs successfully. SVA VLPs mixed with ISA201 adjuvant and ISA201 +Poly(I:C) adjuvant produced higher levels of neutralizing antibody, specific antibody (total IgG, IgG1, IgG2a and IgG2b) and cytokines in the T cells. And there was no significant difference between SVA VLPs+ 201 group and SVA VLPs+Poly(I:C)+ 201 group. Pigs immunized with high dose of SVA VLPs mixed with ISA201 adjuvant could produce higher titers of neutralizing antibody and SVA-specific antibody. Furthermore, the protection rates of SVA VLPs-H and SVA VLPs-L were 100% and 80%, and the viral load of SVA VLPs-H group is the lowest in all SVA VLPs groups. It is the first time to develop the SVA VLPs using the baculovirus expression vector system, which may lay the foundation for the research and development of SVA vaccine.

Experimental Seneca Valley virus infection in sows and their offspring.

Neonatal mortality has been increasingly reported on swine breeding farms experiencing swine idiopathic vesicular disease (SIVD) outbreaks, which can be accompanied by lethargy, diarrhea, and neurologic signs in neonates. Seneca Valley Virus (SVV), or Senecavirus A, has been detected in clinical samples taken from pigs with SIVD. Experimental SVV inoculation has caused vesicular disease in pigs, particularly during the stages from weaning to finishing. However, it remains crucial to investigate whether SVV directly contributes to the increase in neonatal mortality rates. The following study was conducted to chronicle the pathogenesis of SVV infection in sows and their offspring. Ten sows were intranasally inoculated with 4.75 × 107 plaque-forming units of the virus per sow either late in gestation (n = 5) or within fourteen days of farrowing (n = 5). Each sow replicated SVV following intranasal inoculation, but only one out of ten sows developed a vesicular lesion on the snout. Evidence of transplacental infection was observed in two litters, and an additional two litters became infected following parturition out of five litters from sows inoculated in late gestation. No clinical signs were observed in the infected neonates. Likewise, no clinical signs were observed in the other five litters inoculated after farrowing, although each piglet did replicate the challenge virus. In this study, the experimental challenge of SVV did not result in neonatal mortality in contrast to observations in the field; however, it has shed light on the pathogenesis of the virus, the transmission of SVV between sows and their offspring, and host immune response that can help shape control measures in the field.

Construction and characterization of a full-length infectious clone of an emerging senecavirus A strain.

Senecavirus A (SVA) is an emerging virus that causes vesicular disease in pigs. Construction of a full-length SVA cDNA clone is crucial for understanding its replication and pathogenesis. Here, we successfully constructed a CMV-promoter-driven infectious cDNA clone of the SVA isolate SVA/GX/CH/2018, which we named rSVA GX01. Sequence comparison between the pSVA GX01 and the parental isolate (SVA/GX/CH/2018) revealed three single-nucleotide differences. Four-week-old piglets were experimentally infected with either the parental virus or the cloned virus. The results showed that the cloned rSVA GX01 displayed weak pathogenicity in 4-week-old pigs compared to the parental virus SVA CH-GX-01-2018. The infectious clone of SVA will serve as a valuable tool for studying the viral replication cycle and for functional analysis of the viral genome.

Direct detection of canine picornavirus complete coding sequence in wastewater using long-range reverse-transcriptase polymerase chain reaction and long-read sequencing.

We describe four complete coding sequence (cCDS) of canine picornavirus from wastewater in Arizona, USA detected by coupling cCDS single-contig (∼7.5 kb) reverse-transcriptase polymerase chain reaction (RT-PCR) and low-cost long-read high-throughput sequencing. For viruses of medical/veterinary importance, this workflow expands possibilities of wastewater based genomic epidemiology for exploring virus evolutionary dynamics especially in low-resource settings.

Detection and diversity of gastrointestinal viruses in wastewater from Caracas, Venezuela, 2021-2022.

Gastrointestinal viruses (GIV) are an important cause of childhood morbidity and mortality, particularly in developing countries. Their epidemiological impact in Venezuela during the COVID-19 pandemic remains unclear. GIV can also be detected in domestic sewage. Ninety-one wastewater samples from urban areas of Caracas collected over 12 months and concentrated by polyethylene-glycol-precipitation, were analyzed by multiplex reverse-transcription-PCR for rotavirus/calicivirus/astrovirus and enterovirus/klassevirus/cosavirus, and monoplex-PCR for adenovirus and Aichi virus. The overall frequency of virus detection was 46.2%, fluctuating over months, and peaking in the rainy season. Adenoviruses circulated throughout the year, especially type F41, and predominated (52.7%) over caliciviruses (29.1%) that peaked in the rainy months, rotaviruses (9.1%), cosaviruses (5.5%), astroviruses and enteroviruses (1.8%). Aichi-virus and klassevirus were absent. Rotavirus G9/G12, and P[4]/P[8]/P[14] predominated. The occurrence of GIV in wastewater reflects transmission within the population of Caracas and the persistence of a potential public health risk that needs to be adequately monitored.

Metatranscriptomic data mining together with microfluidic card uncovered the potential pathogens and seasonal RNA viral ecology in a drinking water source.

AIMS: Despite metatranscriptomics becoming an emerging tool for pathogen surveillance, very little is known about the feasibility of this approach for understanding the fate of human-derived pathogens in drinking water sources. METHODS AND RESULTS: We conducted multiplexed microfluidic cards and metatranscriptomic sequencing of the drinking water source in a border city of North Korea in four seasons. Microfluidic card detected norovirus, hepatitis B virus (HBV), enterovirus, and Vibrio cholerae in the water. Phylogenetic analyses showed that environmental-derived sequences from norovirus GII.17, genotype C of HBV, and coxsackievirus A6 (CA6) were genetically related to the local clinical isolates. Meanwhile, metatranscriptomic assembly suggested that several bacterial pathogens, including Acinetobacter johnsonii and V. cholerae might be prevalent in the studied region. Metatranscriptomic analysis recovered 349 species-level groups with substantial viral diversity without detection of norovirus, HBV, and CA6. Seasonally distinct virus communities were also found. Specifically, 126, 73, 126, and 457 types of viruses were identified in spring, summer, autumn, and winter, respectively. The viromes were dominated by the Pisuviricota phylum, including members from Marnaviridae, Dicistroviridae, Luteoviridae, Potyviridae, Picornaviridae, Astroviridae, and Picobirnaviridae families. Further phylogenetic analyses of RNA (Ribonucleic Acid)-dependent RNA polymerase (RdRp) sequences showed a diverse set of picorna-like viruses associated with shellfish, of which several novel picorna-like viruses were also identified. Additionally, potential animal pathogens, including infectious bronchitis virus, Bat dicibavirus, Bat nodavirus, Bat picornavirus 2, infectious bursal disease virus, and Macrobrachium rosenbergii nodavirus were also identified. CONCLUSIONS: Our data illustrate the divergence between microfluidic cards and metatranscriptomics, highlighting that the combination of both methods facilitates the source tracking of human viruses in challenging settings without sufficient clinical surveillance.

AAACH is a conserved motif in a cis-acting replication element that is artificially inserted into Senecavirus A genome.

Cis-acting replication element (cre) is required for generating a diuridylylated VPg that acts as a protein primer to initiate the synthesis of picornaviral genome or antigenome. The cre is a stem-loop structure, dependent of different picornaviruses, located in different genomic regions. The AAACA motif is highly conserved in the apical loop of cre among several picornaviral members, and plays a key role in synthesizing a diuridylylated VPg. We previously demonstrated that senecavirus A (SVA) also possesses an AAACA-containing cre in its genome. Its natural cre (Nc), if functionally inactivated through site-directed mutagenesis (SDM), would confer a lethal impact on virus recovery, whereas an artificial cre (Ac) is able to compensate for the Nc-caused functional inactivation, leading to successful rescue of a viable SVA. In this study, we constructed a set of SVA cDNA clones. Each of them contained one functionally inactivated Nc, and an extra SDM-modified Ac. Every cDNA clone had a unique SDM-modified Ac. The test of virus recovery showed that only two SVAs were rescued from their individual cDNA clones. They were AAACU- and AAACC-containing Ac genotypes. Both viruses were serially passaged in vitro for analyzing their viral characteristics. The results showed that both AAACU and AAACC genotypes were genetically stable during twenty passages, implying when the Nc was functionally inactivated, SVA could still use an AAACH-containing Ac to complete its own replication cycle.