Malnutrition and infection lead to poor prognosis and heavy financial burden of patients with chronic heart failure.

Background: Chronic heart failure (CHF) is a major public health concern, as it is associated with poor prognosis and heavy financial burden. In recent years, there has been increasing interest in medications for CHF in China, but few studies pay attention to the effects of nutrition and infection. Methods and results: This was a retrospective study collected patients with CHF admitted to the Department of Cardiology of Qilu Hospital of Shandong University from January 2017 to May 2018. Patients were classified according to the prognosis and the financial burden. Through comparison and regression analysis, we found that the factor associated with worse prognosis were decreased heart rate, albumin and prealbumin; ß-blockers and mineralocorticoid receptor antagonism (MRA) were the factor improved the prognosis of patients with CHF; the factor overburdening financial condition were infection, decreased prealbumin, high Alanine aminotransferase (ALT), usage of recombinant human brain natriuretic peptide (rhBNP) and Levosimendan; aspirin and Sacubitril/Valsartan were the factor releasing financial burden of patients with CHF. Then, we grouped by Controlling Nutritional Status (CONUT) score, which enabled evaluation of the patient's protein reserve and immune defenses. Patients in the malnutrition group had higher infection ratios, longer hospital stays, and greater hospital expenses than the normal group. The improvement ratios of therapeutic outcomes in the moderate or severe malnutrition group were lower than in the normal and mild malnutrition group. Conclusion: Malnutrition and infection caused poor prognosis and increased financial burden of patients with CHF. The high CONUT score indicated the CHF patient's unfavorable prognosis and heavy financial burden.

Construction of an HRP-streptavidin bound antigen and its application in an ELISA for porcine circovirus 2 antibodies.

A fusion protein SBP-Cap∆41, consisting of Cap∆41 (without 41 amino acids at the N-terminus) protein of porcine circovirus 2 (PCV2) and a streptavidin binding peptide (SBP), was constructed. This fusion protein binds to HRP-labeled streptavidin (HRP-SA) through high affinity between SBP and SA, forming an HRP-streptavidin bound antigen (Hsb-Ag) with both immunoreactivity and enzymatic activity, which can be used in a double-antigen sandwich ELISA for detection of PCV2 antibodies. Comparison of the characteristics of the HSb-Cap∆41 and chemical conjugates of the recombinant Cap∆41 protein showed that the HSb-Cap∆41 based double-antigen sandwich ELISA (HBDS-ELISA) had higher specificity and sensitivity. Use of the HBDS-ELISA detected PCV2-IgG in 9 injected pigs as early as 10 days p.i., 3 days earlier than both a double-antigen sandwich ELISA (DS-ELISA) based on a chemically conjugated antigen, and a commercial indirect ELISA kit.

Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera.

Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species.

Identification and characterization of multiple porcine astrovirus genotypes in Hunan province, China.

Astroviruses (AstVs) can infect a variety of hosts, including mammalian and avian species, and are commonly associated with enteric infections. Recently, mammalian AstVs have been linked to extra-intestinal manifestations, including neurologic disorders in humans, cattle and minks, demonstrating zoonotic potential. So far, five porcine AstV (PAstV) genotypes have been identified, with PAstV1, PAstV2, PAstV3 and PAstV5 implicated in cross-species transmission. Our knowledge about PAstV epidemiology in China is still limited. In this study, two duplex differential RT-PCR assays were developed to investigate the distribution and prevalence of PAstV1, PAstV2, PAstV4 and PAstV5. Two hundred eighteen samples were collected from 33 farms and pigs with known diarrhea status in nine regions of Hunan province in China. Specifically, 126 small intestines, 51 fecal swabs, 20 lungs, 19 spleens and two kidneys were obtained. PAstVs were detected in all nine regions and in 81.8% (27/33) of the pig farms investigated. The overall prevalence of PAstV was 46.3% (101/218), with PAstV5 as the predominant type, with a positive rate of 24.8% (54/218). The prevalence of PAstV4, PAstV1 and PAstV2 was 16.1% (35/218), 14.7% (32/218) and 10.1% (22/218), respectively. Besides being present in intestines and fecal swabs, PAstV RNA was also detected in lungs, spleens and kidneys. Sequencing revealed a high level of genetic divergence within each genotype, and a higher positive rate of PAstV5 was associated with pigs with diarrhea compared to pigs without diarrhea. This study revealed for the first time that PAstV4 is circulating in China, and that PAstV5 is the dominant genotype in pig herds in Hunan province in China.

Visible-light-induced photocatalytic azotrifluoromethylation of alkenes with aryldiazonium salts and sodium triflinate.

The efficient visible light photocatalytic azotrifluoromethylation of alkenes with aryldiazonium salts and sodium triflinate is described, which gave the corresponding trifluoromethylated azo compounds in generally good yields. The trifluoromethylated azo products can be easily transformed into useful heterocycles and nitrogen-containing building blocks.

Characterization of Seven New Polystyrene Plates Binding Peptides from a Phage-Displayed Random 12-Peptide Library.

A random 12-peptide library was screened against Erysipelothrix rhusiopthiae and porcine circovirus 2 recombinant Cap protein and the selected peptides were used for detecting the corresponding pathogens quickly and effectively. To our surprise, seven peptides, P1 (WHWNAP WWNGVY), P2 (FHWTWQFPYTST), P3 (GAMHLPWHMGTL), P4 (HWNIWWQHHPSP), P5 (HFFKWHTRTNDQ), P6 (HFFRWHPSAHLG) and P7 (HFAYWWNGVRGP) with the characteristics of polystyrene plate (PS) binding target-unrelated peptides (TUPs), were selected from the library. It has been found that P2 and P4 shared common motif of plastic binding peptide, moreover, P2, P3, P5 and P7 have been isolated repeatedly in other research groups using different targets. Then, the seven peptide phage clones were identified as the PS binding TUP phages by phage-ELISA and elution titration, particularly, P1 and P2 showed strong PS binding affinity which can not be inhibited by usual blocking buffers. In addition, all of the phages were not propagation-related TUP, but P3 showed the similar propagation rate with M13KE (vector phage). We also found that the seven PS-TUPs are rich in W, H, F, P and G, particularly, both W and H are contained in all PS-TUPs. It deduced that they may play a potential role in peptide binding to plastic. Although it is difficult to eliminate the TUP phages in phage display completely, these PS-TUPs can be used to exclude the false positive peptides rapidly and effectively and help us to obtain truly interesting peptides more accurately.

Phototandem catalysis: efficient synthesis of 3-ester-3-hydroxy-2-oxindoles by a visible light-induced cyclization of diazoamides through an aerobic oxidation sequence.

An unprecedented phototandem catalysis based on a single iridium photocatalyst has been successfully developed. This powerful strategy consists of two mechanistically distinct catalytic cycles, namely, photocatalytic energy transfer (ET) and single electron transfer (SET). The novel protocol allows a rapid and efficient construction of biologically and synthetically important 3-ester-3-hydroxy-2-oxindole derivatives from readily available diazoamides through a cyclization/aerobic oxidation sequence under very mild conditions.

Seroprevalence of Toxoplasma gondii infection in sows in Hunan province, China.

Toxoplasma gondii infections are prevalent in animals and humans worldwide. Although the prevalence of T. gondii has been reported in many animals in China, little is known of T. gondii infection in sows. Antibodies to T. gondii in sows in Hunan province, subtropical China, were examined using indirect hemagglutination test (IHAT). Overall, 31.3% (373/1191) of the examined sows were seropositive for T. gondii. Among 11 representative regions of Hunan province, the seroprevalence ranged from 14.8% to 45.1%. In addition, the T. gondii seroprevalence was higher in summer (37.4%) and autumn (34.9%) than in spring (24.6%) and winter (23.9%). Regarding different antibody titers, the seroprevalence ranged from 1.8% (titer ≥ 1: 1024) to 17.4% (titer = 1:64). The findings of the present investigation revealed the high seroprevalence of T. gondii in sows in Hunan province, China, which poses a potential risk for T. gondii infection in humans and animals in this province. Therefore, effective measures should be taken to prevent and control toxoplasmosis of pigs in this province. This is the first report of the comprehensive survey of T. gondii seroprevalence in sows in Hunan Province, subtropical China.

High seroprevalence of Chlamydia infection in sows in Hunan province, subtropical China.

Chlamydia spp. are Gram-negative obligate intracellular bacteria, which are responsible for significant public health problems in humans and have major economic impact on animals. In the present study, the seroprevalence of Chlamydia infection in sows in Hunan province, subtropical China, was examined using indirect hemagglutination assay (IHA). Antibodies to Chlamydia were detected in 747 of 1,191 (62.7%, 95% CI 60-65.5) serum samples (IHA titer ≥ 1:16). The Chlamydia seroprevalence ranged from 35% (95% CI 25.7-44.4) to 77.1% (95% CI 69.1-85.2) among different regions in Hunan province, and the differences were statistically significant (P < 0.01). In addition, the seroprevalence of Chlamydia infection in sows was higher in summer (75.7%, 95% CI 71.3-80) and spring (63.2%, 95% CI 57.5-68.8) than in autumn (56.9%, 95% CI 51.5-62.3) and winter (48.6%, 95% CI 42-55.3), and the differences were statistically significant (P < 0.01). The results of the present investigation indicated the high seroprevalence of Chlamydia infection in sows in Hunan province, subtropical China, which poses a potential risk for human infection with Chlamydia in this province. This is the first report of Chlamydia seroprevalence in sows over the last two decades in Hunan province, subtropical China.

Epitope screening of the PCV2 Cap protein by use of a random peptide-displayed library and polyclonal antibody.

Capsid protein (Cap), the only structural protein of porcine circovirus 2 (PCV2), is involved in the host protective response and is a target for vaccine development. To find a rapid and easy way to fully map the antigenic epitopes of Cap, purified Cap-specific polyclonal antibodies were used to screen a random heptapeptide phage display library. After three rounds of screening, twenty phage clones that had binding activity to Cap-specific antibodies (tested by phage ELISA) were sequenced. When the inserted amino acid sequences were aligned with the Cap protein sequence, eight core regions in Cap ((50)SRTFGYT(56), (62)VRTPSW(67), (68)AVDMMR(73), (79)FLPPGG(84), (86)SNPRSVPF(93), (102)KVEFWP(107), (119)GSSXXXLDDN(128) and (229)PPLNP(233)) were identified, three of which ((50)SRTFGYT(56), (86)SNPRSVPF(93) and (102)KVEFWP(107)) for the first time. Nine phagetopes representing the eight regions were chosen to immunize Kunming mice. All except minotopes (50)SRTFGYT(56) and (229)PPLNP(233) induced antibodies against PCV2 when injected into Kunming mice.

Transmission of porcine circovirus type 2b (PCV2b) in Kunming mice.

To investigate porcine circovirus type 2b (PCV2b) transmission by contact and vertical infection in Kunming mice (an outbred mouse stock deriving from Swiss albino mice with a high ratio of gene heterozygosis), four mice in cage 6 were inoculated with PCV2b and 25 mice without any treatment were placed into cages 1 to 5 (five mice in each cage). Seven days after being infected, the PCV2-binoculated mice were co-mingled with non-inoculated mice from cages 1 to 5 successively at 7, 14, 21, 28 and 35 days post infection (dpi), respectively, for 3 days. In addition, eleven pregnant mice were injected with PCV2b. Samples were collected from non-inoculated mice and three newborn mice from each litter for PCV2b detection by polymerase chain reaction (PCR) and immunohistochemistry (IHC). The PCR results showed that PCV2b transmission rate among mice in cages 1, 2, 3, 4 and 5 was 0/5, 2/5, 5/5, 5/5 and 1/5, respectively. PCV2b antigen signals generally appeared in most organs of the non-inoculated mice in which viruses were detected by PCR. PCV2b DNA was also detected in newborn mice of PCV2b-infected litters, and viral antigen signals were observed in their organs as well. PCV2b was transmitted in Kunming mice by contact, and it also caused vertical infection through the placenta.

Phylogenetic analysis using E2 gene of classical swine fever virus reveals a new subgenotype in China.

Outbreaks of classical swine fever (CSF) have caused serious economic consequences in China. Phylogenetic analysis based on full-length E2 gene sequences showed that five classical swine fever virus (CSFV) isolates collected from Hunan province in 2011 and 2012, together with seven other isolates from neighboring provinces, Guangdong (5) and Guangxi (2), could be classified as a new subgenotype 2.1c, which may have been endemic in the south of China for at least fourteen years. Subgenotype 2.1c isolates share 90.2-94.9% and 89.9-93.8% nucleotide sequence similarity separately with those of subgenotype 2.1a and 2.1b in E2 gene, which are lower than the nucleotide identities between subgenotype 2.1a and 2.1b (91.1-95.7%). Further analysis based on a partial E2 gene sequence (216 nt) indicated that subgenotype 2.1c isolates are also circulating in Thailand. Alignment of E2 amino acid sequences showed that subgenotype 2.1c isolates exhibit a SPA → TPV substitution at positions 777 and 779 compared with subgenotypes 2.1a and 2.1b.

Small-sized circular genomes similar to genome of porcine circovirus 2.

Circular genomes smaller than and similar to the genome of porcine circovirus 2 were obtained from pig tissues along with the full-length genome of porcine circovirus 2. The 922-, 839-, and 617-nucleotide-long genomes exhibit high homology to the rep gene plus the origin of replication sequence of porcine circovirus 2.

Complete genome sequences of classical Swine Fever virus isolates belonging to a new subgenotype, 2.1c, from hunan province, china.

Two isolates of a new classical swine fever virus (CSFV) subgenotype, 2.1c (HNLY-2011 and HNSD-2012), were recently isolated from pigs in Hunan Province, China. The most significant difference in the amino acid sequences of the polyproteins from subgenotypes 2.1a and 2.1b is an SPA → TPV amino acid substitution at positions 777 and 779 in the E2 protein.

Characterization of the complete mitochondrial genome of Spirocerca lupi: sequence, gene organization and phylogenetic implications.

BACKGROUND: Spirocerca lupi is a life-threating parasitic nematode of dogs that has a cosmopolitan distribution but is most prevalent in tropical and subtropical countries. Despite its veterinary importance in canids, the epidemiology, molecular ecology and population genetics of this parasite still remain unexplored. METHODS: The complete mitochondrial (mt) genome of S. lupi was amplified in four overlapping long fragments using primers designed based on partial cox1, rrnS, cox2 and nad2 sequences. Phylogenetic re-construction of 13 spirurid species (including S. lupi) was carried out using Bayesian inference (BI) based on concatenated amino acid sequence datasets. RESULTS: The complete mt genome sequence of S. lupi is 13,780 bp in length, including 12 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes, but lacks the atp8 gene. The gene arrangement is identical to that of Thelazia callipaeda (Thelaziidae) and Setaria digitata (Onchocercidae), but distinct from that of Dracunculus medinensis (Dracunculidae) and Heliconema longissimum (Physalopteridae). All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The content of A + T is 73.73% for S. lupi, in accordance with mt genomes of other spirurid nematodes sequenced to date. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes by BI showed that the S. lupi (Thelaziidae) is closely related to the families Setariidae and Onchocercidae. CONCLUSIONS: The present study determined the complete mt genome sequence of S. lupi. These new mt genome dataset should provide novel mtDNA markers for studying the molecular epidemiology and population genetics of this parasite, and should have implications for the molecular diagnosis, prevention and control of spirocercosis in dogs and other canids.

RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China.

The prevalence of porcine sapovirus infection in weanling pigs was investigated in Hunan Province, China, between August 2006 and October 2007. A total of 153 diarrheic fecal samples from ten intensive pig farms from ten representative administrative regions in Hunan province were examined for porcine sapoviruses using RT-PCR. Twenty-two of 153 (14.37 %) samples were found to contain porcine sapoviruses. Phylogenetic analysis showed that all the porcine sapovirus isolates in Hunan Province belonged to the porcine sapovirus genogroup III. The results of the present investigation have implications for the control of porcine sapovirus infection in pigs in Hunan Province, China.

Seroprevalence of porcine cytomegalovirus and sapovirus infection in pigs in Hunan province, China.

The seroprevalence of porcine cytomegalovirus (PCMV) and sapovirus (SaV) infections in pigs was investigated in Hunan province, China, between May 2005 and October 2010. A total of 500 pig serum samples collected from 10 representative administrative regions in Hunan province were evaluated for antibodies against PCMV and SaV using enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence of porcine cytomegalovirus and sapovirus in pigs was 96.40% (482/500) and 63.40% (317/500), and the seropositivity of 10 herds we surveyed varied, ranging from 94.74% to 98.48% and 56.36% to 72.50%, respectively. The highest prevalence was found in breeding sows (96.67% for PCMV and 83.33% for SaVs). The results of the present survey indicated that infections with porcine cytomegalovirus and sapovirus are highly prevalent in pigs in Hunan province, China.

Identification of new defective interfering RNA species associated with porcine reproductive and respiratory syndrome virus infection.

In the present study, seven new defective interfering (DI) RNA species of porcine reproductive and respiratory syndrome virus (PRRSV) were identified. RT-PCR, Northern blot and sequence analyses indicated that these DI RNA specie have deletions of 8513-9176 nucleotides located between Nsp1/Nsp2 and Nsp10. Compared with the previous DI RNAs of PRRSV reported, they have three distinct characteristics: much smaller deletion sizes; different nucleotide repeats (2-12nt) used in the junction sites and in-frame deletions. The results further suggested that the similarity-assisted RNA recombination may be the main cause of generation of DI RNAs in PRRSV and probably in other arteriviruses.

Viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b.

The viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b (PCV-2b) were investigated. Seventy special pathogen free mice were divided into 2 groups with 35 mice in each group. The test group (TG) was infected with PCV-2b, the control group (CG) was inoculated with sterile cell cultures. Five mice in each group were sacrificed at 3, 7, 14, 21, 28, 35 and 42 dpi (day post infection), respectively. Necropsies were performed on all mice and tissues were collected for testing by histopathology, immunohistochemistry, transmission electron microscope (TEM) and polymerase chain reaction (PCR). Apoptosis and necrosis in lymphoid organs were observed in virus-infected mice, and became severe from 14 to 28 dpi. The proportion of PCV-2b antigen-positive cells was moderate in lung, heart, thymus, liver or kidney, and low in brain from TG. In spleen and cervical lymph node, the proportions of PCV-2b antigen-positive cells were low to high from 7 to 28 dpi, and moderate from 35 to 42 dpi. PCV-2b DNA was detected in all tissues examined in TG from 7 to 42 dpi. Viral inclusion bodies presented in the cytoplasm of lymphocytes, macrophages, hepatocytes, podocytes, neurocytes, spermatids and uterine epithelial cells in TG. In CG, no viruses and viral lesions were detected. PCV-2b could replicate in mice, and PCV-2b associated lesions in mice were similar to those observed in pigs. The present results indicate that it is possible to use Kunming mouse as an animal model for PMWS research.