Search

Abstract Porcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172–554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.

The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-β in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.

ABSTRACTInfluenza A viruses (IAVs) pose a persistent potential threat to human health because of the spillover from avian and swine infections. Extensive surveillance was performed in 12 cities of Guangxi, China, during 2018 and 2023. A total of 2540 samples (including 2353 nasal swabs and 187 lung tissues) were collected from 18 pig farms with outbreaks of respiratory disease. From these, 192 IAV-positive samples and 19 genomic sequences were obtained. We found that the H1 and H3 swine influenza A viruses (swIAVs) of multiple lineages and genotypes have continued to co-circulate during that time in this region. Genomic analysis revealed the Eurasian avian-like H1N1 swIAVs (G4) still remained predominant in pig populations. Strikingly, the novel multiple H3N2 genotypes were found to have been generated through the repeated introduction of the early H3N2 North American triple reassortant viruses (TR H3N2 lineage) that emerged in USA and Canada in 1998 and 2005, respectively. Notably, when the matrix gene segment derived from the H9N2 avian influenza virus was introduced into endemic swIAVs, this produced a novel quadruple reassortant H1N2 swIAV that could pose a potential risk for zoonotic infection.

The Mammalian orthoreovirus (MRV) infects various mammals, including humans, and is linked to gastrointestinal, respiratory, and neurological diseases. A recent outbreak in Liuzhou, Guangxi, China, led to the isolation of a new MRV strain, GXLZ2301, from fecal samples. This strain replicates in multiple cell lines and forms lattice-like structures. Infected cells exhibit single-cell death and syncytia formation. The virus’s titers peaked at 107.2 TCID50/0.1 mL in PK-15 and BHK cells, with the lowest at 103.88 TCID50/0.1 mL in A549 cells. Electron microscopy showed no envelope with a diameter of about 70 nm. Genetic analysis revealed GXLZ2301 as a recombinant strain with gene segments from humans, cows, and pigs, similar to type 3 MRV strains from Italy (2015-2016). Pathogenicity tests indicated that while the bovine MRV strain did not cause clinical symptoms in mice, it caused significant damage to the gut, lungs, liver, kidneys, and brain. The emergence of this MRV strain may pose a threat to the health of animals and humans, and it is recommended that its epidemiology and recombination be closely monitored.

Astroviruses are single-stranded, positive-sense RNA viruses capable of infecting humans as well as a wide range of mammalian and avian species, with a length of approximately 6.6–7.7 kb. In this study, 139 goat fecal samples collected from the Guangxi province were used for the RT-PCR detection, and two of these were positive for goat astrovirus, with a positivity rate of 1.44% (2/139). The complete genome sequence of an astrovirus strain and the partial genome sequence of a strain astrovirus, named GX WZ 2023 and GX HC 2023, were amplified and sequenced, and their sequence lengths were 6284 nt and 6213 nt, respectively. Among them, the capsid protein of goat astrovirus GX HC 2023 showed the highest amino acid identity of 95.9% with ovine astrovirus GX, which belonged to the MAstV-2 genotype. However, the closest relative of the GX WZ 2023 strain was found to be the caprine astrovirus Sichuan, with a nucleotide sequence identity of 76.8%. The ORF1ab nonstructural protein of this strain showed the highest amino acid identities of 89.2 and 95.8% with the ovine astrovirus S5.1 and caprine astrovirus G5.1 strains, respectively. However, its ORF2 capsid protein has 68.4% amino acid identity with the bovine astrovirus (BAstV) 16 2021 CHN strain and only 21.9–64% amino acid identity with all available strains of goat astrovirus. The GX WZ 2023 strain was recombined with the Chinese (BAstV 16 2021 CHN) and Japanese bovine strains (BAstV JPN 2015) in the ORF2 region. Therefore, the goat astrovirus GX WZ 2023 is proposed as a new member of the family goat astroviridae based on the species classification criteria of the International Committee on Taxonomy of Viruses. These findings enhance our understanding of the prevalence and genetic evolution of goat astrovirus and provide a scientific basis for future studies of these viruses in other animals.

ABSTRACT The interferon (IFN) system is an extremely powerful antiviral response in animal cells. The subsequent effects caused by porcine astrovirus type 1 (PAstV1) IFN activation are important for the host’s response to viral infections. Here, we show that this virus, which causes mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa in piglets, induces an IFN response upon infection of PK-15 cells. Although IFN-β mRNA was detected within infected cells, this response usually occurs during the middle stages of infection, after genome replication has taken place. Treatment of PAstV1-infected cells with the interferon regulatory factor 3 (IRF3) inhibitor BX795 decreased IFN-β expression, whereas the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) inhibitor BAY11-7082 did not. These findings indicate that PAstV induced the production of IFN-β via IRF3-mediated rather than NF-κB-mediated signaling pathways in PK-15 cells. Moreover, PAstV1 increased the protein expression levels of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in PK-15 cells. The knockdown of RIG-I and MDA5 decreased the expression levels of IFN-β and the viral loads and increased the infectivity of PAstV1. In conclusion, PAstV1 induced the production of IFN-β via the RIG-I and MDA5 signaling pathways, and the IFN-β produced during PAstV1 infection inhibited viral replication. These results will help provide new evidence that PAstV1-induced IFNs may protect against PAstV replication and pathogenesis. IMPORTANCE Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs. However, astrovirus-host interactions are less well studied, particularly with respect to their antagonism of IFN. Here, we report that PAstV1 acts via IRF3 transcription pathway activation of IFN-β. In addition, the knockdown of RIG-I and MDA5 attenuated the production of IFN-β induced by PAstV1 in PK-15 cells and increased efficient viral replication in vitro. We believe that these findings will help us to better understand the mechanism of how AstVs affect the host IFN response.

ABSTRACT Since the outbreak caused by a porcine epidemic diarrhea virus (PEDV) variant in 2010, the current epidemic of PEDV genotype 2 (G2) has caused huge economic losses to the pig industry in China. In order to better understand the biological characteristics and pathogenicity of the current PEDV field strains, 12 PEDV isolates were collected and plaque purified during 2017 to 2018 in Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate genetic variations, and they were compared with the reported G2a and G2b strains. Phylogenetic analysis of the S protein showed that the 12 isolates were clustered into the G2 subgroup (with 5 and 7 strains in G2a and G2b, respectively) and that they shared 97.4 to 99.9% amino acid identities. Among them, one of the G2a strains, CH/GXNN-1/2018, which had a titer of 106.15 PFU/mL, was selected for pathogenicity analysis. Although piglets infected with the CH/GXNN-1/2018 strain exhibited severe clinical signs and the highest level of virus shedding within 24 h postinfection (hpi), recovery and decreased virus shedding were seen after 48 hpi, and no piglets died during the whole process. Thus, the CH/GXNN-1/2018 strain had low virulence in suckling piglets. Virus neutralizing antibody analysis showed that the CH/GXNN-1/2018 strain induced cross-protection against both homologous G2a and heterologous G2b PEDV strains as early as 72 hpi. These results are of great significance for understanding PEDV in Guangxi, China, and they provide a promising naturally occurring low-virulent vaccine candidate for further study. IMPORTANCE The current epidemic of porcine epidemic diarrhea virus (PEDV) G2 has caused huge economic losses to the pig industry. Evaluation for low virulence of the PEDV strains of subgroup G2a would be useful for the future development of effective vaccines. In this study, 12 field strains of PEDV were obtained successfully, and they were characterized from Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate antigenic variations. One of the G2a strains, CH/GXNN-1/2018, was selected for pathogenicity analysis, and it showed that the CH/GXNN-1/2018 strain had low virulence in suckling piglets. These results provide a promising naturally occurring low-virulent vaccine candidate for further study.

Water pollution caused by a highly hazardous chemical ammonia and a widespread application nanomaterials-nano titanium dioxide (n-TiO2) in nature water has attracted extensive concern of the world. However, the potential joint effects of the two factors are unknown. Aim to investigate the potential interactive effects of ammonia and n-TiO2 and the behind mechanisms, adult female zebrafish (Danio rerio) were co-exposed for 8 weeks by total ammonia nitrogen (TAN; 0, 3, 30 mg/L) and n-TiO2 (0, 0.1, 1 mg/L) in different combination conditions based on a full-factorial design. The analysis of absorption kinetics confirmed that n-TiO2 could absorb free ammonia (NH3) in aqueous solution and the loss rate of free NH3 increased with the rise of n-TiO2 concentration. Consistent with this, free NH3 concentrations in the gill and liver were higher in the presence of n-TiO2 compared to TAN exposure alone. The increases of MDA and PC concentrations in the gill and liver of fish indicated that TAN and n-TiO2 alone or in combination caused oxidative stress. Simultaneously, the activity and transcription of antioxidant enzymes (T-SOD, CuZn-SOD, Mn-SOD, CAT, GPx and GST) as well as antioxidant GSH contents were extensively inhibited by TAN and n-TiO2 via Nrf2-Keap1 signaling. The significant interactive effects of TAN and n-TiO2 were detected on levels of GSH, GST and gstr1 mRNA in the gill, and on levels of GSH, T-SOD, Mn-SOD, CAT levels as well as gpx1a and keap1 mRNAs in the liver, implying synergistic toxic risk of TAN and n-TiO2. The more severe histopathological alterations and higher IBR analysis in co-treatment groups further proved that the existence of n-TiO2 excavated ammonia-induced toxicity in the gill and liver, especially in liver. In conclusion, ammonia and n-TiO2 have a synergistic toxic risk of fish health because ammonia and n-TiO2 cause oxidative-antioxidative imbalance by inducing ROS overproduction.

In recent years, hunniviruses have been reported in a variety of animal species from many countries. Here, hunnivirus was detected in fecal samples from water buffaloes and named as BufHuV-GX-2106. The samples were inoculated into cultures of MDBK cells supplemented with TPCK trypsin and the BufHuV-GX-2106 strain was stably passaged and replicated. Electron microscopic analysis showed the BufHuV-GX-2106 virus particles were spherical and 20~30 nm in diameter. The complete genome of a plaque purified sample of BufHuV-GX-2106 was determined and analyzed. Genomic analysis revealed that the whole sequence of BufHuV-GX-2106 was ~7,601 nucleotides (nt) in length and consisted of a large open reading frame of 6,759nt, a 5′UTR, a 3'UTR and a poly(A) tail. The complete genome sequence of BufHuV-GX-2106 shares 68-85% nucleotide identities with other known hunnivirus strains, indicating high genetic heterogeneity among these viruses. Phylogenetic analysis showed that BufHuV-GX-2106 belonged to the Hunnivirus A species and was more closely related to ovine hunnivirus than other known viruses of this type. This study describes the first isolation and complete genome sequence of a hunnivirus strain from water buffaloes. In addition, this study will help to understand the mechanisms involved in the pathogenesis of Hunnivirus A among different animal species.

Since 2010, porcine epidemic diarrhea virus (PEDV) has swept across China and spread throughout the country, causing huge economic losses. In this study, 673 diarrhea samples from 143 pig farms in Guangxi during 2017–2022 were collected and detected for PEDV. Ninety-eight strains were selected for S1 gene analyses and these strains were classified into four subgroups (G1b, G2a, G2b and G2c), accounting for 1.02 (1/98), 75.51 (74/98), 16.33 (16/98) and 7.14% (7/98) of the total, respectively. Importantly, an increased number of strains in the G2c subgroup was found from 2019 onwards. Bayesian analysis revealed that Guigang may have been the epicenter of PEDVs in Guangxi. In addition, Guigang was identified as the primary hub from which PEDVs spread via two routes, namely Guigang–Wuzhou and Guigang–Laibin. Moreover, several coinfections of novel PEDV variants bearing large deletions in the partial S1 protein and PEDVs possessing an intact partial S1 protein were found in pigs. Further recombination analyses indicated that two of the strains, 18-GXNN-6 and 19-GXBH-2, originated from intra-genogroup recombination. Together, our data revealed a new profile of PEDV in Guangxi, China, which enhances our understanding of the distribution, genetic characteristics and evolutionary profile of the circulating PEDV strains in China.

Astroviruses (AstVs) are major causative agents of gastroenteritis and have been detected worldwide. Little is known about the prevalence of neurotropic AstVs in Chinese water buffaloes, but a novel species which is associated with encephalitis and meningitis has recently been found. In this study, based on nested RT-PCR, rapid amplification of the 3′-cDNA end (3′-RACE) and next-generation sequencing (NGS), we examined the infection of AstVs in water buffaloes in the Guangxi Province of China. The results showed that the AstV infection was found in 40% (6/15) of the farms examined, and the prevalence of AstV in their feces was 11% (33/297). In addition, two near-full-length and two complete open reading frame 2 (ORF2) genes of AstVs from fecal sources were sequenced. Phylogenetic analysis of the ORF2 sequences indicated three lineages of BufAstVs, BufAstV lineage 1 was close related to the BoAstV, lineage 2 was related to the BufAstVs, and lineage 3 was classified as novel AstVs, which had a close relationship with the neurotropic/neurovirulent AstVs strains found in bovine, ovine, and musks. Moreover, genomic a recombination between the BufAstV and BoAstV strains was identified. This is a novel study reporting the genetic diversity of BufAstV infection in China especially found the similar neurotropic strains from fecal sources of water buffaloes, and it also provides details of the epidemiology, genetic recombination, and interspecies transmission of BoAstV and BufAstV in water buffaloes from the Guangxi Province of China.

Farmers involved in the lucrative pork trading business between China and Southeast Asian countries should be aware of a recently discovered novel porcine deltacoronavirus (PDCoV) in Guangxi province, China. A PDCoV strain, CHN/GX/1468B/2017, was isolated from the small intestinal contents of piglets with diarrhea from this region, with a titer of 1 × 108.0 TCID50/mL on LLC-PK cells. The full-length genome sequence consists of 25,399 nt as determined by next-generation sequencing and this was deposited in the GenBank (accession number MN025260.1). Genomic analysis showed that CHN/GX/1468B/2017 strain had 96.9~99.4% nucleotide homology with other 87 referenced PDCoV strains from different areas, and contained 6 and 9-nt deletions at positions 1,733~1,738 and 2,804~2,812, respectively, in the ORF1a gene. Phylogenetic analyses based on the whole gene sequence as well as S protein and ORF1a/1b protein sequences all showed that this strain was closely related to the Southeast Asia strain. When 7-day-old piglets were inoculated orally with the CHN/GX/1468B/2017 strain, they developed severe diarrhea, with a peak of fecal viral shedding at 4 days post-infection. Although no death or fever were observed, the CHN/GX/1468B/2017 strain produced a wide range of tissue tropism, with the main target being the intestine. Importantly, the VH:CD ratios of the jejunum and ileum in infected piglets were significantly lower than controls. These results indicate that CHN/GX/1468B/2017, isolated in China, is a novel PDCoV Southeast Asia-like strain with distinct genetic characteristics and pathogenicity. This finding enriches the international information on the genetic diversity of PDCoV.

Enterovirus G (EV-G) infects porcine populations worldwide and the infections are generally asymptomatic, with the insertion of the papain-like cysteine protease gene (PLCP) increasing the potential public health threats. However, the genetic and pathogenic characteristics of EV-G itself are not fully understood as yet. In the present study, one EV-G strain, named CH/17GXQZ/2017, was isolated and purified from piglets with diarrheic symptoms from the Guangxi Province, China. This strain produced stable cytopathic effects on Marc-145 cells with a titer of 5 × 106 PFU/mL. The spherical enterovirus particles with diameters of 25–30 nm were observed by using transmission electron microscopy. The whole genome sequence of the CH/17GXQZ/2017 strain consists of 7,364 nucleotides, and the phylogenetic tree based on the amino acid sequences of VP1 indicated this strain was clustered to the G1 genotype. Seven-day-old piglets were inoculated orally with the CH/17GXQZ/2017 strain in order to evaluate its pathogenicity. Although none of the infected piglets died during the experiment, clinical neurological symptoms were observed manifesting as mild hyperemia and Nissl bodies vacuolization in the cerebrum. In addition, the infection with the CH/17GXQZ/2017 strain decelerated the weight gain of suckling piglets significantly. This study demonstrates that CH/17GXQZ/2017 is pathogenic to neonatal piglets and advance knowledge on the biological characteristics, evolution and pathogenicity of EV-G.

Abstract Background Porcine astroviruses (PAstVs) are common in pigs worldwide. There are five distinct lineages with each lineage representing a different ancestral origin. Recently, multiple reports have demonstrated the evidence of extra-intestinal infection of PAstVs, but little is known about viremia. Results In this study, a total of 532 fecal samples and 120 serum samples from healthy pigs were collected and tested from 2013 to 2015 in Guangxi province, China; of these 300/532 (56.4%) and 7/120 (5.8%) of fecal samples tested positive for PAstVs, respectively. Our study revealed that there was wide genetic diversity and high prevalence of the virus in the pig population. All five of the known PAstVs genotypes (1–5) prevailed in the pig population of Guangxi province and were distributed in all age groups of pigs, from suckling piglets to sows, with PAstV2 (47.7%), PAstV1 (26.2%) and PAstV5 (21.5%) seen predominantly. Phylogenetic analysis of partial ORF1b and partial capsid sequences from fecal and serum samples revealed that they were divided into the five lineages. Among these genotypes, based on partial ORF2 genes sequencing 23 strains were grouped as PAstV1, including 6 serum-derived strains, and were regarded as the causative agents of viremia in pigs. Conclusions Due to the information regarding the types of PAstV in blood is limit. This is the first report for the presence of PAstV1 in blood and PAstV3 in the feces of nursery pigs of China. This study provides a reference for understanding the prevalence and genetic evolution of PAstVs in pigs in Guangxi province, China. It also provides a new perspective for understanding of the extra-intestinal infection of PAstVs in pigs.

Abstract Background Porcine circovirus 2-associated disease (PCVAD) is acknowledged as one of the most economically important diseases for the swine industry worldwide. The aim of this study was to characterize and determine the genetic diversity of PCV2 in the porcine population of Guangxi, China. Methods The full length genome and open reading frame 2 (ORF2) of 95 PCV2 strains collected from the tissues and sera of pigs that had either died as a result of PCVAD or did not exhibit disease symptoms were analyzed. Results The results of multiple sequence alignments showed that there is considerable diversity among the PCV2 ORF2 sequences. Phylogenetic analyses based on the complete genome showed that current PCV2 strains in this study could be divided into PCV2a (1/95), PCV2b (39/95), PCV2d (43/95), PCV2e (10/95) and PCV2h (2/95). Among the 5 sub-genotypes, PCV2b was dominant in the porcine population from 2002 to 2008. The newly identified sub-genotype, PCV2d, was seen from 2003 and has increased every year. PCV2b and PCV2d formed two predominant genetic groups circulating in southern China between 2009 and 2013 and the sub-genotype PCV2d has become the dominant virus in China since 2014. Conclusions This study reveals the complex genetic diversity of PCV2 and improves our understanding regarding the epidemiological trends of PCV2 sub-genotypes in China.

ABSTRACTHost switch events of influenza A viruses (IAVs) continuously pose a zoonotic threat to humans. In 2013, swine-origin H1N1 IAVs emerged in dogs soon after they were detected in swine in the Guangxi province of China. This host switch was followed by multiple reassortment events between these H1N1 and previously circulating H3N2 canine IAVs (IAVs-C) in dogs. To evaluate the phenotype of these newly identified viruses, we characterized three swine-origin H1N1 IAVs-C and one reassortant H1N1 IAV-C. We found that H1N1 IAVs-C predominantly bound to human-type receptors, efficiently transmitted via direct contact in guinea pigs and replicated in human lung cells. Moreover, the swine-origin H1N1 IAVs-C were lethal in mice and were transmissible by respiratory droplets in guinea pigs. Importantly, sporadic human infections with these viruses have been detected, and preexisting immunity in humans might not be sufficient to prevent infections with these new viruses. Our results show the potential of H1N1 IAVs-C to infect and transmit in humans, suggesting that these viruses should be closely monitored in the future.

Since its recent appearance in China, the NADC30-like strains of porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) have caused an expanding epidemic, and this has further expanded the genetic diversity of PRRSV. In this study, three NADC30-like strains—GXFCG20210401, GXQZ20210403 and GXNN20210506—were isolated from pig serum samples obtained in Guangxi, and their genomes were sequenced. A comparative analysis of the whole genomes showed that the three strains were most similar to NADC30 (88.3–88.7%). In particular, the non-structural protein coding regions (nsp1, nsp4-5, nsp7-8 and nsp9) showed the highest similarities to JXA1, and the ORF2a-ORF5 regions showed the highest similarities to NADC34. The three strains had same discontinuous deletions of 111+1+19 amino acids in the nsp2 region, which were similar to the NADC30-like strains. Phylogenetic tree analysis based on the ORF5 gene showed that the three PRRSV isolates were divided into lineage 1.5 along with the representative NADC34-like strains, but they were classified as NADC30-like strains with respect to the whole genome and nsp2 evolutionary trees. Recombinant analysis revealed complex recombination patterns in the genomes of the three strains, which likely originated from multiple recombination events among JXA1-like, NADC30-like and NADC34-like strains. The results from animal experiments showed that the GXQZ20210403 strain was 20% lethal to piglets and caused more severe clinical reactions than GXFCG20210401, and both recombinant strains were similar in terms of pathogenicity to the previously reported NADC34 strains. This study demonstrates that NADC34-like strains of PRRSV have been circulating in the southern provinces of China and have exchanged genomes with several other indigenous strains. In addition, differences in recombination patterns may cause different clinical pathogenicity and indicate the importance of the surveillance and preventive control of recombinant strains.

Porcine astroviruses (PAstVs) are prevalent in pigs worldwide, and five genotypes have been reported to circulate in China. However, little is known about the coinfection status of PAstVs. For differential and simultaneous diagnoses of these five genotypes of PAstVs, a multiplex RT-PCR method was established on the basis of the ORF2 gene of type 1 PAstV, and the ORF1ab genes of type two to five PAstVs. This quintuple PCR system was developed through optimization of multiplex PCR and detection sensitivity and specificity. The results showed that this multiplex RT-PCR method could specifically detect all the five PAstV genotypes without cross-reaction to any other major viruses circulating in Chinese pig farms. The detection limit of this method was as low as 10 pg of standard plasmids of each PAstV genotype. In addition, a total of 275 fecal samples collected from different districts of Guangxi, China, between April 2019 and November 2020, were tested by this newly established multiplex RT-PCR. Moreover, the sensitivity and specificity of monoplex and multiplex RT-PCR methods were compared by detecting the same set of clinical positive samples. The results revealed that PAstV1 (31/275), PAstV2 (49/275), PAstV3 (36/275), PAstV4 (41/275), and PAstV5 (22/275) were all detected, and dual (PAstV1+PAstV2, PAstV1+PAstV3, PAstV2+PAstV3, PAstV2+PAstV4, PAstV3+PAstV4, and PAstV4+PAstV5) or triple genotypes (PAstV1+PAstV2+PAstV3 and PAstV2+PAstV3+PAstV4) of coinfections were also unveiled in this study. The detection result of multiplex PCR was consistent with that of monoplex PCR. Compared with monoplex PCR, this multiplex PCR method showed obvious advantages such as time and cost efficiency and high sensitivity and specificity. This multiplex RT-PCR method offered a valuable tool for the rapid and accurate detection of PAstV genotypes circulating in pig herds and will facilitate the surveillance of PAstV coinfection status.

Getah virus (GETV) has caused many outbreaks in animals in recent years. Monitoring of the virus and its related diseases is crucial to control the transmission of the virus. In the summer of 2018, we conducted routine tests on clinical samples from different pig farms in Guangxi province, South China, and isolated and characterized a GETV strain, named GX201808. Cytopathic effects were observed in BHK-21 cells inoculated with GX201808. The expression of E2 protein of GETV could be detected in virus-infected cells by indirect immunofluorescence assays. Electron microscopic analysis showed that the virus particles were spherical and ~70 nm in diameter with featured surface fibers. The multistep growth curves showed the virus propagated well in the BHK-21 cells. Molecular genetic analysis revealed that GX201808 belongs to Group 3, represented by Kochi-01-2005 isolated in Japan in 2005, and it clustered closely with the recently reported Chinese strains isolated from pigs, cattle, and foxes. A comparison of the identities of nucleotides and amino acids in the coding regions demonstrated that the GX201808 showed the highest amino acid identity (99.6%) with the HuN1 strain, a highly pathogenic isolate resulting in an outbreak of GETV infection in swine herds in Hunan province in 2017. In the present study, GETV was identified and isolated for the first time in Guangxi province of southern China, suggesting that future surveillance of this virus should be strengthened.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been used as a gene expression vector in the development of vaccines. Most of these recombinant PRRSV vectors express only a single foreign gene through either an internal insertion in the hypervariable region of nsp2 or expression cassette and some of these recombinant vectors are genetically unstable. Here, we combined internal insertion in nsp2 and expression cassette methods to generate a novel recombinant PRRSV stably expressing the red fluorescence protein (RFP) and the green fluorescence protein (GFP) genes. Biological characteristic analysis of the recombinant PRRSV carrying the two marker genes, rGX-RFP-GFP, showed that it displayed similar growth kinetics and yet it yielded less infectious viruses when compared to the parental virus rGXAM. Co-expression of both the RFP and GFP was observed using confocal fluorescence microscopy when the rGX-RFP-GFP viruses infected MARC-145 cells. Furthermore, the PRRSV-based two-marker gene expression vector is genetically stable during 20 serial passages in MARC-145 cells. These data demonstrate that it is possible to express two interested immunogens from a single PRRSV vector.

Porcine reproductive and respiratory syndrome virus (PRRSV) has had a devastating impact on the pig industry in China, and monitoring its genetic diversity is important for epidemiological surveillance and understanding its evolution. Here, we determine the complete genome sequences of two PRRSV strains, GXYL1403 and GXNN1839. Comparative, phylogenetic, and recombination detection program analyses show that the two isolates are recombinant strains with large-fragment amino acid deletions in nsp2. GXYL1403 possesses a unique deletion region of 124 amino acids in nsp2, and GXNN1839 contains a deletion of 131 amino acids in nsp2 as compared with VR2332. Further analysis of the full-length sequence suggests that GXYL1403 is a natural recombinant between sublineages 8.1 (CH-1a like) and 8.3 (JXA1-like). The recombination site of GXYL1403 is located in nsp9–nsp12 (8961nt−11181nt). GXNN1839 is a natural recombinant between the lineage 5 (VR-2332-like) and lineage 1 (NADC30-like) strains. The recombination events occurred in nsp9 (7872nt-8162nt) and in ORF2 (12587nt−13282nt) in the genome of GXNN1839. These results provide new evidence that PRRSV strains circulating in the environment have undergone recombination among the different lineages or sublineages of field strains, and these add to our understanding of RNA combination events that occur in PRRSV.

Porcine epidemic diarrhea virus (PEDV) is one of the major enteric pathogens, causing severe enteric disease, resulting in enormous economic losses. The ORF3 gene encodes an accessory protein which is related to the infectivity and virulence of PEDV. In this study, 33 PEDV positive field samples were collected from Guangxi, from 2017 to 2019, and the genetic diversity of ORF3 was investigated. Thirty-eight strains of ORF3 were obtained, and these were composed of five strains of ORF3 named Guangxi naturally truncated strains that were 293 bp in length, with continuous deletions from 172 to 554 bp. The Guangxi naturally truncated strains encoded a truncated protein of 89 amino acids, which had clustered into a new group referred to as Group 3, and these might be involved in the variations of virulence. Three genotypes (G1-1 subgroup, G1-3 subgroup, and Group 3) existed simultaneously in Guangxi based on the genetic and evolutionary analysis of the ORF3 gene. The sequence information in the current study will hopefully facilitate the establishment of a diagnostic method that can differentiate the PEDV field stains. Continued surveillance will be useful for monitoring PEDV transmission. Differentiation of the ORF3 genes in PEDV field strains can help us to choose an appropriate PEDV vaccine candidate in the future and prevent outbreaks of PED more effectively.

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

Coinfection caused by various genotypes of porcine epidemic diarrhea virus (PEDV) is a new disease situation. We previously reported the coexistence of PEDV strains containing different ORF3 genotypes in China. In this study, the PEDV strains 17GXCZ-1ORF3d and 17GXCZ-1ORF3c were isolated and plaque-purified from the same piglet, which had a natural large deletion at the 172–554 bp position of the ORF3 gene or possessed a complete ORF3 gene, respectively. Meanwhile, 17GXCZ-1ORF3d had >99% nt identity with 17GXCZ-1ORF3c in the 5′UTR, ORF1a/1b, S, E, M, N and 3′UTR regions but only demonstrated low nucleotide identities (80.5%) in the ORF3 gene. To elucidate the pathogenicity, 7-day-old piglets were infected. Piglets infected with these two PEDV strains exhibited severe clinical signs and shed the virus at the highest level within 96 hpi. Compared with the piglets inoculated with the 17GXCZ-1ORF3c strain, the piglets inoculated with the 17GXCZ-1ORF3d strain had higher mortality rates (75% vs. 50%), an earlier onset of clinical signs with a significantly higher diarrhea score, lower VH:CD ratios and a higher percentage of PEDV-positive enterocytes. This study is the first to report PEDV coinfections with different ORF3 genotypes, and a PEDV strain with a large deletion in the ORF3 gene might have the advantage of a potential genetic marker, which would be useful during vaccine development.

Abstract Background Astroviruses (AstVs) have been reported to infect and cause gastroenteritis in most animal species. Human AstVs were regarded the causative agent of viral diarrhea in children. In dogs, little is known about the epidemiology and clinical significance of AstV infection. Findings In this study, we collected and tested 253 rectal swabs from pet dogs; of which 64 samples (25.3%) tested positive for AstVs with diarrhea and 15 more samples (5.9%) also was identified as AstVs, however without any clinical signs. Phylogenetic analysis of 39 partial ORF1b sequences from these samples revealed that they are similar to AstVs, which can be subdivided into three lineages. Interestingly, out of the 39 isolates sequenced, 16 isolates are shown to be in the Mamastrovirus 5/canine astrovirus (CAstV) lineage and the remaining 23 isolates displayed higher similarities with known porcine astrovirus (PoAstV) 5 and 2. Further, analysis of 13 capsid sequences from these isolates showed that they are closely clustered with Chinese or Italy CAstV isolates. Conclusions The findings indicate that CAstVs commonly circulate in pet dogs, and our sequencing results have shown the genomic diversity of CAstVs leading to increasing number of clusters.

ABSTRACT The capacity of influenza A viruses (IAVs) to host jump from animal reservoir species to humans presents an ongoing pandemic threat. Birds and swine are considered major reservoirs of viral genetic diversity, whereas equines and canines have historically been restricted to one or two stable IAV lineages with no transmission to humans. Here, by sequencing the complete genomes of 16 IAVs obtained from canines in southern China (Guangxi Zhuang Autonomous Region [Guangxi]) in 2013 to 2015, we demonstrate that the evolution of canine influenza viruses (CIVs) in Asian dogs is increasingly complex, presenting a potential threat to humans. First, two reassortant H1N1 virus genotypes were introduced independently from swine into canines in Guangxi, including one genotype associated with a zoonotic infection. The genomes contain segments from three lineages that circulate in swine in China: North American triple reassortant H3N2, Eurasian avian-like H1N1, and pandemic H1N1. Furthermore, the swine-origin H1N1 viruses have transmitted onward in canines and reassorted with the CIV-H3N2 viruses that circulate endemically in Asian dogs, producing three novel reassortant CIV genotypes (H1N1r, H1N2r, and H3N2r [r stands for reassortant]). CIVs from this study were collected primarily from pet dogs presenting with respiratory symptoms at veterinary clinics, but dogs in Guangxi are also raised for meat, and street dogs roam freely, creating a more complex ecosystem for CIV transmission. Further surveillance is greatly needed to understand the full genetic diversity of CIV in southern China, the nature of viral emergence and persistence in the region’s diverse canine populations, and the zoonotic risk as the viruses continue to evolve. IMPORTANCE Mammals have emerged as critically underrecognized sources of influenza virus diversity, including pigs that were the source of the 2009 pandemic and bats and bovines that harbor highly divergent viral lineages. Here, we identify two reassortant IAVs that recently host switched from swine to canines in southern China, including one virus with known zoonotic potential. Three additional genotypes were generated via reassortment events in canine hosts, demonstrating the capacity of dogs to serve as “mixing vessels.” The continued expansion of IAV diversity in canines with high human contact rates requires enhanced surveillance and ongoing evaluation of emerging pandemic threats.

Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV). Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.

Editorial to the Special Issue: Towards a New Science of Information – Proceedings of the Fourth International Conference on the Foundations of Information Science, Beijing, August 21–24, 2010

Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Despite several studies showing increased susceptibility to secondary bacterial infections following IAV infection, information on the direct effect of S. pneumoniae on IAV in vitro is unknown. This is an important area of investigation as S. pneumoniae is a common commensal of the human upper respiratory tract, present as an important coinfecting pathogen with IAV infection. A recent study showed that S. pneumoniae enhances human metapneumovirus infection in polarized bronchial epithelial cells in vitro. The aim of the current study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication using a standard immunofluorescence assay (IFA). For this study we used four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus. Our results suggested that pretreatment of MDCK cells with 7.5×10(6) colony-forming units (CFUs) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (0.5 to 4 hr). But, pretreatment of cell lines with 7.5×10(5) and lower CFUs of S. pneumoniae had no detectable effect on either the morphology of cells or on the IAV replication. However, unlike in epithelial cell lines, due to influence of secreted host factors the effect of pneumococci on IAV replication may be different during coinfections in vivo in the human upper respiratory tract, and in vitro with primary human polarized bronchial epithelial cells.

Background: Coronavirus disease 2019 (COVID-19) is resurgent in the UK and health and economic costs of the epidemic continue to rise. There is a need to understand the health and economic costs of different courses of action. Methods: We combine modelling, economic analysis and a user-friendly interface to contrast the impact and costs of different testing strategies: two levels of testing within the current test-trace-isolate (TTI) strategy (testing symptomatic people, tracing and isolating everyone) and a strategy where TTI is combined with universal testing (UT; i.e. additional population testing to identify asymptomatic cases). We also model effective coverage of face masks. Results: Increased testing is necessary to suppress the virus after lockdown. Partial reopening accompanied by scaled-up TTI (at 50% test and trace levels), full isolation and moderately effective coverage of masks (30% reduction in overall transmission) can reduce the current resurgence of the virus and protect the economy in the UK. Additional UT from December 2020 reduces the epidemic dramatically by Jan 2021 when combined with enhanced TTI (70% test-trace levels) and full isolation. UT could then be stopped; continued TTI would prevent rapid recurrence. This TTI+UT combination can suppress the virus further to save ~20,000 more lives and avoid ~£90bn economic losses, though costs ~£8bn more to deliver. We assume that all traced and lab-confirmed cases are isolated. The flexible interface we have developed allows exploration of additional scenarios, including different levels of reopening of society after the second lockdown in England as well as different levels of effective mask coverage. Conclusions: Our findings suggest that increased TTI is necessary to suppress the virus and protect the economy after the second lockdown in England. Additional UT from December 2020 reduces the epidemic dramatically by Jan 2021 and could then be stopped, as continued TTI would prevent rapid recurrence.

This report provides a brief account of the major reforms to higher education in China since 1949 and background reforms since 1978, focusing on recent trends and perspectives.