Epidemiology of Chikungunya virus isolates 2016-2018 in Pakistan.

The chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which has infected millions of people in Africa, Asia, Americas, and Europe since it remerged in India and Indian Ocean regions in 2005-2006. The purpose of this study was to evaluate the genetic diversity and evolutionary changes in CHIKV from 2016 to 2018 in Pakistan. Blood specimens were collected and processed following the Centers for Disease Control and Prevention Trioplex Protocol. Sequencing and phylogenetic analysis of complete coding sequence of representative isolates from the CHIKV outbreak was carried out during December 2016 to July 2018, a total of 1549 samples were received, out of which 50% (n = 774) were found positive for CHIKV RNA. Mean age of chikungunya positive patients was 31.8 ± 15.7 years and most affected were between 21 and 40 years of age. The Pakistan CHIKV strains clustered with the Indian Ocean sublineage of East/Central/South African with cocirculation of some variants In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Key substitutions in the neutralizing epitopes site and a few changes indicative of adaptive to other insect cells were also detected in Pakistani strains. This study provides the emerging trend of CHIKV in the country for early identification of potential variants of high virulence and preventive measures for vector borne disease especially in the endemic areas.

SEC14L2 enables pan-genotype HCV replication in cell culture.

Since its discovery in 1989, efforts to grow clinical isolates of the hepatitis C virus (HCV) in cell culture have met with limited success. Only the JFH-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations. We hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. To identify the missing factors, we transduced Huh-7.5 human hepatoma cells with a pooled lentivirus-based human complementary DNA (cDNA) library, transfected the cells with HCV subgenomic replicons lacking adaptive mutations, and selected for stable replicon colonies. This led to the identification of a single cDNA, SEC14L2, that enabled RNA replication of diverse HCV genotypes in several hepatoma cell lines. This effect was dose-dependent, and required the continuous presence of SEC14L2. Full-length HCV genomes also replicated and produced low levels of infectious virus. Remarkably, SEC14L2-expressing Huh-7.5 cells also supported HCV replication following inoculation with patient sera. Mechanistic studies suggest that SEC14L2 promotes HCV infection by enhancing vitamin E-mediated protection against lipid peroxidation. This provides a foundation for development of in vitro replication systems for all HCV isolates, creating a useful platform to dissect the mechanisms by which cell culture-adaptive mutations act.

Innate Immune Responses in Serum and Cerebrospinal Fluid From Neonates and Infants Infected With Parechovirus-A3 or Enteroviruses.

BACKGROUND: Parechovirus (PeV)-A3 and enteroviruses (EV) are the most common viruses causing sepsis and meningoencephalitis in neonates and young infants. Clinical manifestations of PeV-A3 infection are more severe than those of EV infection, and no pleocytosis with a positive polymerase chain reaction (PCR) result for PeV-A3 in cerebrospinal fluid (CSF) are characteristic findings. We hypothesized that innate immune responses to PeV-A3 and EV are distinct in serum and CSF. METHODS: We evaluated 22 cytokines/chemokines in serum and CSF from PeV-A3- or EV-infected patients younger than 4 months in Niigata, Japan, from 2015 through 2018. Infection was diagnosed with real-time PCR followed by sequencing. Febrile neonates and infants with sepsis-like syndrome who had negative bacterial culture and viral PCR for both PeV-A and EV were also included (non-PeV-A/EV patients). RESULTS: Among 192 febrile patients, we evaluated 16 PeV-A3-infected, 15 EV-infected, and 8 non-PeV-A/EV patients. Serum pro-/anti-inflammatory cytokine/chemokine levels were higher in PeV-A3-infected patients than in EV-infected patients (P < .02). Although most cytokine/chemokine were elevated in CSF from EV-infected patients, levels were low or undetectable in PeV-A3-infected and non-PeV-A/EV patients (P < .001). CONCLUSIONS: Distinct cytokine/chemokine patterns in serum and CSF may explain the different clinical manifestations of PeV-A3-infected and EV-infected neonates and young infants.

Aptamer-Based Field-Effect Transistor for Detection of Avian Influenza Virus in Chicken Serum.

Early diagnosis of the highly pathogenic H5N1 avian influenza virus (AIV) is significant for preventing and controlling a global pandemic. However, there is no existing electrical biosensor for detecting biomarkers for AIV in clinically relevant samples such as chicken serum. Herein, we report the first use of an aptamer-functionalized field-effect transistor (FET) as a label-free sensor for AIV detection in chicken serum. A DNA aptamer is employed as a sensitive and selective receptor for hemagglutinin (HA) protein, which is a biomarker for AIVs. This aptamer is immobilized on a gold microelectrode that is connected to the gate of a reusable FET transducer. The specific binding of the target protein results in a change in the surface potential, which generates a signal response of the FET transducer. We hypothesize that a conformational change in the DNA aptamer upon specific binding of HA protein may alter the surface potential. The signal of the aptamer-based FET biosensor increased linearly with the increase in the logarithm of HA protein concentration in a dynamic range of 10 pM to 10 nM with a detection limit of 5.9 pM. The selectivity of the biosensor for HA protein was confirmed by employing relevant interfering proteins. The proposed biosensor was successfully applied to the selective detection of HA protein in a chicken serum sample. Owing to its simple and low-cost architecture, portability, and sensitivity, the aptamer-based FET biosensor has potential as a point-of-care diagnosis of H5N1 AIVs in clinical samples.

Complement-Mediated Neutralization of a Potent Neurotropic Human Pathogen, Chandipura Virus, Is Dependent on C1q.

Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens, including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae, is a deadly human pathogen known to cause fatal encephalitis, especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and, thus, is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum, we have demonstrated that CHPV neutralization is C3, C4, and C1q dependent and independent of factor B, suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches, we showed (i) a direct association of C1q to CHPV, (ii) deposition of complement proteins C3b, C4b, and C1q on CHPV, and (iii) virus aggregation. Depletion of C8, an important component of the pore-forming complex of complement, had no effect on CHPV, further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV.IMPORTANCE Chandipura virus is a clinically important human pathogen of the Indian subcontinent. The rapidity of death associated with CHPV infection in addition to the absence of an effective vaccine or therapeutics results in poor clinical prognosis. The biology of the virus and its interaction with the host immune system, including the complement system, are understudied. Our investigation reveals the susceptibility of CHPV to fluid phase complement and also dissects the pathway involved and the mechanism of virus neutralization. Direct binding of C1q, an important upstream component of the classical pathway of complement to CHPV, and the strong dependency on C1q for virus neutralization highlight the significance of identifying such interactions to better understand CHPV pathogenesis and devise strategies to target this deadly pathogen.

Mx1 in Hematopoietic Cells Protects against Thogoto Virus Infection.

Myxovirus resistance 1 (Mx1) is an interferon-induced gene that encodes a GTPase that plays an important role in the defense of mammalian cells against influenza A and other viruses. The Mx1 protein can restrict a number of viruses independently of the expression of other interferon-induced genes. Mx genes are therefore considered to be an important part of the innate antiviral immune response. However, the possible impact of Mx expression in the hematopoietic cellular compartment has not been investigated in detail in the course of a viral infection. To address this, we performed bone marrow chimera experiments using congenic B6.A2G Mx1+/+ and B6.A2G Mx1-/- mice to study the effect of Mx1 expression in cells of hematopoietic versus nonhematopoietic origin. Mx1+/+ mice were protected and Mx1-/- mice were susceptible to influenza A virus challenge infection, regardless of the type of bone marrow cells (Mx1+/+ or Mx1-/- ) the animals had received. Infection with Thogoto virus, however, revealed that Mx1-/- mice with a functional Mx1 gene in the bone marrow compartment showed reduced liver pathology compared with Mx1-/- mice that had been grafted with Mx1-/- bone marrow. The reduced pathology in these mice was associated with a reduction in Thogoto virus titers in the spleen, lung, and serum. Moreover, Mx1+/+ mice with Mx1-/- bone marrow failed to control Thogoto virus replication in the spleen. Mx1 in the hematopoietic cellular compartment thus contributes to protection against Thogoto virus infection.IMPORTANCE Mx proteins are evolutionarily conserved in vertebrates and can restrict a wide range of viruses in a cell-autonomous way. The contribution to antiviral defense of Mx1 expression in hematopoietic cells remains largely unknown. We show that protection against influenza virus infection requires Mx1 expression in the nonhematopoietic cellular compartment. In contrast, Mx1 in bone marrow-derived cells is sufficient to control disease and virus replication following infection with a Thogoto virus. This indicates that, in addition to its well-established antiviral activity in nonhematopoietic cells, Mx1 in hematopoietic cells can also play an important antiviral function. In addition, cells of hematopoietic origin that lack a functional Mx1 gene contribute to Thogoto virus dissemination and associated disease.

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes.

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

Detection of novel coronavirus (SARS-CoV-2) RNA in peripheral blood specimens.

The latest outbreak of pneumonia caused by SARS-CoV-2 presents a significant challenge to global public health and has a major impact on clinical microbiology laboratories. In some situations, such as patients in coma condition, the oropharyngeal or nasopharyngeal sampling is seldom feasible, and blood sampling could be an alternative. In the current article, a comprehensive literature search has been conducted for detecting coronavirus disease 2019 (COVID-19) using plasma or serum samples. To date, twenty-six studies have used SARS-CoV-2 nucleic acid in plasma or serum (RNAaemia) to diagnose COVID-19. The pros and cons are discussed in this article. While the detection of SARS-CoV-2 viral load in respiratory specimens is commonly used to diagnose COVID-19, detecting SARS-CoV-2 RNA in plasma or serum should not lose sight and it could be considered as an alternative diagnostic approach.

Alternative pathway of complement activation has a beneficial role against Chandipura virus infection.

The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/"tick-over" mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.

In-depth serum virome analysis in patients with acute liver failure with indeterminate etiology.

In clinical virome research, whole-genome/transcriptome amplification is required when starting material is limited. An improved method, named "template-dependent multiple displacement amplification" (tdMDA), has recently been developed in our lab (Wang et al. in BioTechniques 63:21-25. https://doi.org/10.2144/000114566, 2017). In combination with Illumina sequencing and bioinformatics pipelines, its application in virome sequencing was explored using a serum sample from a patient with chronic hepatitis C virus (HCV) infection. In comparison to an amplification-free procedure, virome sequencing via tdMDA showed a 9.47-fold enrichment for HCV-mapped reads and, accordingly, an increase in HCV genome coverage from 28.5% to 70.1%. Eight serum samples from acute patients liver failure (ALF) with or without known etiology were then used for virome sequencing with an average depth at 94,913x. Both similarity-based (mapping, NCBI BLASTn, BLASTp, and profile hidden Markov model analysis) and similarity-independent methods (machine-learning algorithms) identified viruses from multiple families, including Herpesviridae, Picornaviridae, Myoviridae, and Anelloviridae. However, their commensal nature and cross-detection ruled out an etiological interpretation. Together with a lack of detection of novel viruses in a comprehensive analysis at a resolution of single reads, these data indicate that viral agents might be rare in ALF cases with indeterminate etiology.

Development of Dengue Virus Serotype-Specific NS1 Capture Assays for the Rapid and Highly Sensitive Identification of the Infecting Serotype in Human Sera.

Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes, we have applied an innovative approach for the generation and selection of serotype-specific anti-NS1 mAbs. To elicit mAbs against conformational epitopes, NMRI mice were immunized with living HEK 293 transfectants expressing the native target Ags in multiple display on the cell surface. For each serotype, three different NS1 sequence variants were sequentially used for immunization of mice, hybridoma selection, and capture assay development, respectively. Selection of optimal combinations of capturing and detecting mAbs yielded highly sensitive and specific NS1 serotyping ELISAs (st-ELISAs) for the four serotypes. st-ELISA testing of 41 dengue patient sera showed a 100% concordance with the serotype determined by serotype-specific reverse transcriptase real-time quantitative PCR. The respective NS1 variants could be detected for ∼10 d after the onset of illness. Ab-dependent enhancement of DENV infections may be associated with a specific range of pre-existing anti-DENV serological Ab titers. Testing of patient sera with the developed st-ELISAs will not only be useful for epidemiological studies and surveillance, but it may also help to develop and validate assays that can distinguish protective versus enhancing Ab responses for risk assessment for the development of severe dengue disease in individual patients.

High genetic diversity and recombination events of porcine astrovirus strains identified from ill and asymptomatic pigs in 2017, Hunan Province, China.

Astroviruses (AstV) are associated with enteric and systemic disease in mammals and birds. Astroviruses have received increased attention recently as they have been found to be associated with sporadic neurologic disease in mammals including humans. In pigs, porcine astrovirus (PoAstV) can be widely detected and has been grouped in five genotypes (PoAstV1 to PoAstV5). In the present study, we detected multiple PoAstVs in serum samples, nasal swabs, and fecal swabs collected from pigs suffering from respiratory disease or diarrhea but also from asymptomatic pigs, indicating a wide tissue tropism of the identified PoAstV genotypes. Coinfection of different genotypes in the same pig was commonly observed, and within an individual pig a high genetic diversity was observed for viruses belonging to the same PoAstV genotype. Two complete genomes of PoAstV2-WG-R2/2017 and PoAstV4-WG-R2/2017 were successfully obtained and characterized, with genome sizes of 6396 and 6643 nucleotides, respectively. The PoAstV2-WG-R2/2017 genome showed identities of 67.2-77.4% to other known PoAstV2 genomes, and the PoAstV4-WG-R2/2017 genome showed identities of 72.8-80.5% to other known PoAstV4 genomes. The predicted spike domain of open reading frame 2 (ORF2) of these strains showed the highest genetic heterogeneity, with amino acid identities of 13.7-70.9% for PoAstV2-WG-R2/2017 to other known PoAstV2 strains, and identities of 24.4-63.3% for the PoAstV4-WG-R2/2017 to other known PoAstV4 strains. Possible recombination events were identified in each of the two sequences. Two subclades of PoAstV2 and three subclades of PoAstV4 were defined in the present analyses. The obtained data provide further evidence for extraintestinal infectivity of PoAstVs, and confirmed the high genetic diversity of PoAstVs and the coinfection potential of different PoAstV types in a single pig.

Characterization of the viral genomes present in commercial batches of horse serum obtained by high-throughput sequencing.

Horses are often used as blood donors for commercial horse serum (HS) production and to manufacture biologicals. HS is an alternative for fetal bovine serum (FBS) used as a supplement for cell culture and vaccine production. Furthermore, HS is also frequently obtained in order to produce antisera toxins and pathogens. The advent of high-throughput sequencing (HTS) has promoted changes in virus detection, since previous knowledge of targets is not required. Thus, the present study aimed to describe the virome of five different batches of commercial HS from New Zealand (three batches) and Brazil and the United States (one batch each) using HTS. Each HS pool were processed and sequenced using an Illumina MiSeq platform. Sequences-related to viruses belonging to the Flaviviridae, Herpesviridae, and Parvoviridae families were detected. Particularly, equine hepacivirus (EqHV), equine pegivirus (EPgV), and Theiler's disease-associated virus (TDAV) were more frequent found in the batches analyzed. The presence of viral genomes in cell culture sera illustrates that these commercial sera can contain a mixture of different viruses and, therefore, can be regarded as potentially infectious for susceptible hosts. Moreover, the innocuity of commercial HS is important for the efficiency and security of diagnostics and the production of biological products.

Genotypic analysis of S segment of Crimean-Congo hemorrhagic fever virus in Turkey.

The Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne virus, whose viral RNA consists of S, L, and M segments. The virus is migrating through the ticks with animals and migratory birds and the geographical distribution can be investigated based on genetic analysis. To better understand the connection between the seropositivity and the mortality rate, the key factor could be the temporal and spatial analysis of the different strains. In this study, serum samples (n = 26) were selected from CCHFV RNA-positive patients and subjected to sequence analysis of the gene regions encoding the S segments. According to the neighbor-joining analysis, the obtained partial sequences were linked to the European strain. The strains were closely related to Turkey-Kelkit06, Turkey 200310849 viruses, and viruses from Russia and Kosovo. The comparison with previously analyzed isolates from the GenBank showed 95%-99% sequence similarity. The isolates in phylogenetic branches were divided into two groups. AST, platelet, and APTT levels were found significantly higher in Group 2 compared to Group 1. Nucleotide differences can be prognostic factor in CCHF disease. Increasing CCHF cases not originating from local isolates were circulating strains imported from different neighboring countries of Turkey. The results show new evidence to the emerging threat of the CCHF disease.

Detection of novel tick-borne pathogen, Alongshan virus, in Ixodes ricinus ticks, south-eastern Finland, 2019.

The newly identified tick-borne Alongshan virus (ALSV), a segmented Jingmen virus group flavivirus, was recently associated with human disease in China. We report the detection of ALSV RNA in Ixodes ricinus ticks in south-eastern Finland. Screening of sera from patients suspected for tick-borne encephalitis for Jingmen tick virus-like virus RNA and antibodies revealed no human cases. The presence of ALSV in common European ticks warrants further investigations on its role as a human pathogen.

Human immunodeficiency virus in cadavers: A review.

Millions of people are infected with human immunodeficiency virus (HIV); however, limited research focuses on post-mortem HIV detection. Post-mortem HIV testing is vital because medical records are not always available, and the HIV status can be unknown. The aims of this study were to review the available literature and determine the most efficient HIV test for post-mortem samples, the optimal tissue or bodily fluid to be tested, and the duration that HIV remains reliably detectable. A literature search was conducted using PubMed and Google Scholar. Terms were related to HIV (HIV detection, HIV testing, HIV prevalence) and deceased individuals (post-mortem, cadaver, deceased, organ donor). Inclusion criteria included English studies, or articles with at least an English abstract, while review articles were excluded. From this literature search, 43 studies were applicable. These studies most commonly used enzyme-linked immunosorbent assay and Western blot as screening and confirmation tests, respectively. As for the optimal tissue or bodily fluid, serum remained the golden standard, while testing skin seemed promising. HIV remains detectable in the body up to 58 days after death, although few studies tested samples after 48 h. Knowledge of the HIV status can be beneficial in the case of accidental exposure and can create a range of possible research opportunities on the effects of HIV in different organ systems. This review outlined several gaps in the current literature and future studies should investigate these gaps because this information can be relevant to numerous professions. Clin. Anat. 32:603-610, 2019. © 2019 Wiley Periodicals, Inc.

Serological Evidence for the Circulation of Ebolaviruses in Pigs From Sierra Leone.

Many human ebolavirus outbreaks have been linked to contact with wildlife including nonhuman primates and bats, which are assumed to serve as host species. However, it is largely unknown to what extent other animal species, particularly livestock, are involved in the transmission cycle or act as additional hosts for filoviruses. Pigs were identified as a susceptible host for Reston virus with subsequent transmission to humans reported in the Philippines. To date, there is no evidence of natural Ebola virus (EBOV) infection in pigs, although pigs were shown to be susceptible to EBOV infection under experimental settings. To investigate the potential role of pigs in the ecology of EBOV, we analyzed 400 porcine serum samples from Sierra Leone for the presence of ebolavirus-specific antibodies. Three samples reacted with ebolavirus nucleoproteins but had no neutralizing antibodies. Our results (1) suggest the circulation of ebolaviruses in swine in Sierra Leone that are antigenically related but not identical to EBOV and (2) could represent undiscovered ebolaviruses with unknown pathogenic and/or zoonotic potential.

PREDICTIVE VALUE OF SERUM IL-17A AND IP-10 FOR EVALUATION OF LIVER FIBROSIS PROGRESSION IN PATIENTS WITH HBV/HIV CO-INFECTION.

The problem of HBV and HCV infections in addition to the HIV-infection in sub-Saharan African countries remains important due to the high prevalence and mortality after fast progressing fibrogenesis and development of hepatocellular carcinoma. Despite of the large number of investigations on diagnostics and prediction of the disease course, the exact role of the proinflammatory influence of IP-10 and IL-17A on the fibrogenesis during HIV/HBV-co-infection is still unknown. The aim of the study was to investigate IP-10 and IL-17A concentration in blood serum among HIV/HBV patients to consider their potential role in improvement of diagnostics of liver fibrosis progression. 53 HIV/HBV patients of Lewanika General Hospital (West Zambia) and 21 healthy blood donors were checked for serological markers, liver biopsy and IP-10, IL-17A in blood serum. The obtained results were analyzed by statistical package SPSS 12.0. Mean IP-10 was 753,6 pg/ml among HIV/HBV co-infected patients with F3-4 and it was reliably higher than in F1-2 patients and healthy responders (р=0,005). This group had also higher level of IL-17A (37,54 pg/ml) than comparison groups (р=0,032). We found out strong correlation between increasing IP-10 (r=0,6), IL-17A (r=0,52) and fibrotic severity (р<0,05). High IP-10, IL-17A amount increases the risk of F3-4 formation in HIV/HBV patients.

Comprehensive Molecular Approach for Characterization of Hepatitis E Virus Genotype 3 Variants.

Autochthonous hepatitis E virus genotype 3 (HEV-3) infections in industrialized countries are more frequent than previously assumed. HEV-3 is zoonotic and the causal pathogen of chronic hepatitis E. According to the latest classification of the family Hepeviridae, 10 designated HEV-3 subtypes (HEV-3a to HEV-3j) and 7 unassigned HEV-3 subtypes are proposed. In order to identify and characterize the HEV-3 variants in circulation, we developed a molecular approach combining a sensitive HEV-specific real-time reverse transcription-PCR (RT-PCR) targeting the overlapping region of HEV ORF2 and ORF3 (the ORF2/3 region) and two newly designed consensus nested RT-PCRs targeting the HEV ORF1 and ORF2 genes, respectively. Since complete genome sequences are required for new HEV-3 subtype assignment, we implemented a straightforward approach for full-length HEV-3 genome amplification. Twenty-nine human serum samples and six human feces samples from chronic hepatitis E patients were selected for evaluation of the system. Viral loads ranged from 1 × 104 to 1.9 × 1010 copies/ml of serum and from 1.8 × 104 to 1 × 1012 copies/g of feces. Sequence and phylogenetic analyses of partial ORF1 and ORF2 sequences showed that HEV strains had considerable genetic diversity and clustered into the HEV-3c (29/35), HEV-3e (2/35), HEV-3f (2/35), and unassigned HEV-3 (2/35) subtypes. Moreover, from these strains, three full-length HEV-3 genome sequences were generated and characterized. DE/15-0030 represents a typical HEV-3c strain (95.7% nucleotide identity to wbGER27), while DE/15-0031 and SW/16-0282 have <89.2% homology to known HEV-3 strains and are phylogenetically divergent, indicating novel HEV-3 subtypes. In summary, our approach will significantly facilitate the detection, quantification, and determination of HEV-3 strains and will thus help to improve molecular diagnostics and our knowledge of HEV diversity and evolution.

Natural history of serum HBV-RNA in chronic HBV infection.

Virus-like particles encapsulating HBV-RNA represent a serum biomarker for assessing viral replication activity in clinical practice. However, baseline levels of serum HBV-RNA and their associations with viral replicative intermediates and liver disease in phases of chronic hepatitis B remain unknown. In this cross-sectional study, 102 patients were categorized into immune-tolerant (IT), HBeAg-positive immune active (HBeAg+IA), inactive carrier (IC) and HBeAg-negative immune active (HBeAg-IA) phases. HBV-RNA in serum samples and in 66 paired liver biopsies were quantified and correlated with serum ALT levels, histopathological scores and the levels of other viral replicative intermediates. Mean levels of serum HBV-RNA differed among phases, with the highest levels among IT (6.78 ± 0.83 log10 copies mL-1 ) patients, followed by HBeAg+IA (5.73 ± 1.16 log10 copies mL-1 ), HBeAg-IA (4.52 ± 1.25 log10 copies mL-1 ) and IC (2.96 ± 0.40 log10 copies mL-1 ) patients. Serum HBV-RNA levels correlated with HBV DNA in all phases, although correlations with other viral replicative intermediates weakened or disappeared when cases were stratified into phases. Distinct compositions of viral products were found among phases: the ratio of HBsAg to serum HBV-RNA was highest in IC patients, while the ratio of serum HBV-RNA to intrahepatic HBV-RNA and the ratio of intrahepatic HBV-DNA to intrahepatic HBV-RNA were significantly higher in IT patients. In conclusion, baseline levels of HBV-RNA and the composition of viral replicative intermediates differ significantly across the natural course of chronic HBV infection. These findings shed light on the nature of viral replication and pathogenesis of disease among different phases of chronic HBV infection.