Comparison of gut viral communities in diarrhoea and healthy dairy calves.

Calf diarrhoea has been a major cause of economic losses in the global dairy industry. Many factors, including multiple pathogen infections, can directly or indirectly cause calf diarrhoea. This study compared the faecal virome between 15 healthy calves and 15 calves with diarrhoea. Significantly lower diversity of viruses was found in samples from animals with diarrhoea than those in the healthy ones, and this feature may also be related to the age of the calves. Viruses belonging to the families Astroviridae and Caliciviridae that may cause diarrhoea in dairy calves have been characterized, which revealed that reads of caliciviruses and astroviruses in diarrhoea calves were much higher than those in healthy calves. Five complete genomic sequences closely related to Smacoviridae have been identified, which may participate in the regulation of the gut virus community ecology of healthy hosts together with bacteriophages. This research provides a theoretical basis for further understanding of known or potential enteric pathogens related to calf diarrhoea.

Bartonella quintana type IV secretion effector BepE-induced selective autophagy by conjugation with K63 polyubiquitin chain.

Bartonella effector proteins (named Beps) are substrates of VirB type IV secretion system for translocation into host cells evolved in Bartonella spp. Among these, BepE has been shown to protect cells from fragmentation effects triggered by other Beps and to promote in vivo dissemination of bacteria from the dermal site of inoculation to the bloodstream. Bacterial pathogens secreted effectors to modulate the interplay with host autophagy, either to combat autophagy to escape its bactericidal effect or to exploit autophagy to benefit intracellular replication. Here, we reported a distinct phenotype that selective autophagy in host cells is activated as a countermeasure, to attack BepE via conjugation with K63 polyubiquitin chain on BepE. We found that ectopic expression of Bartonella quintana BepE specifically induced punctate structures that colocalised with an autophagy marker (LC3-II) in host cells, in addition to filopodia and membrane ruffle formation. Two tandemly arranged Bartonella Intracellular Delivery (BID) domains in the BepE C-terminus, where ubiquitination of sister pairs of lysine residues was confirmed, were essential to activate host cell autophagy. Multiple polyubiquitin chain linkages of K27, K29, K33, and K63 were found to be conjugated at sites of K222 and K365 on BepE, of which K63 polyubiquitination on BepE K365 determined the selective autophagy (p62/SQSTM1 positive autophagy) independent of the PI3K pathway. Colocalisation of BepE with LAMP1 confirmed the maturation of BepE-induced autophagosomes in which BepE were targeted for degradation. Moreover, host cells employed selective autophagy to counter-attack BepE to rescue cells from BepE-induced endocytosis deficiency.

Proteome Analysis Reveals Syndecan 1 Regulates Porcine Sapelovirus Replication.

Porcine sapelovirus A (PSV) is a single stranded, positive-sense, non-enveloped RNA virus that causes enteritis, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs. Research on PSV infection and interaction with host cells is unclear. In this study, we applied tandem mass tag proteomics analysis to investigate the differentially expressed proteins (DEPs) in PSV-infected pig kidney (PK)-15 cells and explored the interactions between PSV and host cells. Here we mapped 181 DEPs, including 59 up-regulated and 122 down-regulated DEPs. Among them, osteopontin (SPP1), induced protein with tetratricopeptide repeats 5 (IFIT5), ISG15 ubiquitin-like modifier (ISG15), vinculin (VCL), and syndecan-1 (SDC1) were verified significantly changed using RT-qPCR. Additionally, overexpression of SDC1 promoted PSV viral protein (VP)1 synthesis and virus titer, and silencing of SDC1 revealed the opposite results. Our findings show that SDC1 is a novel host protein and plays crucial roles in regulating PSV replication.

Entry of sapelovirus into IPEC-J2 cells is dependent on caveolae-mediated endocytosis.

BACKGROUND: Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, are a significant cause of enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. However, the life cycle of PSV on the molecular level is largely unknown. METHODS: Here, we used chemical inhibitors, RNA interference, and overexpression of dominant negative (DN) mutant plasmids to verify the roles of distinct endocytic pathways involved in PSV entry into porcine small intestinal epithelial cell line (IPEC-J2). RESULTS: Our experiments indicated that PSV infection was inhibited when cells were pre-treated with NH4Cl or chloroquine. Inhibitors nystatin, methyl-ß-cyclodextrin, dynasore and wortmannin dramatically reduced PSV entry efficiency, whereas the inhibitors chlorpromazine and EIPA had no effect. Furthermore, overexpression caveolin DN mutant and siRNA against caveolin also decreased virus titers and VP1 protein synthesis, whereas overexpression EPS15 DN mutant and siRNA against EPS15 did not reduce virus infection. CONCLUSIONS: Our findings suggest that PSV entry into IPEC-J2 cells depends on caveolae/lipid raft mediated-endocytosis, that is pH-dependent and requires dynamin and PI3K but is independent of clathrin and macropinocytosis.

Viral metagenomics reveals significant viruses in the genital tract of apparently healthy dairy cows.

The virome in genital tract secretion samples collected from 80 dairy cattle in Shanghai, China, was characterized. Viruses detected included members of the families Papillomaviridae, Polyomaviridae, Hepeviridae, Parvoviridae, Astroviridae, Picornaviridae, and Picobirnaviridae. A member of a new species within the genus Dyoxipapillomavirus and six circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viral genomes were fully sequenced and phylogenetically analyzed. The prevalence of bovine polyomaviruses 1 and 2 was measured by PCR to be 10% (8/80) and 6.25% (5/80), respectively. PCR screening also indicated that the novel papillomavirus ujs-21015 and bovine herpesvirus 6 were present in three and two out of the 80 samples, respectively.

Plasma virome of cattle from forest region revealed diverse small circular ssDNA viral genomes.

BACKGROUND: Free-range cattle are common in the Northeast China area, which have close contact with farmers and may carry virus threatening to cattle and farmers. METHODS: Using viral metagenomics we analyzed the virome in plasma samples collected from 80 cattle from the forested region of Northeast China. RESULTS: The virome of cattle plasma is composed of the viruses belonging to the families including Parvoviridae, Papillomaviridae, Picobirnaviridae, and divergent viral genomes showing sequence similarity to circular Rep-encoding single stranded (CRESS) DNA viruses. Five such CRESS-DNA genomes were full characterized, with Rep sequences related to circovirus and gemycircularvirus. Three bovine parvoviruses belonging to two different genera were also characterized. CONCLUSION: The virome in plasma samples of cattle from the forested region of Northeast China was revealed, which further characterized the diversity of viruses in cattle plasma.

Molecular characterization of a porcine teschovirus HuN-1 isolate proliferating in PK-15 cell.

BACKGROUND: Porcine teschoviruses (PTVs) are small non-enveloped viruses with single-stranded, positive sense genomic RNA, belonging to the family Picornaviridae. Natural infections of teschoviruses are limited to pigs. RESULTS: In this study, a PTV HuN-1 was found that it could be proliferated in PK-15 cell, and it came from the pig fecal samples from Hunan province, in central China. The complete genome of the HuN-1 was amplified by RT-PCR and sequenced. The complete genome of HuN-1 isolate is 7098 nt, which shares the highest sequence identity (85.9%) with the PTV 8 strain of Jilin/2003/2 and Fuyu/2009/2. The HuN-1 isolate contains only one ORF (from 320 to 7039 nt) coding a 2240 amino acid polyprotein. Aligned sequences show that more mutations occurred in the structural region than in the nonstructural region. Phylogenetic analysis showed that HuN-1 isolate did not clustered with the hitherto reported strains, according to P1 sequences, forming a subgroup in the PTV cluster. CONCLUSION: In this study, complete genome of PTV HuN-1 was cloned and sequenced. Detection and characterization of further PTV strains from different geographic areas are important to understand the worldwide distribution and heterogeneity (serotype) of PTVs and their association with symptomatic infections in pigs.

Compared proteomic analysis of 8- and 32-week-old postnatal porcine ovaries.

Pigs share many anatomical and physiological features with humans, offering a unique and viable model for biomedical research. Tandem mass tag method followed by mass spectrometry analysis was utilized to identify peptides (47,405), proteins (14,701), and protein groups (7634) in ovaries of 8- and 32-week-old postnatal Banna miniature pigs. After annotation and analysis by Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology, the proteins were identified as being involved in hormone metabolic pathways and maintenance, proliferation, and regulation of stem cells. In addition, we found 638 differentially expressed proteins between ovaries of 8- and 32-week-old postnatal Banna miniature pigs. We used Interactive Pathway Explorer to produce an overview of pig ovarian proteomics. Compared with those of the 8-week-old group, the proteins enriched in metabolism of steroid hormones, metabolism of lipids, and energy metabolism pathway were upregulated in the 32-week-old group, indicating physiological characteristics of sexual maturity. These findings have implications in applications of biomedicine. SIGNIFICANCE OF THE STUDY: Pigs share many anatomical and physiological features with humans, offering a unique and viable model for biomedical research. In this study, we used tandem mass tag quantitative proteomics to describe, for the first time, protein expression patterns of postnatal pig ovaries. Proteins involved in hormone metabolic pathways and maintenance, proliferation, and regulation of stem cells were identified. With further analysis by Interactive Pathway Explorer, proteins enriched in metabolism of steroid hormones, metabolism of lipids, and energy metabolism pathway were upregulated in the 32-week-old group, indicating physiological characteristics of sexual maturity. These findings have implications in applications of biomedicine.

Lymphatic Circulation Disseminates Bartonella Infection Into Bloodstream.

The hallmark of Bartonella infection is long-lasting intraerythrocytic parasitism. However, the process of Bartonella bacteremia is still enigmatic. In the current study, we used Bartonella tribocorum to determine how Bartonella invasion into the bloodstream from dermal inoculation might occur. Bartonella was poorly phagocytized by peritoneal macrophages in vitro. Intracellular Bartonella survived and replicated in macrophages at an early stage of infection. Intracellular Bartonella inhibited spontaneous cell death of macrophages. They also inhibited Salmonella-induced pyroptosis and mildly reduced inflammasome activation through an unidentified mechanism. A rat model confirmed that Bartonella was also inadequately phagocytized in vivo, because numerous free-floating bacilli were observed in lymph collected from thoracic duct drainage as early as 2 hours after inoculation. Lymphatic fluid drainage in the bloodstream significantly reduced the bacterial load in the bloodstream. These findings illustrated a potential route by which Bartonella invade bloodstream from dermal inoculation before they are competent to infect erythrocytes.

Excretion of infectious hepatitis E virus into milk in cows imposes high risks of zoonosis.

UNLABELLED: Hepatitis E virus (HEV) represents the main cause of acute hepatitis worldwide. HEV infection in immunocompromised patients involves a high risk for the development of chronic hepatitis. Because HEV is recognized as a zoonotic pathogen, it is currently believed that swine is the primary reservoir. However, this is not sufficient to justify the strikingly high seroprevalence of HEV in both developing and Western countries. Thus, this study aimed to identify new zoonotic sources that bear a high risk of transmission to humans. We collected fecal, blood, and milk samples of cows in a typical rural region of Yunnan Province in southwest China, where mixed farming of domestic animals is a common practice. HEV RNA was quantified by quantitative real-time polymerase chain reaction, and the whole genome was sequenced. HEV infectivity was assessed in rhesus macaques. We found a high prevalence of active HEV infection in cows as determined by viral RNA positivity in fecal samples. Surprisingly, we discovered that HEV is excreted into milk that is produced by infected cows. Phylogenetic analysis revealed that all HEV isolates from cow/milk belong to genotype 4 and subtype 4h. Gavage with HEV-contaminated raw and even pasteurized milk resulted in active infection in rhesus macaques. Importantly, a short period of boiling, but not pasteurization, could completely inactivate HEV. CONCLUSION: Infectious HEV-contaminated cow milk is recognized as a new zoonotic source that bears a high risk of transmission to humans; these results call attention to understanding and establishing proper measurement and control of HEV zoonotic transmission, particularly in the setting of mixed farming of domestic animals. (Hepatology 2016;64:350-359).

Genomic organization and recombination analysis of a porcine sapovirus identified from a piglet with diarrhea in China.

BACKGROUND: Sapovirus (SaV), a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. To date, both intra- and inter-genogroup recombinant strains have been reported in many countries except for China. Here, we report an intra-genogroup recombination of porcine SaV identified from a piglet with diarrhea of China. METHODS: A fecal sample from a 15-day-old piglet with diarrhea was collected from Shanghai, China. Common agents of gastroenteritis including porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine SaV, porcine norovirus, and porcine epidemic diarrhea virus were detected by RT-PCR or PCR method. The complete genome of porcine SaV was then determined by RT-PCR method. Phylogenetic analyses based on the structural region and nonstructural (NS) region were carried out to group this SaV strain, and it was divided into different genotypes based on these two regions. Recombination analysis based on the genomic sequence was further performed to confirm this recombinant event and locate the breakpoint. RESULTS: All of the agents showed negative results except for SaV. Analysis of the complete genome sequence showed that this strain was 7387 nt long with two ORFs and belonged to SaV GIII. Phylogenetic analyses of the structural region (complete VP1 nucleotide sequences) grouped this strain into GIII-3, whereas of the nonstructural region (RdRp nucleotide sequences) grouped this strain into GIII-2. Recombination analysis based on the genomic sequence confirmed this recombinant event and identified two parental strains that were JJ259 (KT922089, GIII-2) and CH430 (KF204570, GIII-3). The breakpoint located at position 5139 nt of the genome (RdRp-capsid junction region). Etiologic analysis showed the fecal sample was negative with the common agents of gastroenteritis, except for porcine SaV, which suggested that this recombinant strain might lead to this piglet diarrhea. CONCLUSIONS: P2 strain was an intra-genogroup recombinant porcine SaV. To the best of our knowledge, this study would be the first report that intra-genogroup recombination of porcine SaV infection was identified in pig herd in China.

Identification and genomic characterization of a novel species of feline anellovirus.

Here, a novel feline anellovirus strain (named FelineAV621 and GenBank no. KX262893) was detected in two cats with diarrhea. The complete genome of FelineAV621 is 2409 nt long with a G+C content of 56.67 %, including three open reading frames (ORFs). Phylogenetic analysis based on the amino acid sequence of the putative capsid protein (ORF1) indicated that FelineAV621 belonged to a novel anellovirus species inside a clade containing the seal anellovirus, canine TTVs, and porcine TTVs, but was distant from all the previous feline anelloviruses.

New serotypes of porcine teschovirus identified in Shanghai, China.

Teschoviruses are widely endemic and commonly found in pig fecal samples. In this study, we collected fecal specimens from various pig herds and genotyped them based on the VP1 gene. Of 322 samples, 276 were positive, giving a PTV infectivity rate of 85.7 %. PTV4 was the most common serotype found in Shanghai, followed by PTV8 and PTV10. Interestingly, Some Shanghai strains belonging to a new PTV serotype were also isolated. In phylogenetic analysis, PTV SH8 did not correspond to any known serotype. PTV4 and PTV6 showed similar levels of sequence identity to PTV SH8. These data suggest that PTV SH8 is a new serotype, distinct from the new serotype PTV wild boar/WB2C-TV/2011/HUN, which clusters with PTV SH2, SH10, and SH25.

Molecular characterization and phylogenetic analysis of the genome of porcine torovirus.

In this study, we amplified and sequenced the first genome of porcine torovirus (PToV SH1 strain). The genome was found to be 28,301 bp in length, sharing 79 % identity with Breda virus. It mainly consists of replicase (20,906 bp) and structural genes: spike (4,722 bp), membrane (702 bp), hemagglutinin-esterase (1,284 bp), and nucleocapsid (492 bp). Sequence alignments and structure prediction suggest genetic differences among toroviruses, mainly in NSP1 (papain-like cysteine proteinase domain). Rooted phylogenetic trees were constructed based on the 3C-like proteinase and RNA-dependent RNA polymerase genes. PToV, Berne virus and Breda virus were clustered together, forming a separate branch from white bream virus that was distant from that of the coronaviruses.

Reston virus in domestic pigs in China.

Historically, Reston virus (RESTV) has been found to be associated with outbreaks of disease only in nonhuman primates. Its spread to domestic pigs was reported for the first time in 2008. In this study, we report the discovery, molecular detection, and phylogenetic analysis of Reston virus (RESTV) in domestic pigs in China. A total of 137 spleen specimens from pigs that died after showing typical clinical signs of porcine reproductive and respiratory syndrome (PRRS), and for which infection with porcine reproductive and respiratory syndrome virus (PRRSV) was confirmed by RT-PCR, were collected from three farms in Shanghai from February to September 2011. Of these samples, 2.92 % (4/137) were found to be positive for RESTV. All of the positive piglets were under the age of 8 weeks and were co-infected with PRRSV. Sequences were found that shared 96.1 %-98.9 % sequence similarity with those of two RESTV variants that had been discovered previously in domestic pigs and cynomolgus macaques from the Philippines. We therefore conclude that RESTV was present in domestic pigs in Shanghai, China.

Molecular detection of genogroup I and II picobirnaviruses in pigs in China.

In this study, a total of 187 stool specimens were collected from a pig farm in Hunan province of China, from November 2011 to June 2012. 39 (20.9 %) stool specimens were positive for picobirnaviruses using reverse transcription-polymerase chain reaction. Among 39 stool specimens, 84.6 % (33/39) were identified to be genogroup I (prototype 1-CHN-97), 38.5 % (15/39) belonged to genogroup II (prototype 4-GA-91), and 23.1 % (9/39) of which showed the evidence of genogroup I picobirnavirus were also positive for genogroup II picobirnaviruses. Picobirnaviruses exist in pigs which were divided into five groups according to the age and physiological status. Nineteen representative strains of genogroup I picobirnaviruses and eleven strains of genogroup II picobirnaviruses detected in this study were selected to analyze their phylogenetic relationships with other picobirnaviruses reference strains. The phylogenetic tree analysis suggested the prevalence of multiple picobirnaviruses in pigs in China.

Depolymerization of cytokeratin intermediate filaments facilitates intracellular infection of HeLa cells by Bartonella henselae.

Bartonella henselae is capable of invading epithelial and endothelial cells by modulating the function of actin-dependent cytoskeleton proteins. Although understanding of the pathogenesis has been increased by the development of an in vitro infection model involving endothelial cells, little is known about the mechanism of interaction between B. henselae and epithelial cells. This study aims to identify the binding candidates of B. henselae in epithelial cells and explores their effect on B. henselae infection. Pull-down assays and mass spectrometry analysis confirmed that some of the binding proteins (keratin 14, keratin 6, and F-actin) are cytoskeleton associated. B. henselae infection significantly induces the expression of the cytokeratin genes. Chemical disruption of the keratin network by using ethylene glycol tetraacetic acid promotes the intracellular persistence of B. henselae in HeLa cells. However, cytochalasin B and phalloidin treatment inhibits B. henselae invasion. Immunofluorescent staining demonstrates that B. henselae infection induces an F-actin-dependent rearrangement of the cytoskeleton. However, we demonstrated via immunofluorescent staining and whole-mount cell electron microscopy that keratin intermediate filaments are depolymerized by B. henselae. The results indicate that B. henselae achieves an intracellular persistence in epithelial cells through the depolymerization of cytokeratin intermediate filaments that are protective against B. henselae invasion.

Complete genome sequence of a new-genotype porcine norovirus isolated from piglets with diarrhea.

Noroviruses (NoVs) are members of the family Caliciviridae and are emerging enteric pathogens of humans and animals. So far, porcine NoVs have been detected exclusively in fecal samples from adult swine without clinical signs. Here we report the genome sequence of a NoV strain isolated from piglets with diarrhea. Experimental infection of miniature pigs with this porcine NoV-positive fecal sample confirmed that this strain can cause diarrhea in piglets. A phylogenetic tree based on the predicted amino acid sequence of the complete capsid region showed that this strain is separate from known porcine GII strains (GII-11, GII-18, and GII-19), constituting the sole member of a new branch.

Complete genome sequence of a novel porcine enterovirus strain in China.

The porcine enteroviruses (PEVs) belong to the family Picornaviridae. We report a complete genome sequence of a novel PEV strain that is widely prevalent in pigs at least in central and eastern China. The complete genome consists of 7,390 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 812 and 7318 and encodes a 2,168-amino-acid polyprotein. Phylogenetic analysis based on the 3CD and VP1 regions reveals that this PEV strain belongs to a species of PEV9 but may represent a novel sero-/genotype in CPE group III. We also report the major findings from bootscan analysis based on the whole genomes of PEVs in the present study and those available in GenBank.

Complete genome of a novel porcine astrovirus.

Astroviruses have been widely described in mammalian and avian species. Here, we report a complete genome sequence of a novel porcine astrovirus (PoAstV) isolated from a porcine fecal sample in China. The genome consists of 6,611 nucleotides, excluding the 3' poly(A) tail, and has two open reading frames (ORFs). ORF1 maps between nucleotide positions 19 and 4211 and encodes a 1,396-amino-acid (aa) polyprotein precursor consisting of nonstructural protein and putative RNA-dependent RNA polymerase, and ORF2 maps between nucleotide positions 4202 and 6531 and encodes a 775-aa polyprotein which is a capsid precursor protein. The genome sequence of the virus was distinct enough from those of the known PoAstVs to be considered a novel sequence. Phylogenetic analysis based on the predicted amino acid sequence of the complete capsid region showed that this strain may be a novel porcine astrovirus.