Exploratory application of a cannulation model in recently weaned pigs to monitor longitudinal changes in the enteric microbiome across varied porcine reproductive and respiratory syndrome virus (PRRSV) infection statuses.

Introduction: The enteric microbiome and its possible modulation to improve feed conversion or vaccine efficacy is gaining more attention in pigs. Weaning pigs from their dam, along with many routine procedures, is stressful. A better understanding of the impact of this process on the microbiome may be important for improving pig production. The objective of this study was to develop a weaner pig cannulation model, thus allowing ileum content collection from the same pig over time for 16S rRNA sequencing under different porcine reproductive and respiratory syndrome virus (PRRSV) infection statuses. Methods: A total of 15 3-week-old pigs underwent abdominal surgery and were fitted with an ileum cannula, with ileum contents collected over time. In this pilot study, treatment groups included a NEG-CONTROL group (no vaccination, no PRRSV challenge), a POS-CONTROL group (no vaccination, challenged with PRRSV), a VAC-PRRSV group (vaccinated, challenged with PRRSV), a VAC-PRO-PRRSV group (vaccinated, supplemented with a probiotic, challenged with PRRSV), and a VAC-ANTI-PRRSV group (vaccinated, administered an antibiotic, challenged with PRRSV). We assessed the microbiome over time and measured anti-PRRSV serum antibodies, PRRSV load in serum and nasal samples, and the severity of lung lesions. Results: Vaccination was protective against PRRSV challenge, irrespective of other treatments. All vaccinated pigs mounted an immune response to PRRSV within 1 week after vaccination. A discernible impact of treatment on the diversity, structure, and taxonomic abundance of the enteric microbiome among the groups was not observed. Instead, significant influences on the ileum microbiome were observed in relation to time and treatment. Discussion: The cannulation model described in this pilot study has the potential to be useful in studying the impact of weaning, vaccination, disease challenge, and antimicrobial administration on the enteric microbiome and its impact on pig health and production. Remarkably, despite the cannulation procedures, all vaccinated pigs exhibited robust immune responses and remained protected against PRRSV challenge, as evidenced by the development of anti-PRRSV serum antibodies and viral shedding data.

Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle.

In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats.

Bovine Rhinitis B Virus Variant as the Putative Cause of Bronchitis in Goat Kids.

A diagnostic investigation into an outbreak of fatal respiratory disease among young goats in Iowa, USA revealed bronchitis lesions of unknown etiology and secondary bacterial bronchopneumonia. Hypothesis-free metagenomics identified a previously unreported picornavirus (USA/IA26017/2023), and further phylogenetic analysis classified USA/IA26017/2023 as an aphthovirus related to bovine rhinitis B virus. Viral nucleic acid was localized to lesions of bronchitis using in situ hybridization. This marks the first report of a picornavirus putatively causing respiratory disease in goats and highlights the potential for cross-species transmission of aphthoviruses.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024.

We report highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milk production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus-infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to-mammal transmission.

Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing.

(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics in vitro; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligible effect. The sgRNAs targeting ORF30 showed the strongest effect on the suppression of EHV-1 replication, with a reduction in viral genomic copy numbers and infectious progeny virus output. Next-generation sequencing identified variants with deletions in the specific cleavage site of selective sgRNAs. Moreover, we evaluated the combination between different sgRNAs and found that the dual combination of sgRNAs targeting ORF30 and ORF7 significantly suppressed viral replication to lower levels compared to the use of a single sgRNA, suggesting a synergic effect; (4) Conclusion: data demonstrate that sgRNA-guided CRISPR/Cas9 can be used to inhibit EHV-1 replication in vitro, indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1.

Coding-complete genome sequences of two strains of canine pneumovirus derived from dogs with upper respiratory disease in the United States.

Canine pneumovirus was detected by RT-qPCR in 2022 from nasal swabs collected from two dogs with upper respiratory disease in a shelter in Louisiana, United States. The genomes from the designated strains CPnV USA/LA/2022/124423 and USA/LA/2022/123696 were sequenced and show the closest similarity to the pneumonia virus of mice J3666.

ArcA positively regulates the expression of virulence genes and contributes to virulence of porcine Shiga toxin-producing enterotoxigenic Escherichia coli.

IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in humans and animals, leading to death and huge economic loss worldwide. Thus, elucidation of ETEC's pathogenic mechanisms will provide powerful data for the discovery of drugs serving as prevention or therapeutics against ETEC-caused diarrheal diseases. Here, we report that ArcA plays an essential role in the pathogenicity and virulence regulation in ETEC by positively regulating the expression of several key virulence factors including F18 fimbriae, heat-labile and heat-stable toxins, Shiga toxin 2e, and hemolysin, under microaerobic conditions and in vivo. Moreover, we found that positive regulation of several virulence genes by ArcA requires a global repressor H-NS (histone-like nucleoid structuring), implying that ArcA may exert positive effects by antagonizing H-NS. Collectively, our data established a key role for ArcA in the pathogenicity of porcine ETEC and ETEC strains isolated from human infections. Moreover, our work reveals another layer of regulation in relation to oxygen control of virulence factors in ETEC.

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Development, Evaluation, and Clinical Application of PRRSV-2 Vaccine-like Real-Time RT-PCR Assays.

In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAXTM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a CT cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.

Genomic characterization of Streptococcus equi subspecies zooepidemicus from a 2021 outbreak in Indiana with increased sow mortality.

IMPORTANCE: This study highlights a Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) strain isolated from an outbreak in Indiana, which resulted in mortality events among a swine herd in 2021. The Indiana outbreak strain was found to be genetically and phylogenetically distant to a strain isolated from the 2019 outbreaks in Ohio and Tennessee, which caused high swine mortality. We also discovered multiple unique genetic features in the Indiana outbreak strain, including distinct S. zooepidemicus genomic islands, and notable S. zooepidemicus virulence genes-many of which could serve as biomarkers for the diagnosis of this strain. These findings provide significant insights into monitoring and potentially preventing severe outbreaks caused by the Indiana outbreak strain in the future.

NhaA facilitates the maintenance of bacterial envelope integrity and the evasion of complement attack contributing to extraintestinal pathogenic Escherichia coli virulence.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for severe bloodstream infections in humans and animals. However, the mechanisms underlying ExPEC's serum resistance remain incompletely understood. Through the transposon-directed insertion-site sequencing approach, our previous study identified nhaA, the gene encoding a Na+/H+ antiporter, as a crucial factor for infection in vivo. In this study, we investigated the role of NhaA in ExPEC virulence utilizing both in vitro models and systemic infection models involving avian and mammalian animals. Genetic mutagenesis analysis revealed that nhaA deletion resulted in filamentous bacterial morphology and rendered the bacteria more susceptible to sodium dodecyl sulfate, suggesting the role of nhaA in maintaining cell envelope integrity. The nhaA mutant also displayed heightened sensitivity to complement-mediated killing compared to the wild-type strain, attributed to augmented deposition of complement components (C3b and C9). Remarkably, NhaA played a more crucial role in virulence compared to several well-known factors, including Iss, Prc, NlpI, and OmpA. Our findings revealed that NhaA significantly enhanced virulence across diverse human ExPEC prototype strains within B2 phylogroups, suggesting widespread involvement in virulence. Given its pivotal role, NhaA could serve as a potential drug target for tackling ExPEC infections.

The role of AJB35136 and fdtA genes in biofilm formation by avian pathogenic Escherichia coli.

BACKGROUND: Infections caused by avian pathogenic Escherichia coli (APEC) result in significant economic losses in poultry industry. APEC strains are known to form biofilms in various conditions allowing them to thrive even under harsh and nutrient-deficient conditions on different surfaces, and this ability enables them to evade chemical and biological eradication methods. Despite knowing the whole genome sequences of various APEC isolates, little has been reported regarding their biofilm-associated genes. A random transposon mutant library of the wild-type APEC IMT 5155 comprising 1,300 mutants was analyzed for biofilm formation under nutrient deprived conditions using Videoscan technology coupled with fluorescence microscopy. Seven transposon mutants were found to have reproducibly and significantly altered biofilm formation and their mutated genes were identified by arbitrary PCR and DNA sequencing. The intact genes were acquired from the wild-type strain, cloned in pACYC177 plasmid and transformed into the respective altered biofilm forming transposon mutants, and the biofilm formation was checked in comparison to the wild type and mutant strains under the same conditions. RESULTS: In this study, we report seven genes i.e., nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 and fdtA of APEC with significant contribution to biofilm formation. Reintroduction of AJB35136 and fdtA, reversed the altered phenotype proving that a significant role being played by these two O-antigen related genes in APEC biofilm formation. Presence of these seven genes across nonpathogenic E. coli and APEC genomes was also analyzed showing that they are more prevalent in the latter. CONCLUSIONS: The study has elucidated the role of these genes in APEC biofilm formation and compared them to adhesion expanding the knowledge and understanding of the economically significant pathogens.

Molecular characterization of Glaesserella parasuis strains circulating in North American swine production systems.

BACKGROUND: Glaesserella parasuis is the causative agent of Glässer's disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted. RESULTS: In silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages (LI, LII, and LIII), with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency;  blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%).   CONCLUSION: This study showed the use of WGS to type G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control.

Diagnostic investigation of Mycoplasma hyorhinis as a potential pathogen associated with neurological clinical signs and central nervous system lesions in pigs.

Mycoplasma hyorhinis (M. hyorhinis) is a commensal of the upper respiratory tract in swine with the typical clinical presentations of arthritis and polyserositis in postweaning pigs. However, it has also been associated with conjunctivitis and otitis media, and recently has been isolated from meningeal swabs and/or cerebrospinal fluid of piglets with neurological signs. The objective of this study is to evaluate the role of M. hyorhinis as a potential pathogen associated with neurological clinical signs and central nervous system lesions in pigs. The presence of M. hyorhinis was evaluated in a clinical outbreak and a six-year retrospective study by qPCR detection, bacteriological culture, in situ hybridization (RNAscope®), and phylogenetic analysis and with immunohistochemistry characterization of the inflammatory response associated with its infection. M. hyorhinis was confirmed by bacteriological culture and within central nervous system lesions by in situ hybridization on animals with neurological signs during the clinical outbreak. The isolates from the brain had close genetic similarities from those previously reported and isolated from eye, lung, or fibrin. Nevertheless, the retrospective study confirmed by qPCR the presence of M. hyorhinis in 9.9% of cases reported with neurological clinical signs and histological lesions of encephalitis or meningoencephalitis of unknown etiology. M. hyorhinis mRNA was confirmed within cerebrum, cerebellum, and choroid plexus lesions by in situ hybridization (RNAscope®) with a positive rate of 72.7%. Here we present strong evidence that M. hyorhinis should be included as a differential etiology in pigs with neurological signs and central nervous system inflammatory lesions.

A recombinant porcine reproductive and respiratory syndrome virus type 2 field strain derived from two PRRSV-2-modified live virus vaccines.

A porcine reproductive and respiratory syndrome virus (PRRSV) type 2 (PRRSV-2) isolate was obtained from lung samples collected from a 4.5-month-old pig at a wean-to-finish site in Indiana, USA, although no gross or microscopic lesions suggestive of PRRSV infection were observed in the lung tissue. Phylogenetic and molecular evolutionary analyses based on the obtained virus sequences indicated that PRRSV USA/IN105404/2021 was a natural recombinant isolate from Ingelvac PRRS® MLV and Prevacent® PRRS, which are PRRSV-2-modified live virus vaccines commercially available in the United States. This study is the first to report the detection of a PRRSV-2 recombinant strain consisting entirely of two modified live virus vaccine strains under field conditions. Based on clinical data and the absence of lung lesions, this PRRSV-2 recombinant strain was not virulent in swine, although its pathogenicity needs to be confirmed by clinical trials.

Outbreak of Pathogenic Streptococcus equi subsp. zooepidemicus in Guinea Pigs Farms of The Andean Region.

Streptococcus zooepidemicus is an emerging zoonotic pathogen involved in septicemic infections in humans and livestock. Raising guinea pigs in South America is an important economic activity compared to raising them as pets in other countries. An outbreak of severe lymphadenitis was reported in guinea pigs from farms in the Andean region. S. zooepidemicus was isolated from multiple cervical and mandibular abscesses. Isolate was characterized by multilocus sequence typing and phylogenetic analysis. This is the first molecular characterization of a highly pathogenic strain, showing major important virulence factors such as the M-like protein genes szP and mlpZ, the fimbrial subunit protein gene fszF, and the protective antigen-like protein gene spaZ. Additionally, this guinea pig strain was phylogenetically related to equines but distant from zoonotic and pig isolates reported in other countries.

Genomic Island-Encoded Histidine Kinase and Response Regulator Coordinate Mannose Utilization with Virulence in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

Analysis of Recombinant Characteristics Based on 949 PRRSV-2 Genomic Sequences Obtained from 1991 to 2021 Shows That Viral Multiplication Ability Contributes to Dominant Recombination.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases affecting the pig-raising industry. The PRRS virus (PRRSV) has high genetic diversity, partly owing to viral recombination. Some individual recombinant type 2 PRRSV (PRRSV-2) strains have been detected; however, the sequence composition characteristics of recombination hot spots and potential driving forces for recombinant PRRSV-2 are still unreported. Therefore, all available genomic sequences of PRRSV-2 (n = 949, including 29 genomes sequenced in this study) from 11 countries from 1991 to 2021 were collected and analyzed. The results revealed that the dominant major recombinant parent has been converted from lineage 3 (L3) to L1 since 2012. The recombination hot spots were located at nucleotides (nt) 7900 to 8200 (in NSP9, encoding viral RNA-dependent RNA polymerase) and nt 12500 to nt 13300 (in ORF2-ORF4, mean ORF2 to ORF4); no AU-rich characteristics were found in the recombination hot spots. Based on infectious clones of L1 and L8 PRRSV-2, recombinant PRRSVs were generated by switching complete or partial NSP9 (harboring the recombination hot spot). The results showed that recombinant PRRSVs based on the L1 backbone, but not the L8 backbone, acquired a higher replication capacity in pig primary alveolar macrophages. These findings will help to understand the reason behind the dominance of L1-based recombination in PRRSV-2 strains and provide new clues for an in-depth study of the recombination mechanism of PRRSV-2. IMPORTANCE Recombination is an important driver of the genetic shifts that are tightly linked to the evolution of RNA viruses. Viral recombination contributes substantially to the emergence of new variants, alterations in virulence, and pathogenesis. PRRSV is genetically diverse, partly because of extensive recombination. In this study, we analyzed interlineage recombination based on available genomic sequences of PRRSV-2 from 1991 to 2021. The study revealed the temporal and geographical distribution of recombinant PRRSVs and the recombination hot spot's location and showed that artificially constructed recombinant PRRSVs (harboring a high-frequency region) had more viral genomic copies than their parental virus, indicating that dominant recombination was shaped by a tendency to benefit viral replication. This finding will enrich our understanding of PRRSV recombination and provide new clues for an in-depth study of the recombination mechanism.

Characterization of PRRSV in clinical samples and the corresponding cell culture isolates.

Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in cell culture is a primary means of obtaining virus isolates for autogenous vaccine production and other applications. However, it has not been well characterized whether cell culture isolate and the virus in clinical sample are equivalent. This study compared PRRSV ORF5 sequences from 1024 clinical samples (995 PRRSV-2, 26 PRRSV-1, and three PRRSV-1 and PRRSV-2 PCR-positive) and their isolates in MARC-145 and/or ZMAC cells. For three PRRSV-1 and PRRSV-2 PCR-positive clinical samples, both PRRSV-1 and PRRSV-2 were isolated in ZMAC cells, whereas either PRRSV-1 or PRRSV-2, but not both, was isolated in MARC-145 cells, with isolate sequences matching the respective viruses in clinical samples. Twenty-six PRRSV-1 and most of 995 PRRSV-2 PCR-positive clinical samples had matching viral ORF5 sequences with their cell culture isolates. However, 14 out of 995 PRRSV-2 cases (1.4%) had nonmatching viral sequences between clinical samples and MARC-145 isolates, although viral sequences from clinical samples and ZMAC isolates matched. This is concerning because, if the MARC-145 isolate is directly used for autogenous vaccine production without sequencing confirmation against the virus in the clinical sample, it is possible that the produced autogenous vaccine does not include the desired wild-type virus strain found on the farm and instead contains vaccine-like virus. Vaccine-specific PCR and next-generation sequencing performed on six selected cases indicated presence of ≥2 PRRSV-2 strains (mixed infection) in such clinical samples. In summary, PRRSV ORF5 sequences from clinical samples and cell culture isolates matched each other for majority of the cases. However, PRRSV sequences between clinical sample and MARC-145 cell culture isolate could occasionally be different when the clinical sample contains ≥2 PRRSV-2 strains. Characterizing PRRSV sequences from clinical samples and cell culture isolates should be conducted before using isolates for producing autogenous vaccines or other applications.

High Genetic Diversity of Porcine Sapovirus From Diarrheic Piglets in Yunnan Province, China.

As one of the most important enteric viruses, sapovirus (SaV) can infect humans and a variety of animals. Until now, 19 SaV genogroups have been identified, among which 4 from human (GI, GII, GIV, and GV) and 8 from swine (GIII, GV-GXI). Porcine sapovirus (PoSaV) GIII has been prevalent in China; however, the status of PoSaV infection in Yunnan province remains unknown. In this study, 202 fecal samples were collected from piglets associated with outbreaks of acute diarrhea in Yunnan between January and May 2020. PoSaV detection revealed that the total PoSaV infection rate in Yunnan was 35.2%, with 21 PoSaV strains determined and phylogenetically analyzed. The phylogenetic tree analyses demonstrated that twenty PoSaV strains belonged to GIII and fell into five genotypes, whereas one PoSaV strain (YNQB) belonged to GV. Sequence alignments revealed deletions in VP2 region in 10 of the 20 GIII strains, as well as deletions and insertions in VP1 region of the GV strain (YNQB). Furthermore, genomic recombination analyses showed that two GIII strains (YNAN and YNJD) were recombinants, closely related to reference sequences MK965898 and LC215880, MK965898 and FJ387164, respectively. In summary, PoSaV-GIII strains were identified in Yunnan in 2020, and for the first time, a PoSaV-GV strain was identified from China, whereas the comprehensive analyses illustrated high genetic diversity of Yunnan PoSaV strains. This study may shed new light on the current PoSaV infections in Yunnan and pave the way toward further control of the PoSaV infections in China.