Inhibition of Porcine Endogenous Retrovirus by Multi-Targeting Micro RNA Against Long Terminal Region.

BACKGROUND: There might be much benefit in xenotransplantation, however, the risk of infections across species barriers remains, especially porcine endogenous retrovirus (PERV). To date, many attempts have been made to knock down active PERVs by inhibitory RNA (RNAi) and micro RNA (miRNA), which target different genes of PERV. There are a few studies that have explored whether targeting promoter regions of PERV could exert an inhibition effect. METHODS: miRNAs were automatically selected based on an online program BLOCK-iT RNAi Designer. The inhibition efficiency between miRNAs was compared based on their inhibition of different PERV genes: long terminal repeats (LTR), gag, and pol. Both relative quantitative real-time polymerase chain reaction (PCR) and C-type reverse transcriptase activity were performed. RESULTS: The results demonstrated that miRNA targeting the LTR region degraded the target sequence, and simultaneously inhibited the mRNA expression of both gag and pol genes of PERV. The LTR1, LTR2, and dual LTR1 + LTR2 miRNA inhibited 76.2%, 22%, and 76.8% of gag gene expression, respectively. Similarly, the miRNA was found to knock down the pol gene expression of 69.8%, 25.5%, and 77.7% for single targeting miRNA (LTR1 and LTR2) and multi-targeting miRNA (LTR1 + LTR2), respectively. A stable PK15 clone constitutively expressed dual LTR1 + LTR2 miRNA and exhibited higher inhibitory up to 82.8% and 92.7% of the expressions of the gag and pol genes, respectively. Also, the result of co-cultivation of dual LTR1 + LTR2 miRNA transfected PK15 cell with a human cell line inhibited expression of LTR, gag, and pol genes of PERV. CONCLUSIONS: In conclusion, this study suggested that the LTR might be an alternative target for gene silencing of PERV, and that multi-targeting miRNA had better inhibitory effect than single- targeting miRNA. In an in vitro model, the presence of miRNA was able to reduce PERV infectivity in a human cell line.

Detection and Phylogenetic Analysis of Porcine Deltacoronavirus in Korean Swine Farms, 2015.

This study applied molecular-based method to investigate the presence of porcine deltacoronavirus (PDCoV) in 59 commercial pig farms in South Korea. The results of RT-PCR screening on a relatively large collection of faeces samples (n = 681) from January 2013 to March 2015 did not reveal the presence of PDCoV until the end of 2014. However, on March 2015, PDCoV-positive samples (SL2, SL5) were detected from SL swine farm in Gyeongbuk province. The phylogenetic trees based on the complete spike- and nucleocapsid protein-coding genes showed that SL2 and SL5 closely related to the US PDCoV strains rather than those in China. Thought Korean strains of PDCoV isolated in 2014 (KNU14.04) and in 2015 (SL2 and SL5) grouped within US PDCoV cluster, the reconstruction of ancestral amino acid changes suggested that they are different.

Evolutionary Dynamics of a Highly Pathogenic Type 2 Porcine Reproductive and Respiratory Syndrome Virus: Analyses of Envelope Protein-Coding Genes.

Porcine reproductive and respiratory syndrome virus (PRRSV) has long been an economically devastating swine viral disease. The recent emergence of a highly pathogenic type 2 PRRSV with high mobility and mortality in China, spreading in Vietnam, Laos, and Thailand has placed neighbouring countries at risk. This study applied a codon-based extension of the Bayesian relaxed clock model and the fixed effects maximum-likelihood method to investigate and compare the evolutionary dynamics of type 2 PRRSV for all of known structural envelope protein-coding genes. By comparing the highly pathogenic type 2 PRRSV clade against the typical type 2 PRRSV clade, this study demonstrated that the highly pathogenic clade evolved at high rates in all of the known structural genes but did not display rapid evolutionary dynamics compared with typical type 2 PRRSV. In contrast, the ORF3, ORF5 and ORF6 genes of the highly pathogenic clade evolved in a qualitatively different manner from the genes of the typical clade. At the population level, several codons of the sequence elements that were involved in viral neutralization, as well as codons that were associated with in vitro attenuation/over-attenuation, were predicted to be selected differentially between the typical clade and the highly pathogenic clade. The results of this study suggest that the multigenic factors of the envelope protein-coding genes contribute to diversifying the biological properties (virulence, antigenicity, etc.) of the highly pathogenic clade compared with the typical clade of type 2 PRRSV.

Phylogenetic and phylogeographic analyses of porcine circovirus type 2 among pig farms in Vietnam.

This study demonstrated the prevalence of Porcine circovirus type 2 (PCV2) among pig farms in Vietnam. Analyses of the genome, capsid protein and phylogeny classified all 30 Vietnamese PCV2 strains as the PCV2b genotype, belonging to the clusters of 1A, 1B, 1C and recombinant forms. Each viral genome was 1767 nucleotides long and shared 96.0-100% nucleotide sequence identity. The amino acid substitutions in the capsid protein of the Vietnamese PCV2 strains were in immunodominant regions, and the majority of strains (24/30) contained a lysine extension at the C-terminus. Bayesian phylogeographic analysis revealed epidemic links of the PCV2 recombinant cluster within and among countries, which supports a circulating recombinant form of PCV2. Further analysis by the Jameson-Wolf antigenic index indicated antigenic alterations at important sites in the capsid protein (sites 131-133) among the recombinant cluster and the other clusters of PCV2b.

A Bayesian phylogeographical analysis of type 1 porcine reproductive and respiratory syndrome virus (PRRSV).

Understanding viral transmission is an important factor for the effective prevention one of the most devastating swine diseases, porcine reproductive and respiratory syndrome. Focusing on molecular epidemiology of type 1 PRRSV, this study analysed a large ORF5 dataset collected worldwide from 1991 to 2012 using a coalescent-based Bayesian Markov chain Monte Carlo approach. The results suggested that the virus diversified into unique subpopulations in Russia & Belarus and Italy approximately 100 years ago. Previously unreported consecutive diffusions of the virus were identified, which showed that some countries, such as Spain and Germany, acted as distribution sources to some extent. This study also provided statistical evidence for the existence of an ORF5-based phylogeographical structure of type 1 PRRSV, in which the virus tended to cluster by geographical locations more tightly than expected by chance. In contrast to this tight geographical structure, the evolution of the ORF5 gene, based on mapping of non-synonymous/synonymous substitutions, was best described by a non-homogeneous process that could be implicated as a mechanism for viral immune evasion.

Epidemiologic and phylogenetic characteristics of porcine reproductive and respiratory syndrome viruses in conventional swine farms of Jeju Island as a candidate region for PRRSV eradication.

Jeju island is the biggest island in Korea. The imports of pigs or their relatives from mainland Korea to this island has been banned since 1998. With this unique geographical and epidemiological context, epidemiology of porcine reproductive and respiratory syndrome virus (PRRSV) was investigated on the island. While all investigated farms showed 100% of seropositive rate for PRRSV, pigs on 37.2% (16/43) of the farms had viremia with type II PRRSV. The seropositive and viremia-positive rates for PRRSV in 30- to 60-day-old pigs were significantly higher in the western area ('swine farm complex' area) than the eastern area ('Scattered swine farm' area) of Jeju island. When 21 ORF5 sequences obtained from viremic sera were phylogenetically analysed, lineage 5 and Kor (newly termed in this study) of type II PRRSV were only found in Jeju island without changes from a previous report (2002-2003). Because other lineages of type II PRRSV (lineage 1 and 3) and type I PRRSV have recently emerged in mainland Korea, the banning of pigs' movement might be effective to protect the island from the introduction of these new PRRSV genotypes. Under this epidemiological condition (no introduction of new strains except for the modified-live vaccine (MLV) strain), the positive selection sites were analysed based on ORF5 of the virus. The amino acid 58 of GP5 (located on the hypervariable region) was predicted as a strong positive selection site. Although 51.2% (22/43) of the investigated farms had applied MLV, field strains of type II PRRSV were still circulating with strong positive selection. However, the restricted population of type II PRRSV (lineage 5 and Kor) without introduction of type I PRRSV or the other lineages of type II PRRSV indicate that the island has an effective quarantine system, which would allow PRRSV eradication.

Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei.

Due to the need to track and monitor genetic diversity, the genome of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) strain KLV-2010-01 in cultured Litopenaeus vannamei shrimp that originated from the first Korean outbreak in 2010 was sequenced and analyzed. The genome, with a length of 3914 nucleotides, was sequenced from the Korean IHHNV. The genome encoded three large and overlapping open reading frames: ORF1 (NS-1) of 2001 bp, ORF2 (NS-2) of 1092 bp and ORF3 (capsid protein) of 990 bp. The overall organization, size and predicted amino acid sequence of the three ORFs in Korean IHHNV were highly similar to those of members of the infectious IHHNV group, and the most closely related strains were IHHNVs described from Ecuador and Hawaii. Additionally, phylogenetic analysis showed that the Korean IHHNV was clustered with lineage III in the infectious IHHNV group and was most similar to IHHNV isolates from Ecuador, China and Taiwan.

Randomised feasibility trial of pre-exposure rabies vaccination with DTP-IPV in infants.

BACKGROUND: Pre-exposure vaccination against rabies generally simplifies treatment and could be especially beneficial to children in countries where the disease is enzootic. We studied the feasibility of administering to infants pre-exposure rabies vaccination with combined diphtheria, tetanus, whole-cell pertussis, and inactivated poliomyelitis vaccine (DTP-IPV). METHODS: 84 Vietnamese infants were randomly assigned to groups that received three doses of DTP-IPV vaccine at 2, 3, and 4 months of age alone (n = 43) or with two doses of purified Vero cell rabies vaccine (PVRV) at 2 and 4 months (n = 41). The safety and immunogenicity data of the groups were compared. FINDINGS: All infants in both groups developed protective antibody concentrations against diphtheria, tetanus, pertussis, and polio. All infants who received the PVRV vaccine developed protective antibody concentrations against rabies. No serious adverse effects were reported, nor did systemic reactions differ between groups. INTERPRETATION: Administration of PVRV with DTP-IPV proved safe, and elicited what are presumed to be protective antibody concentrations to all antigens in all 41 infants. Confirmation of these results could lead to integration of pre-exposure rabies vaccination into Expanded Programme on Immunisation (EPI) sessions in selected countries where rabies is enzootic.

Se, Zn, Mg, Ca, K, cholesterol, and creatine kinase concentrations in men during the 12 days after an acute myocardial infarction.

We determined concentrations of selenium, zinc, magnesium, calcium, potassium, total and high-density lipoprotein cholesterol, and total creatine kinase (EC 2.7.3.2) in plasma (P) and (or) erythrocytes (E) of 32 men at the time of hospital admission for acute myocardial infarction and then on subsequent days 2, 3, 6, 10, and 12. During these 12 days, only P-selenium and high-density lipoprotein cholesterol concentrations remained unchanged. For the other variables, individual comparison of means indicated the dates of significant changes in concentration (p less than 0.05).

[Halogenated inhalation anesthesia with a semiclosed circle system]. / Anesthésie halogénée utilisant un respirateur muni d'un circuit-filtre.

Thirty-six patients were anaesthetized for a maxillofacial surgical procedure. Ventilation was controlled by a ventilator (Kontron ABT 4100) with a semi-closed circle system. The flow of fresh gases was 1,200 ml.min-1. The vaporizer for the halogenated anaesthetic agent was placed out of the circle before the ventilator. Halothane was used for maintenance of anaesthesia and isoflurane for induced hypotension in orthognathic surgery. The inspired concentration of the halogenated agent was monitored by an analyser. A linear correlation between the delivered and the inhaled concentration of halogenated agent was established, the latter never reaching the delivered concentration. Monitoring the inspired oxygen concentration was required, so as to maintain a constant value. Carbon dioxide absorption by soda lime was also studied. The known advantages were: substantial economies in nitrous oxide and halogenated agents, prevention of contamination of the operating theatre, humidification and warning of the inspired gases. The use of such a system with the vaporizer out of the circle was safe, all the more so as the concentration of inhaled halogenated agents could be predicted.

Low lead doses and atherogenic diet in rabbits: biochemical results in blood.

Changes in concentrations of blood lead, cholesterol, triacylglycerols, GOT and GPT enzymes, phosphorus, proteins, plasma calcium and magnesium, and erythrocyte magnesium were studied for 25 weeks in 4 groups of 6 rabbits each receiving different diets: I (controls), II (water with 9.66 mumol/l of lead), III (atherogenic) and IV (atherogenic + 9.66 mumol/l of lead). Differences observed, with respect to the diets, for lead, cholesterol, enzymes, proteins and erythrocyte magnesium were evident from the 42nd day on. Low lead doses raised the levels of cholesterol and triacylglycerols in group II, whereas triacylglycerols were lower in the plasma of group IV in comparison with group III and were accumulated in the aorta. The elevation of enzymes in groups III and IV indicated heart and liver dysfunctions. There was a significant drop in erythrocyte magnesium in groups III and IV, which was especially marked in the latter group, with an interaction between the two diets.

Concentrations of lead, magnesium, calcium, zinc and cadmium in twenty rabbit tissues after exposure to low lead doses and atherogenic diet.

Four groups of 6 rabbits were subjected to the following diets for 25 weeks: I (controls), II (water with 9.66 mumol/l of lead), III (atherogenic) and IV (atherogenic + 9.66 mumol/l of lead). Lead, magnesium, calcium, zinc and cadmium were then analyzed in 20 dry tissues. At the level of the arteries, veins and skin a tendency was observed toward increased lead concentration in rabbits of groups II and IV, elevated calcium levels in groups III and IV (a tenfold increase of calcium in the aortas), and a higher concentration of cadmium in animals of group II. There was a significant reduction in lead and magnesium concentrations in the liver of animals in groups III and IV because of fibrosis and overabundance of fatty liver cells. The lead level in the liver of animals in group II had slightly increased. Lead concentrations were higher in the kidneys and spleen in groups II and IV. Cadmium levels were significantly lower in the liver, kidneys, adrenal glands and spleen of animals in groups III and IV, whereas in group II they were particularly increased in the liver and kidneys. Very little significant interaction between the two diets was noted. Rabbits in groups III and IV showed notable histopathological alterations in aorta, carotid and femoral arteries, left ventricle and liver. Extrapolation from rabbit to man would be inadvisable.