Genotype D4 Research Articles

Measles outbreak in the sanitary area of Guadalajara (Spain): difficulty in microbiological diagnosis in the era of its elimination

IntroductionIn Spain, like in other countries where endemic measles has been eliminated, there is a need for available diagnostic tolos for confirming any cases in order to prevent and control its transmission. We describe the different microbiological tests used for the diagnosis of measles during an outbreak that occurred in 2019 in the province of Guadalajara (Spain). MethodsSerological and molecular tests were performed at the Microbiology laboratory of the Guadalajara University Hospital and at the National Center for Microbiology of the Carlos III Health Institute (Majadahonda, Spain). Patient data were obtained from the surveillance system. ResultsA total of 43 patients had a laboratory diagnosis of measles: 29 cases by PCR (pharyngeal exudate or urine) and positive specific IgM, 11 cases by PCR, and 3 cases only by a positive IgM. Genotype D8 was identified in 35 confirmed cases and genotype A in two that were discarded as post-vaccination cases. PCR was positive in the acute sera of 11 out of 14 patients with a negative IgM. Eleven confirmed cases had recieved one or two vaccine doses. Twelve adult patients were hospitalizated, all of them with a diagnostic of hepatitis. ConclusionsThe combination of molecular tests and the presence of specific IgM is necessary for a correct diagnosis of measles and also to classify patients with a breakthrough infection or vaccine failures (primary or secondary). Genotyping is essential for the correct classification of the patients in the context of a measles elimination program.

Frequency of Hepatitis B Markers in Systemic Lupus Erythematosus Patients in Iran.

Background:Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. Hepatitis B virus is the causative agent for chronic, acute, cirrhosis, and hepatocellular carcinoma. SLE patients with chronic or occult hepatitis B infection undergoing immunosuppressive drugs may become reactive and develop fatal hepatitis. Therefore, this study was conducted to determine HBV markers in SLE patients before the administration of immunosuppressive drugs in Ahvaz city, Iran. Materials and methods:The sera of 92 SLE patients were tested for HBs Ag and anti-HBc using ELISA, HBV DNA (by Nested PCR) testes. Real-time PCR was performed for the patients with positive anti-HBc and negative HBsAg. The positive HBV DNA samples were checked for HBV genotype and HBV subtypes. Results: Among the 92 SLE patients, three (3.3%) were males and 89 (96.7%) females . The patients’ ages ranged from 14 to 70 years [mean age of 38.9±10.1]. Three of 92 (3.26%) subjects [2/3 males and 1/89 female] were positive for HBsAg, anti-HBc Ab, and HBV DNA detected with PCR (p=0.000003)]. Five of 89 (5.61%) subjects [1 male and 4/88 females were only positive for anti-HBc and negative for HBs Ag, HBV DNA(PCR) using Real-time PCR (p=0.05). The results of the nucleotide data and phylogenetic tree showed all three HBV patients were genotype D1. The results of amino acid sequencing revealed all three HBV patients were HBV subtype ayw2. Conclusion:This study proved that 3.26% of SLE patients were positive for overt HBV infection (positive for anti-HBc, HBsAg and HBV-DNA using PCR). All the three isolated HBV were genotype D1 and subtype ayw2. The fact that 5.61% of the patients were only positive for anti-HBc characterized the occult hepatitis B infection (OBI) although further investigation is needed. To prevent HBV or OBI reactivation for SLE patients before immunosuppression treatment, HBV markers including anti-HBc, HBsAg, HBV-DNA should be implemented using PCR and Real-time PCR .

Characterization of Vaccine Breakthrough Cases during Measles Outbreaks in Milan and Surrounding Areas, Italy, 2017-2021.

Despite the existence of an effective live-attenuated vaccine, measles can appear in vaccinated individuals. We investigated breakthrough measles cases identified during our surveillance activities within the measles/rubella surveillance network (MoRoNet) in Milan and surrounding areas (Northern Italy). Between 2017 and 2021, we confirmed measles virus (genotypes B3 or D8) infections in 653 patients and 51 of these (7.8%) were vaccinees. Among vaccinated individuals whose serum was available, a secondary failure was evidenced in 69.4% (25/36) of cases while 11 patients (30.6%) were non-responders. Non-responders were more frequently hospitalized and had significantly lower Ct values in both respiratory and urine samples. Median age and time since the last immunization were similar in the two groups. Importantly, we identified onward transmissions from vaccine failure cases. Vaccinees were involved in 20 outbreaks, in 10 of them they were able to transmit the virus, and in 8 of them, they were the index case. Comparing viral hemagglutinin sequences from vaccinated and non-vaccinated subjects did not show a specific mutation pattern. These results suggest that vaccination failure was likely due to the poor immune response of single individuals and highlights the importance of identifying breakthrough cases and characterizing their clinical and virologic profiles.

Mind your Ps: A probabilistic model to aid the interpretation of molecular epidemiology data.

SummaryBackgroundAssessing relatedness of pathogen sequences in clinical samples is a core goal in molecular epidemiology. Tools for Bayesian analysis of phylogeny, such as the BEAST software package, have been typically used in the analysis of sequence/time data in public health. However, they are computationally-, time-, and knowledge-intensive, demanding resources that many laboratories do not have available or cannot allocate frequently.MethodsTo evaluate a faster and simpler alternative method to support the routine interpretation of sequence data for epidemiology, we obtained sequences for two regions in the measles virus genome, N-450 and MF-NCR, from patient samples of genotypes B3, D4 and D8 taken between 2011 and 2017 in the UK and Romania. A mathematical model incorporating time, possible shared ancestry and the Poisson distribution describing the number of expected substitutions at a given time point was developed to exclude epidemiological relatedness between pairs of sequences. The model was validated against the commonly used Bayesian phylogenetic method using an independent dataset collected in 2017–19.FindingsWe demonstrate that our model, using time and sequence information to predict whether two samples may be related within a given time frame, minimises the risk of erroneous exclusion of relatedness. An easy-to-use implementation in the form of a guide and spreadsheet is provided for convenient application.InterpretationThe proposed model only requires a previously calculated substitution rate for the locus and pathogen of interest. It allows for an informed but quick decision on the likelihood of relatedness between two samples within a time frame, without the need for phylogenetic reconstruction, thus facilitating rapid epidemiological interpretation of sequence data.FundingThis work was funded by the United Kingdom Health Security Agency (UKHSA). The World Health Organization European Regional Office funded Aurora Fernández-García and Mihaela Lazar training visits to UKHSA.

Molecular identification and characterization of measles virus genotypes circulating in Afghanistan, 2008-2018.

The measles virus (MV) remains a leading cause of morbidity and mortality in children under 5 years of age. Molecular identification of circulating wild-type MV) strains is a vital component of the measles elimination program. We received 159 oral swab samples from Afghanistan during 2008-2018. Viral RNA was extracted, followed by one-step RT-PCR and positive amplicons were subject to sequencing for genotype identification. Out of 159 total samples, 52% (83/159) were detected positive by RT-PCR. Genotype D4 was identified from 2.4% (2/83), genotype H1, 4.8% (4/83),and genotype B3, 92.7% (77/83) cases, respectively.

Decreased hepatitis B virus vaccine response among HIV-positive infants compared with HIV-negative infants in Botswana.

We sought to determine vaccine antibody titres and the prevalence of hepatitis B surface antigen (HBsAg) in both HIV-positive and HIV-negative infants born to HIV-positive mothers in Botswana. This was a retrospective cross-sectional study using 449 archived dried blood spot samples from both HIV-positive and HIV-negative infants collected between 2016 and 2018. We screened dried blood spot samples for HBsAg and determined hepatitis B surface antibody titres. We determined hepatitis B virus (HBV) genotypes by amplifying 415 base-pairs of the surface region. HIV-positive infants mounted a significantly lower immune response to the HBV vaccine (P < 0.001). Furthermore, a lower proportion of HIV-positive infants had protective hepatitis B surface antibody titres (74.5%) than HIV-negative infants (89.2%) (P < 0.001). HIV-positive infants were older and 50.9% of them had completed vaccination (P = 0.018). Of the 449 infant samples tested, three (0.67%) were positive for HBsAg. Of the three HBsAg-positive infants, two had protective titres (>10 mIU/ml). Two of the three HBV-positive infants were infected with genotype D3 and had no drug-resistance or escape mutations. Vaccine response was lower among HIV-positive infants compared with HIV-negative infants. HBV infections were observed in both HIV-positive and HIV-negative infants in Botswana. Studies to investigate additional preventive strategies to reduce HBV mother-to-child transmission are recommended.

Епідемічні реалії кору на Тернопільщині

Кір є інфекцією, керованою засобами вакцинації, що продовжує залишатись величезною соціально-економічною проблемою для Тернопільщини. У роботі проаналізовано матеріали звітної документації Головного управління Держсанепідслужби та дитячих інфекційних стаціонарів у Тернопільській області. Захворюваність на кір, що фіксувалася за час незалежності країни, значно перевищувала загальноукраїнські показники, зберігала типову для довакцинального періоду періодичність епідемологічних підйомів. В усі проаналізовані пікові періоди діти шкільного віку були найбільш вразливою щодо кору віковою категорією. Виявлено стійку тенденцію до зміщення захворюваності на старший шкільний вік та в бік дорослого населення. Серед захворілих фіксувався високий відсоток щеплених. Лабораторно підтверджено появу вірусу кору генотипу D4 під час останнього спалаху захворювання в області. Аналіз спалахів кору відображає потребу у поглибленні вивчення особливостей становлення поствакцинального протикорового імунітету в сучасних умовах з урахуванням потреби в дослідженні певних імунологічних феноменів.

Investigation of Hepatitis C Virus (HCV) Genotypes by "Reverse Hybridisation Strip Assay" and DNA Sequence Analysis Methods

Hepatitis C Virus (HCV) is a virus that is estimated to infect approximately 185 million people worldwide and causes 350 000 deaths each year. The target in HCV treatment is the sustained virological response (SVR), and the most important virological factors determining SVR are genotype and baseline HCV-RNA levels. In this study, it was aimed to compare the HCV genotypes and subtypes detected by the "line probe assay (LIPA)" method with sequence analysis. HCV genotypes and subtypes were investigated by line probe assay (LIPA) (NLM analytica, Italy) and sequence analysis methods in 212 patients with chronic HCV diagnosis and with HCV RNA viral load of 10⁴ IU/ml and above. 5'UTR and core regions were studied in LIPA method and NS5B gene region was studied in the sequencing method. After amplifying 340 bp region in the NS5B gene region with the hemi-nested PCR method, sequence analysis (ABI 3500 Genetic Analyzer [Applied Biosystems, USA]) was performed. Statistical analysis of the study was determined by using TURCOSA Analytics program. In the study, HCV genotypes and subtypes that were determined in 212 patients by LIPA method were; 40 (18.8%) genotype 1a, 97 (45.75%) genotype 1b, 7 (3.3%) genotype 2, 12 (5.6%) genotype 2a/c, 2 (0.94%) genotype 2b, 19 (8.96%) genotype 3, 16 (7.55%) genotype 4, 1 (0.47%) genotype 4a, 15 (7.5%) genotype 4c/d, 1 (0.47%) genotype 4h and 2 (0.94%) genotype 5. The HCV genotypes and subtypes determined in 212 patients by sequence analysis were; 20 (9.43%) genotype 1a, 118 (55.6%) genotype 1b, 16 (7.55%) genotype 2a, 4 (1.89%) genotype 2b, 1 (0.47%) genotype 2k, 21 (9.91%) genotype 3a, 2 (0.94%) genotype 4a, 28 (13.21%) genotype 4d and 2 (0.94%) genotype 5a. The difference in results between the two methods was found to be statistically significant (p< 0.001). According to the viral load-genotype relationship, the highest viral load was detected in genotype 1a patients (p< 0.001). In conclusion, in order to determine HCV genotypes and subtypes in our study, LIPA and sequence analysis methods were compared and the genotype compatibility between the two methods were determined as 97.5% on the basis of genotype and 19% on the basis of subtype. Since the direct-acting antiviral (DEA) treatment protocol in chronic HCV patients is planned according to the genotype/subtype determination, the inconsistency of the results obtained in the LIPA method, which is routinely widely used, with the sequence analysis method is remarkable, and it was concluded that these results should be supported by studies containing more samples.

The Emergence of Hepatitis C Virus Genotype 4d Among Human Immunodeficiency Virus-Infected Patients in Mexico City: A Molecular Epidemiological Study.

The recent detection of hepatitis C virus genotype 4 infection in human immunodeficiency virus-infected patients prompted performing molecular characterization of these isolates. All the Mexican isolates belonged to a subcluster within the 4d group and shared a common ancestor with a French isolate. The estimated timing of introduction in Mexico City was as recent as December 2015.

Surveillance, Epidemiology and Impact of EV-A71 Vaccination on Hand, Foot, and Mouth Disease in Nanchang, China, 2010-2019.

After the first national-scale outbreak of Hand, foot, and mouth disease (HFMD) in China, a national surveillance network was established. Here we described the epidemiology and pathogenic profile of HFMD and the impact of EV-A71 vaccination on pathogen spectrum of enteroviruses in the southeastern Chinese city of Nanchang during 2010–2019. A total of 7,951 HFMD cases from sentinel hospitals were included, of which 4,800 EV-positive cases (60.4%) were identified by real-time RT-PCR. During 2010–2012, enterovirus 71 (EV-A71) was the main causative agent of HFMD, causing 63.1% of cases, followed by 19.3% cases associated with coxsackievirus A16 (CV-A16). Since 2013, the proportion of other enteroviruses has increased dramatically, with the sub genotype D3 strain of Coxsackievirus A6 (CV-A6) replacing the dominance of EV-A71. These genetically diverse native strains of CV-A6 have co-transmitted and co-evolved in Nanchang. Unlike EV-A71 and CV-A16, most CV-A6 infections were concentrated in autumn and winter. The incidence of EV-A71 infection negatively correlated with EV-A71 vaccination (r = −0.990, p = 0.01). And severe cases sharply declined as the promotion of EV-A71 vaccines. After 2-year implementation of EV-A71 vaccination, EV-A71 is no longer detected from the reported HFMD cases in Nanchang. In conclusion, EV-A71 vaccination changed the pattern of HFMD epidemic, and CV-A6 replaced the dominance of EV-A71 over time.

Measles and rubella in the North-West of Russia in period of elimination

Aim: To evaluate the epidemic situation on measles and rubella and to carry out the laboratory diagnostics of these infections in the North-West of Russia in the period of elimination.Materials and Methods. In 2016-2019 a total of 534 blood serum samples from patients with measles diagnosis, 210 – from patients with rubella diagnosis and 1251 – from patients with exanthema were studied by ELISA using “VectoMeasles-IgM” and “EIA-Rubella-IgM” test-systems.Results. Measles incidence in NWR varied from 0 to 0.96 per 100,000 in 2016 and 2019, respectively. Among measles patients the proportion of children and adults changed in 2018-2019, adults constituted 65.9%. Patients not vaccinated against measles and those with the unknown vaccination status were mainly involved in epidemic process (85%). High level of patients with revaccination status was revealed in 2018 (18.4%). These patients demonstrated high levels of IgG (&gt;3.0 IU/ml) as well as high percentage of IgG avidity (&gt;94.3%) on 4-7th day of rash onset thus evidencing secondary immune response. Molecular studies of the samples from patients revealed the circulation of D8 and B3 genotypes of measles virus. Strains of D8 and B3 genotypes were represented by 3 and 4 variants, respectively. Rubella incidence did not exceed 0.13 per 100,000. Strains of rubella virus of genotype 1E were detected.Conclusion. The data of annual measles incidence and molecular studies of circulating wild type measles viruses prove the on-going measles elimination process in the NorthWest of Russia. For rubella, the persistent phase of elimination was demonstrated in the region.

Genetic Analysis of the Measles Virus From the Outbreaks in South Korea, 2019.

Three genotypes (B3, D8, and H1) of the measles virus (MeV) have recently caused global outbreaks. In Korea, four measles outbreaks were reported during 2018–2019 and most patients were infants and health care workers in their 20s and 30s. To investigate the genetic characteristics and molecular epidemiology of the outbreaks, we analyzed the sequence of MeVs by targeting the N-450, MF-NCR, and/or H gene regions. Considering their phylogenetic relationships, besides the N-450 and MF-NCR sequences that are commonly used for genotyping MeVs, the MF-NCR-H sequence was related to the dynamics for identifying the transmission of MeVs. Phylogenetic clustering patterns reconstructed from the MF-NCR-H sequence set revealed that genotype D8 caused three of the four outbreaks, while B3 seemed to have induced the fourth outbreak. These results suggest that the MF-NCR-H sequence is useful for rapid confirmation of measles outbreaks and to identify the epidemiological routes of MeVs.

Co-detection of the measles vaccine and wild-type virus by real-time PCR: public health laboratory protocol.

In rare cases vaccination with the measles virus vaccine genotype A (MeVA) may cause a vaccine reaction with clinical signs similar to infection with wild-type measles virus (MeVwt). Rapid differentiation between MeVA and MeVwt infection is important for taking adequate public health measures. Recently, a few MeVA real-time reverse-transcription quantitative PCR methods (RT-qPCRs) were described that can distinguish between MeVA and MeVwt. However, detection of MeVA does in theory not exclude infection with MeVwt. In the present study, we established a protocol for determination of co-infections with MeVA and MeVwt. To this end, MeVA RT-qPCRs were used in combination with the routine measles virus (MeV) RT-qPCR, and the results suggested that the differences between the RT-qPCR Ct values (delta Ct, ∆Ct) could be used as criteria. Subsequently, we tested samples from vaccine-associated measles cases that were confirmed by genotyping. In addition, experimental mixtures of MeVA and MeVwt were tested in different concentrations. All tested MeVA clinical samples had ∆Ct ≤3.6. The results of experimental mixtures showed a mean ∆Ct ≤2.8 for genotype A alone and >3.2 when combined with either genotype B3 or D8. The results of a receiver operator characteristic analysis indicated that the optimum ∆Ct for use as a cut-off value was 3.5, while with ∆Ct values of 2.9 and 3.7 sensitivity and specificity were respectively 1.00. Thus, ∆Ct could be used to exclude the presence of MeVwt if MeVA is detected and ∆Ct is <2.9, while ∆Ct >3.7 were highly suggestive of co-infection and ≥2.9 ∆Ct <3.7 warranted additional confirmation, such as next-generation sequencing. This RT-qPCR-based protocol could be used for the exclusion of infection with MeVwt in cases with vaccine-associated measles reaction, crucial for the timely implementation of public health prevention and control measures.

Biological Characterization of Hepatitis B virus Genotypes: Their Role in Viral Replication and Antigen Expression.

Hepatitis B virus (HBV) inter-host evolution has resulted in genomic diversification reflected in the existence of nine genotypes (A-I) and numerous subgenotypes. There is growing evidence that genotypes influence HBV natural history, clinical outcomes, and treatment response. However, the biological characteristics underlying these differences have not yet been established. By transfecting HuH-7 cells with unit-length constructs of genotypes A2, B2, C1, D1, and F1b, we identified major differences in HBV replicative capacity and antigen expression across genotypes. Genotypes B2 and F1b showed a 2-fold increase in cccDNA levels compared to the other genotypes (p<0.005). Genotype A2 expressed the lowest pgRNA levels, with a 70-fold decrease in relation to the other genotypes (p<0.0001), while genotype B2 showed the lowest Precore RNA levels, with a 100-fold reduction compared to genotype A2 (p<0.0001). The highest intracellular HBV DNA levels were observed for genotype B2 and the lowest for genotypes A2 and C1 (p<0.0001). Regarding antigen expression, genotype F1b secreted the highest HBsAg levels and genotype D1 the lowest (p<0.0001), while genotypes A2 and B2 showed the highest intracellular HBsAg levels (p<0.0001). Interestingly, genotype C1 secreted the highest HBeAg levels, while genotype A2 showed the highest intracellular levels (p<0.0001). Finally, the analysis of the intra/extracellular antigen ratios revealed that most genotypes retained intracellularly 5–20% of the antigens, except the genotype A2 that retained 50% of the total expressed antigens. In conclusion, this study provides new insights into the biological characteristics of HBV genotypes, being the first study to comparatively analyze European (A and D) and Asian (B and C) genotypes with the Latin American (F) genotype. The differences in HBV replication and antigen expression might contribute to understand the differential role of genotypes in pathogenesis.

Amino acid substitutions in core and HBsAg regions of hepatitis B virus in patients with monoinfection and HBV/HIV-coinfection in the Republic of Guinea

The aim of this study was to characterize the genetic variants of HBV currently circulating in the Republic of Guinea, based on the nucleotide sequences of the complete virus genome, and to analyze clinically significant mutations in the Core and HBsAg regions during HBV monoinfection and HBV/HIV coinfection.Materials and methods. The study material was represented by 2616 blood serum samples collected from residents of the Republic of Guinea. The subjects were examined for the presence of HBV markers with a qualitative detection of HBsAg, HBs IgG, and HBCore IgG. HBV complete genome nucleotide sequences were obtained for 298 samples including HIV/HBV coinfected patients. Amplification and subsequent sequencing of HBV were performed using nested PCR with pair’s overlapping primers jointly flanking the complete HBV genome (S, P, C, X genes).Results. HBV serological markers were detected in 80.77% samples, while HBsAg was detected in 16.01% of the examined group. HBV DNA we detected in 22.36%. The prevalence of HBsAg-negative HBV in patients with HIV RNA is 45.16%, which is significantly higher than 6.07% found in the group without HIV infection. Phylogenetic analysis of HBV in the examined samples showed that HBV genotype E (75.5%) predominates in the group compared to HBV genotype D1 (9.39%), D2 (4.02%), D3 (6.37%), and A2 (4.7%). In the tested group, the variability of amino acids among the HBV samples was higher in the PreCore/Core region than in the PreS1/PreS2/S region. SHB mutations were detected in 83,89%, Core mutations in 94.29%, PreCore amino acid substitutions in 16.77% of the patients, respectively.The results obtained in this work demonstrate a high prevalence of HBV in the region and indicate the need for further largescale studies of HBV mutations in order to improve strategies for disease control and prevention in the Republic of Guinea.

Brote de sarampión en el área sanitaria de Guadalajara (España): dificultad en el diagnóstico microbiológico en la era de su eliminación

IntroductionIn Spain, like in other countries where endemic measles has been eliminated, there is a need for available diagnostic tools for confirming any cases in order to prevent and control its transmission. We describe the different microbiological tests used for the diagnosis of measles during an outbreak that occurred in 2019 in the province of Guadalajara (Spain). MethodsSerological and molecular tests were performed at the Microbiology laboratory of the Guadalajara University Hospital and at the National Center for Microbiology of the Carlos III Health Institute (Majadahonda, Spain). Patient data were obtained from the surveillance system. ResultsA total of 43 patients had a laboratory diagnosis of measles: 29 cases by PCR (pharyngeal exudate or urine) and positive specific IgM, 11 cases by PCR, and 3 cases only by a positive IgM. Genotype D8 was identified in 35 confirmed cases and genotype A in 2 that were discarded as post-vaccination cases. PCR was positive in the acute sera of 11 out of 14 patients with a negative IgM. Eleven confirmed cases had recieved one or 2 vaccine doses. Twelve adult patients were hospitalizated, all of them with a diagnostic of hepatitis. ConclusionsThe combination of molecular tests and the presence of specific IgG and IgM are necessary for a correct diagnosis of measles and also to classify patients with a breakthrough infection or vaccine failures (primary or secondary). Genotyping is essential for the correct classification of the patients in the context of a measles elimination program.

Infection courses, virological features and IFN-α responses of HBV genotypes in cell culture and animal models

HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment. Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model. HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. Herein, we generated stable cell lines producing HBV of various genotypes that are infectious invitro and invivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models. Stable cell lines producing high-titer cell culture-generated hepatitis B virus (HBV) of various genotypes were established. HBV genotypes showed stable infectivity in both invitro and invivo models, which are valuable tools for antiviral development.

Epidemiological and genetic characterization of measles virus circulating strains at Marseille, France during 2017–2019 measles outbreak

We attempted to establish a molecular investigation by Next Generation sequencing of the measles virus (MeV) strains circulating in Marseille-France during the last outbreak that occurred between 2017 and 2019. The circulating MeV were isolated from clinical samples using cell culture method and whole genomes were sequenced by Illumina Miseq Next Generation. Genotyping and comparative analyses were assessed by phylogenetic reconstructions. Clinical and epidemiological data from cases were also recorded. A total of 110MeV strains were isolated in cell culture. Our analysis based on whole genome sequences of 98 isolates confirmed that 93 strains belonged to the genotype D8 and 5 to the genotype B3. Phylogenetic analyses revealed 4 distinct MeV circulating clones in Marseille. Measles mostly occured in children <5 years-old and in adults 30-50 years-old. Measles infection also occurred in 2 adequately vaccinated cases (2 doses). Among 63 measles cases of whom we had available clinical data informations, a total of 35 patients were hospitalized and 19 developed complications including one death case recorded. Whole Genome Sequencing seems to be a useful tool for more refined genomic characterization of large measles outbreak. Vaccination strategies for measles eradication need to be re-evaluated in the current context.

Genome Sequences of Measles Virus D4 and D8 Genotypes from India.

ABSTRACTThe genomes of 15 measles viruses isolated in 2006 to 2017 from patients <16 years of age with fever and skin rashes from four states and two union territories of India were sequenced. Study genomes were phylogenetically analyzed using 143 Indian and global genomes. The study reconfirms two lineages of D4 isolates and three lineages of D8 isolates from India.

Surveillance and genetic characteristics of imported cases of measles virus of D8 genotype in Hebei province

Objective: To understand the epidemiological and genotypic characteristics of imported cases of measles virus of D8 genotype in Hebei province. Methods: Epidemiological investigation of measles cases in surveillance was carried out. The throat swabs of the measles cases in acute phase were collected for real time RT-PCR identification, measles virus culture and genotype identification. Results: A total of 36 imported measles cases of genotype D8 were detected. The cases were mainly distributed in 8 counties of Handan city. Number of confirmed measles cases in Cheng'an county was the highest, accounting for 58.33% (21/36) of all the reported cases. All patients had fever and rash, and 55.55% (20/36) of the cases were under 2 years old and 86.11% (31/36) of the cases had no immunization history. The children with pneumonia accounted for 44.12% (15/34) of the total children with D8 genotype measles virus infection. The nucleotide and amino acid homologies between the imported measles virus genotype D8 and the WHO reference strain of D8 genotype (D8-Manchester.UNK/30.94) were 98.4%-98.6% and 97.3%, respectively. Compared with the strains of H1 genotype in China, the nucleotide and amino acid homologies were 92.8%-93.1% and 93.3%, respectively. Conclusions: The imported cases of measles virus of D8 genotype might have caused local transmission in Hebei province. Molecular epidemiological surveillance for measles virus needs to be further strengthened. It is necessary to detect and control the epidemic early and improve the coverage rate and timely rate of measles vaccination. It is also important to prevent cross infection in hospitals.