Effect of Mycoplasma synoviae strains isolated from the respiratory and reproductive tracts of chicken on SPF chicken embryos

Mycoplasma synoviae (MS) infections in poultry are an important epidemiological and economic problem in poultry production all over the world. The differences between M. synoviae strains are related to the pathogenicity and the course of the disease. In recent years, the pathogenicity of M. synoviae strains has increased, and some of them are capable of causing serious infections. Both horizontal and vertical transmission routes play an important role in MS infection in flocks. The aim of the study was to determine the impact of infection with selected MS strains obtained from chickens showing a clinical form of MS infection on SPF chicken embryos. Ten strains of M. synoviae were used for this purpose. The strains were isolated from the respiratory tract and the oviduct of chickens with symptoms typical of infection with this pathogen. Genetic material isolated from liquid cultures of these strains was confirmed by molecular (PCR and LAMP) and microbiological methods. The selected M. synoviae strains belonged to six different genotypes. Significant differences in virulence between the strains were demonstrated. In nine infected groups of embryos, M. synoviae strains caused weight loss, and in seven groups they produced anatomopathological changes characteristic of mycoplasma infections. The most pathogenic for SPF chicken embryos turned out to be strains characterized as genotype F isolated from the chicken oviduct and strains of genotype C isolated from the respiratory tract. One strain of genotype H isolated from the respiratory tract showed no pathogenic effect on SPF chicken embryos. The study showed that infections with M. synoviae can have a significant impact on the production of chicken chicks in commercial hatcheries and the economy of the poultry industry.

A novel subgenotype C6 Enterovirus A71 originating from the recombination between subgenotypes C4 and C2 strains in mainland China

Recombination plays important roles in the genetic diversity and evolution of Enterovirus A71 (EV-A71). The phylogenetics of EV-A71 in mainland China found that one strain DL71 formed a new subgenotype C6 with unknown origin. This study investigated the detailed genetic characteristics of the new variant. DL71 formed a distinct cluster within genotype C based on the genome and individual genes (5′UTR, VP4, VP1, 2A, 2B, 2C, 3D, and 3′UTR). The average genetic distances of the genome and individual genes (VP3, 2A, 2B, 2C, 3A, 3C, and 3D) between DL71 and reference strains were greater than 0.1. Nine recombination events involving smaller fragments along DL71 genome were detected. The strains Fuyang-0805a (C4) and Tainan/5746/98 (C2) were identified as the parental strains of DL71. In the non-recombination regions, DL71 had higher identities with Fuyang-0805a than Tainan/5746/98, and located in the cluster with C4 strains. However, in the recombination regions, DL71 had higher identities with Tainan/5746/98 than Fuyang-0805a, and located in the cluster with C2 strains. Thus, DL71 was a novel multiple inter-subgenotype recombinant derived from the dominant subgenotype C4 and the sporadic subgenotype C2 strains. Monitoring the emergence of new variants by the whole-genome sequencing remains essential for preventing disease outbreaks and developing new vaccines.

Serum O-Glycosylated Hepatitis B Surface Antigen Levels in Patients with Chronic Hepatitis B During Nucleos(t)ide Analog Therapy

Serum hepatitis B surface antigen (HBsAg) is a component of hepatitis B virus (HBV) virions and non-infectious subviral particles (SVPs). O-glycosylation of the PreS2 domain of middle HBsAg protein is a distinct characteristic of genotype C HBV virions versus SVPs. We measured serum O-glycosylated HBsAg levels in 47 treatment-naïve patients with genotype C chronic hepatitis B (CHB) at baseline and after 48 weeks of nucleos(t)ide analog (NA) therapy by immunoassay using a monoclonal antibody against the O-glycosylated PreS2 domain of middle HBsAg, and analyzed their correlations with conventional HBV marker levels. At baseline, serum O-glycosylated HBsAg levels were significantly correlated with HBV DNA, HBsAg, and hepatitis B-core related antigen (HBcrAg) levels. HBV DNA and O-glycosylated HBsAg levels were decreased after 48 weeks of NA therapy. The correlation between O-glycosylated HBsAg and HBsAg or HBcrAg levels was lost in patients who achieved undetectable HBV DNA. HBV DNA and RNA were detected in the O-glycosylated HBsAg-binding serum fraction, and the proportion of HBV RNA increased during NA therapy. In conclusion, serum O-glycosylated HBsAg levels change during NA therapy and may reflect combined serum HBV DNA and RNA virion levels, and an O-glycosylated HBsAg-based immunoassay may allow monitoring viral kinetics during NA therapy.

E2 Site Mutations in S Protein Strongly Affect Hepatitis B Surface Antigen Detection in the Occult Hepatitis B Virus

The mechanism of occult hepatitis B infection (OBI) has not yet been fully clarified. Our previous research found that novel OBI-related mutation within S protein, E2G, could cause the hepatitis B surface antigen (HBsAg) secretion impairment, which resulted in intracellular accumulation in OBI of genotype B. Here, to further explore the role of E2 site mutations in the occurrence of OBI, we analyzed these site mutations among 119 OBI strains identified from blood donors. Meanwhile, 109 wild-type HBV strains (HBsAg positive/HBV DNA positive) were used as control group. Furthermore, to verify the E2 site mutations, two conservative 1.3-fold full-gene expression vectors of HBV genotype B and C (pHBV1.3B and pHBV1.3C) were constructed. Then, the E2 mutant plasmids on the basis of pHBV1.3B or pHBV1.3C were constructed and transfected into HepG2 cells, respectively. The extracellular and intracellular HBsAg were analyzed by electrochemical luminescence and cellular immunohistochemistry. The structural characteristics of S proteins with or without E2 mutations were analyzed using relevant bioinformatics software. E2 mutations (E2G/A/V/D) existed in 21.8% (26/119) of OBIs, while no E2 mutations were found in the control group. E2G/A/V/D mutations could strongly affect extracellular and intracellular level of HBsAg (p < 0.05). Notably, unlike E2G in genotype B that could cause HBsAg intracellular accumulation and secretion decrease (p < 0.05), E2G in genotype C could lead to a very significant HBsAg decrease both extracellularly (0.46% vs. pHBV1.3C) and intracellularly (11.2% vs. pHBV1.3C) (p < 0.05). Meanwhile, for E2G/A mutations, the relative intracellular HBsAg (110.7–338.3% vs. extracellular) and its fluorescence intensity (1.5–2.4-fold vs. with genotype-matched pHBV1.3B/C) were significantly higher (p < 0.05). Furthermore, N-terminal signal peptides, with a typical cleavage site for peptidase at positions 27 and 28, were exclusively detected in S proteins with secretion-defective mutants (E2G/A). Our findings suggest that: (1) E2G/A/V/D mutations were confirmed to significantly influence the detection of HBsAg, (2) the underlying mechanism of OBI caused by E2G mutation is quite different between genotype B and genotype C, and (3) E2G/A could produce a N-terminal truncated S protein, which might attribute to the HBsAg secretion impairment in the OBIs.

Viral Hepatitis and Hepatocellular Carcinoma: State of the Art

Viral hepatitis is one of the main causes leading to hepatocellular carcinoma (HCC). The continued rise in incidence of HCC suggests additional factors following infection may be involved. This review examines recent studies investigating the molecular mechanisms of chronic hepatitis and its association with hepatocarcinogenesis. Hepatitis B virus patients with genotype C display an aggressive disease course leading to HCC more than other genotypes. Furthermore, hepatitis B excretory antigen (HBeAg) seems to be a more sensitive predictive tumor marker exhibiting a six-fold higher relative risk in patients with positive HBsAg and HBeAg than those with HBsAg only. Single or combined mutations of viral genome can predict HCC development in up to 80% of patients. Several mutations in HBx-gene are related with higher HCC incidence. Overexpression of the core protein in HCV leads to hepatocellular lipid accumulation associated with oncogenesis. Reduced number and decreased functionality of natural killer cells in chronic HCV individuals dysregulate their surveillance function in tumor and viral cells resulting in HCC. Furthermore, high T-cell immunoglobulin and mucin 3 levels supress CD8+ T-cells, which lead to immunological dysregulation. Hepatitis D promotes HCC development indirectly via modifications to innate immunity, epigenetic alterations and production of reactive oxygen species with the LHDAg being the most highly associated with HCC development. Summarizing the results, HBV and HCV infection represent the most associated forms of viral hepatitis causing HCC. Further studies are warranted to further improve the prediction of high-risk patients and development of targeted therapeutics preventing the transition from hepatic inflammation–fibrosis to cancer.

PSIX-29 Study of the association of mtDNA haplogroups and Ages of 100 days in pigs

The aim of the work was to determine the mtDNA haplogroups and assess their associations with Days_100 in pigs based on sequencing the D-loop region. The research was carried out on Landrace sows (n = 123). To amplification a fragment of mtDNA D-loop conducted PCR using the following primers: F5 ‘ - TGC AAA CCA AAA CGC CAA GT-3’ and R: 3 ‘ - TTT TTG GGG TTT GGC AAG GC-5. Statistical analyzes were performed using Linear mixed model fit by REML (‘lmerModLmerTest’). The studied group of pigs had the largest number of D haplogroups, which were definitely in 66 sows (53.7%). Haplogroup E was identified in 18 sows (14.6%). Among the haplogroups of Asian origin, which include A, B and C, only genotype C was present. Statistical differences for D100 were established between Hap_C and Hap_E. The presented results indicate the influence of mtDNA haplogroups on Days_100 and pigs of hapogroup E showed the best results compared to analogs of haplogroup E. This may be due to the fact that haplotype E is of European origin, and haplotype C is of Asian origin. Breeding commercial European pigs is focused on increasing the growth rate, and this significantly reduces pig keeping costs and increases production efficiency. It should be noted that changes in growth rate are associated with more intense metabolic processes where mitochondria play a significant role. This may be reflected at the genetic level being determined by the nucleotide sequence of mtDNAs and at the haplotype level in particular. This research was funded by Ministry of Science and Higher Education of the Russian Federation (0445-2021-0008).

Duffy blood system and g6pd genetic variants in p. Vivax malaria patients from manaus, amazonas, brazil

Background Over a third of the world’s population lives at risk of potentially severe Plasmodium vivax induced malaria. The unique aspect of the parasite’s biology and interactions with the human host make it harder to control and eliminate the disease. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Duffy-negative blood groups are two red blood cell variations shown to confer protection against malaria. Methods Molecular genotyping of G6PD and Duffy was performed in 225 patients with severe and non-severe malaria randomly admitted at a reference center for infectious disease from Manaus. For G6PD variants characterization of the variants, Real Time PCR (qPCR) was performed, while Duffy genotyping by PCR-RFLP. Results Of the 225 patients, 29 (12.94%) and 43 (19.19%) were carriers of the G6PD c.202G > A and c.376A > G, respectively. For the Duffy genotype (c.-67T > C in the GATA promoter region), 70 (31.11%) were phenotyped as Fy(a + b-), 98 (43.55%) Fy(a + b+), 56 (24.9%) Fy(a-b+) and 1 (0.44%) Fy(a-b-). The FY*01/FY*02 genotype was prevalent in both non-severe and severe malaria. In women, the FY*01/FY*02 allele occurred concomitantly with c.376A > G more frequently in non-severe malaria, while in men, this combination was predominant in severe malaria. Duffy phenotype Fy(a-b+) (p = 0.022) and genotypes FY*01/FY*01 / FY*02/FY*02 (p = 0.015) correlated with high parasitemia density before and after treatment. Conclusions Our results showed only one uncomplicated vivax malaria patient with Duffy phenotype Fy(a-b-). Heterozygous GATA variants did not confer protection against malaria infection in this study. Research on G6PD and Duffy antigen deficiencies has been valuable, particularly when focused on densely populated areas. Our results confirm possible genetic molding mechanisms in vivax malaria in our Amazon region and can help to improve the understanding of the relationship between G6PD deficiency and Duffy genotypes concomitantly in the protection or susceptibility to P. vivax infection. Molecular diagnosis before treatment may be necessary in the Amazonian population, regardless of the diagnosis of uncomplicated or severe malaria.

First report of Taenia laticollis and a new species, “Taenia sp. Eurasian lynx” from the Eurasian lynx (Lynx lynx) in Northwestern China

BackgroundThe Eurasian lynx (Lynx lynx) is a medium-sized wild cat species distributed throughout Eurasia, from Europe to the Far East. There has been no report on Taenia species (Cestoda: Cyclophyllidea) infecting this felid in China.MethodsIn this study, 24 tapeworms were found in two Eurasian lynxes (#1 and #2) in Xinjiang, northwestern China. The tapeworms were identified based on two mitochondrial genetic markers, the cytochrome c oxidase subunit 1 (cox1) and 16S rDNA. This was followed by detailed morphologic characterization.ResultsMolecular and phylogenetic analyses of the cox1 gene revealed that i) a single tapeworm from lynx #2 shared 100% identity with Taenia laticollis genotype C detected in Eurasian lynx in Finland, and ii) the remaining 23 tapeworms, provisionally named here as “Taenia sp. Eurasian lynx”, had two nucleotide substitutions but phylogenetically clustered together. The latter showed only 93.25% sequence identity to T. hydatigena from sheep (Ovis aries) in Slovakia. The scolex morphology is characteristic enough to distinguish “Taenia sp. Eurasian lynx” from other species of its genus.Conclusions“Taenia sp. Eurasian lynx” is a novel tapeworm species found in Eurasian lynx. In addition, T. laticollis was found in this wild felid for the first time in China. The intermediate hosts of T. laticollis and “Taenia sp. Eurasian lynx” should be explored in the near future.

Multilocus drug resistance mutation analysis in 148 HBV patients in northern Henan Province of China

Objectives: To analyze the genotypes and reverse transcriptase mutations gene sites in hepatitis B (HBV) patients in northern Henan Province from 2016 to 2018, which treated by tenofovir dipivoxil(TDF), Lbivudine(LDT), adefovir(ADV), lamivudine (LAM) and entecavir(ETV) analogues, to provide evidences for rational drug use and antiviral therapy for HBV patients. Methods: HBV patients serum DNA was extracted, specific primers were designed for YMDD mutation sites in conservative regions; DNA sequencing of the PCR products was carried out by PCR amplification combined with Dideoxygenation termination sequencing(DDT), and then analyzed genotype and reverse transcriptase mutations gene sites of HBV patients. Results: We collected 148 samples of HBV patients in northern Henan Province from 2016 to 2018; 97.97% of HBV patients were genotype C and 2.03% were genotype B; there are 46 cases of LAM and FTC resistance mutation, respectively; 28 cases of LDT resistance mutation, 10 cases of ADV resistance mutation, 8 cases of ETV resistance mutation, and 1 case of TDF resistance mutation were detected; the mutation rates of drug resistance was 43.45% in HBV patients with genotype C and 33.33% in HBV patients with genotype B. There was no significant difference in the distribution rate between the two genotypes (P>0.05). The most common mutations types were M204M|V and M204M|I. The M204M|V mutation type often occurred in combination of M204M|V+L180M|L+L173V|L and M204M|V+L180M|L and M204M|V+ L180M|L+V173V|M, while M204M|I mutation type was often occurred alone, M204M|V+L180M|L combination appeared alone or in combination. N236+A181 base substitution was the dominant site in ADV resistance. ETV resistance mutation was based on LAM and ETV resistance, mainly were T184 and S202 base substitution; and LDT resistance mutation was M204M|I. Conclusion: In this article, PCR and DDT DNA sequencing were used to detect multisite resistance mutations of HBV reverse transcriptase gene in HBV patients. It was found that most of the genotypes of HBV patients in northern Henan Province were genotype C, the combination of drug resistance mutations was complex, and the mutation rates of multidrug resistance genes was high. There was no significant difference in the distribution rate of HBV genotypes among the detected patients, those data may provide evidences for the detection and confirmation of HBV resistance and rational drug use in HBV patients in the region.

The Influence of rs1859168 Polymorphism on Serum Expression of HOTTIP and Its Target miR-615-3p in Egyptian Patients with Breast Cancer

Background: Polymorphisms of long noncoding RNAs are lately documented as hazardous factors for the development of numerous tumors. Furthermore, the evaluation of noncoding RNAs has emerged as a novel detector of breast cancer patients. We aimed to genotype the HOXA transcript at the distal tip (HOTTIP) rs1859168 and assess its relationship with the levels of the serum HOTTIP and its target miR-615-3p in patients with breast cancer (BC). Methods: One hundred and fifty-one patients with BC, 139 patients with fibroadenoma (FA), and 143 healthy participants were incorporated into the current study. The genotyping of rs1859168 and the measurements of the HOTTIP and miR-615-3p levels were assessed using quantitative real-time PCR. Results: We revealed a significant association between each of the CC genotypes, C allele, dominant and recessive models, and the increased risk of BC (p = 0.013, p < 0.001, p < 0.001, and p < 0.001, respectively) relative to the healthy controls. Similarly, the CC genotype, C allele, and recessive model were observed to be related to the increased incidence of BC with respect to FA (p < 0.001 for all). A significant upregulation of HOTTIP and a marked decrease of miR-615-3p were verified in patients with BC compared to each of the healthy individuals, patients with FA, and the non-BC group (healthy subjects + FA) (p < 0.001 for all). A significant negative correlation was demonstrated between the expression of HOTTIP and miR-615-3p in the serum of patients with BC. The HOTTIP expression was upregulated, while that of miR-615-3p was downregulated in patients with BC who carried the CC genotype with respect to those who carried the AA or AC genotypes (p < 0.05 for all). Conclusions: The genetic variants of rs1859168 are linked to an increased susceptibility to BC. Moreover, HOTTIP and miR-615-3p may be used as novel indicators and targets for the treatment of patients with BC.