VP1 Sequences Research Articles

Molecular Epidemiology and Clinical Features of Enteroviruses-Associated Hand, Foot, and Mouth Disease and Herpangina Outbreak in Zunyi, China, 2019.

Background: Hand, foot and mouth disease (HFMD) and herpangina (HA), two of the most common childhood infectious diseases, are associated with enteroviruses (EVs) infection. The aim of this study was to identify the molecular epidemiology of enterovirus causing HFMD/HA in Zunyi, China, during 2019, and to describe the clinical features of the cases.Methods: We collected the information on demographic and clinical characteristics, laboratory data of laboratory-confirmed EVs associated HFMD/HA cases in Zunyi Medical University Third Affiliated Hospital between March 1 and July 31, 2019. EV types were determined by either one-step real time RT-PCR or partial VP1 gene sequencing and sequence alignment. Phylogenetic analysis of CVA6, CVA2, and CVA5 were established based on the partial VP1 gene sequences by neighbor-joining method. Differences in clinical characteristics and laboratory results of the cases were compared among patients infected with the most prevalent EV types.Results: From 1 March to 31 July 2019, 1,377 EVs associated HFMD/HA inpatients were confirmed. Of them, 4 (0.3%, 4/1,377) were EV-A71-associated cases, 84 (6.1%, 84/1,377) were CVA16-associated cases, and 1,289 (93.6%, 1,289/1,377) were non-EV-A71/CVA16-associated cases. Of the randomly selected 372 non-EV-A71/CVA16 cases, EV types have been successfully determined in 273 cases including 166 HFMD and 107 HA cases. For HFMD cases, the three most common types were CVA6 (80.7%, 134/166), CVA2 (5.4%, 9/166) and CVA5 (3.0%, 5/166); similarly, for HA cases, the three most prevalent serotypes were CVA6 (36.5%, 39/107), CVA2 (21.5%, 23/107) and CVA5 (18.7%, 20/107). Phylogenetic analysis showed that subclade D of CVA5, and subclade E of CVA6 and CVA2 were predominant in Zunyi during the outbreak in 2019. Compared with the cases caused by CVA16, the incidence of high fever and severe infection associated with CVA2, CVA5, and CVA6 was higher.Conclusions: The recent HFMD/HA outbreak in Zunyi is due to a larger incidence of CVA6, CVA2, and CVA5. Novel diagnostic reagents and vaccines against these types would be important to monitor and control EV infections.

Organization of the Structural Protein Region of La Jolla Virus Isolated from the Invasive Pest Insect Drosophila suzukii.

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.

Infectious bursal disease in Nigeria: continuous circulation of reassortant viruses.

Outbreaks of infectious bursal disease (IBD), a highly contagious immunosuppressive disease of young chickens, are still reported globally despite vaccination efforts. This study investigated the genetic characteristics of infectious bursal disease virus (IBDV) from 26 reported outbreaks in 2019 in Nigeria. Nucleotide sequences of VP2 hypervariable (hvVP2) region (n=26) and VP1 (n=23) of Nigerian IBDVs were determined. Our results revealed the detection of reassortant strains with segment A related to very virulent IBDV (vvIBDV) having virulence marker (222A, 242I, 256I, 294I and 299S), whereas their segment B were closely related to previously detected IBDV strains having QEG substitution at positions 145-147. Phylogenetic analysis of the hvVP2 region revealed that all the Nigerian IBDV clustered with vvIBDV (genogroup 3) and were independent of the Asian/European lineage. Interestingly, in the hvVP2, all the viruses had a G-S substitution at residue 254. Additionally, one isolate had an A321T substitution at the PHI loop, which has been suggested to play a key role in antigenicity. Four of the viruses (Bauchi=3 and Plateau=1) had a unique A-T substitution at residue 144 on the VP1 region. We also observed a T174S substitution in nine of the Nigerian viruses from Bauchi and Plateau state that were not found in any outbreak viruses from Oyo and Akwa Ibom. This report demonstrates the circulation of reassortant strains in commercial and backyard poultry farms in Nigeria despite sustained vaccination efforts. Our data suggest that the Nigerian outbreak viruses have mutations that may affect antigenicity and contribute to antigenic drift.

Molecular surveillance of coxsackievirus A16 in southern China, 2008-2019.

A national surveillance system on hand, foot, and mouth disease (HFMD) was launched in 2008 in China. Since then, millions of HFMD cases have been reported each year, with enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), and coxsackievirus A6 (CV-A6) as the major causative pathogens. Long-term surveillance of viral infection rates and genetic changes is essential for understanding the disease epidemiology pattern. Here, we analyzed molecular surveillance data on CV-A16 covering a period of 12 years (2008-2019) in Guangdong, China, one of the regions reporting the largest number of HFMD cases. Full VP1 sequences of 456 strains were determined to examine the genetic diversity and changes in the distribution of CV-A16 variants. Our study revealed an irregular pattern of CV-A16 infections in Guangdong. Different from the cyclic epidemics observed in some Asia-Pacific regions, there was a continuously high CV-A16 infection rate from 2008 to 2014, and after a period of lower epidemic activity in 2015-2017, an upsurge of CV-A16 infection was observed in 2018-2019. Cocirculation of subgenotypes B1a and B1b was observed, but while subgenotype B1a was predominant from 2008 to 2012, it appears to have been replaced by B1b, which has circulated as the predominant subgenotype since 2013. Phylogenetic analysis showed that most of the circulating CV-A16 strains are endemic, with occasional transmission between neighboring regions. The re-emergence of B1a in 2016-2019 in Guangdong was likely the result of introduction(s) from Southeast Asia. These results highlight the importance of continuous molecular surveillance from different areas, which will improve our understanding of the origin of the epidemic and facilitate the development of strategies for HFMD disease control.

Detection of Human Sapoviruses in Sewage in China by Next Generation Sequencing.

Human sapovirus (SaV) is an important causative agent of nonbacterial gastroenteritis in humans. However, little is known about its circulation in China. To study the prevalence and diversity of human SaV genotypes circulating in eastern China, a 3-year environmental surveillance combined with next generation sequencing (NGS) technology was conducted. A total of 36 raw sewage samples were collected from January 2017 to December 2019 in Jinan and processed. Thirty-five (97.22%) samples were positive for human SaV genome in quantitative RT-PCR assay; 33 (91.67%) samples were positive in nested RT-PCR assay on partial capsid VP1 sequence and all amplicons were further analyzed separately by NGS. Among those, ten genotypes belonging to the genogroups of GI, GII, GIV, and GV were identified by NGS, including 4 major genotypes (GI.2, GI.1, GV.1 and GI.3) and 6 uncommon genotypes (GII.5, GII.1, GII.NA1, GII.3, GI.6 and GIV.1). A temporal switch of predominant genotype was observed from GI.2 to GI.1 around June 2019. Local and foreign sequences clustered together in some branches according to phylogenetic analysis, indicating frequent transmission of various lineages in different regions of the world. Environmental surveillance provides a comprehensive picture of human SaV in China. NGS-based environmental surveillance improves our knowledge on human SaV circulating in communities greatly and should be encouraged as a sensitive surveillance tool.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12560-021-09469-x.

Molecular Characterization of Coxsackievirus A24v from Feces and Conjunctiva Reveals Epidemiological Links.

Coxsackievirus A24 variant (CVA24v), the main causative agent of acute hemorrhagic conjunctivitis (AHC), can be isolated from both the eyes and lower alimentary tract. However, the molecular features of CVA24v in feces is not well-documented. In this study, we compared the VP1 and 3C sequences of CVA24v strains isolated from feces during AHC epidemics in Cuba in 1997, 2003, and 2008–2009 with those obtained from conjunctival swabs during the same epidemic period. The sequence analyses of the 3C and VP1 region of stool isolates from the three epidemics showed a high degree of nucleotide identity (ranging from 97.3–100%) to the corresponding conjunctival isolates. The phylogenetic analysis showed that fecal CVA24v isolates from the 1997 and 2003 Cuban outbreaks formed a clade with CVA24v strains isolated from conjunctival swabs in Cuba and other countries during the same period. There were three amino acid changes (3C region) and one amino acid change (VP1 region) in seven CVA24v strains isolated sequentially over 20 days from fecal samples of one patient, suggesting viral replication in the intestine. Despite these substitutions, the virus from the conjunctival swab and fecal samples were genetically very similar. Therefore, fecal samples should be considered as a reliable alternative sample type for the routine molecular diagnosis and molecular epidemiology of CVA24v, also during outbreaks of AHC.

Molecular epidemiological characteristics of echovirus 6 in mainland China: extensive circulation of genotype F from 2007 to 2018

Echovirus 6 (E6) is associated with various clinical diseases and is frequently detected in environmental sewage. Despite its high prevalence in humans and the environment, little is known about its molecular phylogeography in mainland China. In this study, 114 of 21,539 (0.53%) clinical specimens from hand, foot, and mouth disease (HFMD) cases collected between 2007 and 2018 were positive for E6. The complete VP1 sequences of 87 representative E6 strains, including 24 strains from this study, were used to investigate the evolutionary genetic characteristics and geographical spread of E6 strains. Phylogenetic analysis based on VP1 nucleotide sequence divergence showed that, globally, E6 strains can be grouped into six genotypes, designated A to F. Chinese E6 strains collected between 1988 and 2018 were found to belong to genotypes C, E, and F, with genotype F being predominant from 2007 to 2018. There was no significant difference in the geographical distribution of each genotype. The evolutionary rate of E6 was estimated to be 3.631 × 10-3 substitutions site-1 year-1 (95% highest posterior density [HPD]: 3.2406 × 10-3-4.031 × 10-3 substitutions site-1 year-1) by Bayesian MCMC analysis. The most recent common ancestor of the E6 genotypes was traced back to 1863, whereas their common ancestor in China was traced back to around 1962. A small genetic shift was detected in the Chinese E6 population size in 2009 according to Bayesian skyline analysis, which indicated that there might have been an epidemic around that year.

Genogrouping of Infectious Bursal Disease Viruses Circulating in Ethiopian Chickens: Proposal for Assigning Very Virulent Strains in the Country into New Sub Genogroup 3d.

IntroductionIn 2017 infectious bursal disease viruses (IBDVs) were reclassified into genogroups based on nature of clustering on a phylogenetic tree constructed using VP2 gene sequence data rather than according to their pathotype and/or antigenic types. Ethiopian IBD viruses were not reclassified according to the proposed genogrouping.MethodsIn order to genogroup the Ethiopian IBDVs, available VP2 gene sequences data together with reference strain sequences were retrieved from GenBank and genogrouped as recently recommended based on evolutionary tree reconstruction and determination of their clustering on the phylogenetic tree.ResultsThe Ethiopian IBDVs were grouped into genogroups 1 and 3 that antigenically represent classically virulent and very virulent IBDVs, respectively. The genogroup 1 IBDVs were clustered with the vaccine strain while the genogroup 3 viruses were clustered with four known viruses belonging to sub-genogroup 3a and sub-genogroup 3b. Almost half of the Ethiopian IBDVs reported did not cluster with the specific sub-groups of genogroup 3; rather, the isolates were clustered differently suggesting they deserve a different sub-genogroup tentatively proposed as 3d. The two genogroups observed based on clustering on a phylogenetic tree were supported by corresponding deduced amino acid changes in similar positions in VP2 sequences. In addition, virulence marker amino acid genes coupled with second major hydrophilic region (amino acid positions 314–325) were predicted in these sequences that could be responsible for the occurrence of IBD outbreaks.ConclusionA new sub-genogroup of IBDVs, 3d, were observed in the sequences that could be one of the reasons for the frequent occurrence of IBD outbreaks and questions the protective potential of the existing vaccine. To institute disease control in the country, the effectiveness of the vaccine in use needs to be assessed in vivo against both genogroups 1 and 3 viruses and all three sub-genogroup 3 viruses circulating in the country.

Molecular epidemiological analysis of a HFMD epidemic caused by CV-A6

Objective To identify the pathogen that caused the cluster of HFMD epidemic in a middle school of Fuxin City in September 2019, and to find out the homogeneity of the pathogen. Methods The samples were preliminary identified by real-time PCR. Partial VP1 sequences were amplified by nested PCR. The virus type was identified by sequencing and blast comparison. and the homology and phylogenetic analyes of VP1 region were performed by bioinformatics software with CV-A6 reference sequences. Results Samples of 5 cases were tested by real-time fluorescent PCR, and the results showed that the enterovirus universal nucleic acid was positive, and both CV-A16 and EV71 nucleic acids were negative. They were judged to be non-CV-A16 and non-EV71 enterovirus infection. Online BLAST comparison of partial nucleotide sequences of the enterovirus VP1 region showed that samples of 5 cases were all positive for CV-A6 enterovirus, The homology of VP1 nucleotide sequence among 5 samples were 100%. Phylogenetic analysis showed that 5 samples in this study belonged to the CV-A6 D3 subtype which was widely epidemic in China, and they were clustered with the dominant strains in recent years in Liaoning province. Conclusion This cluster of HFMD epidemic is induced by CV-A6. It is closely related to the prevalent CV-A6 strains in Liaoning province in recent years, and no significant variation is detected. 摘要：目的 对2019年9月辽宁省阜新市某中学一起手足口病聚集性疫情的未分型肠道病毒进行病原学鉴定, 分 析其种系发生情况。 方法 使用实时荧光PCR对标本进行初步病原学鉴定, 使用巢式PCR对未分型肠道病毒VP1区 部分核苷酸序列进行扩增和测序, BLAST比对确定肠道病毒型别, 利用生物信息学软件将VP1区全长序列与柯萨奇病 毒A6型(CV-A6)各亚型参考序列进行同源性和系统进化分析。 结果 5份标本经实时荧光PCR检测, 结果 显示肠道 病毒通用型核酸阳性 , CV-A16型和EV71型核酸均为阴性 , 判断为非CV-A16非EV71型肠道病毒感染。对肠道病毒 VP1区部分核苷酸序列进行在线BLAST比对 , 结果 显示5例患者标本均为CV-A6型肠道病毒阳性, 其VP1区核苷酸序 列同源性为100%。系统进化分析显示, 5例标本属于我国广泛流行的CV-A6 D3亚型 , 且与辽宁省近几年的优势流行 株在同一个进化树分支上。 结论 引起此次聚集性手足口病疫情的病原体为CV-A6型肠道病毒, 与近年来辽宁省流 行的CV-A6毒株亲缘关系密切, 未发生明显变异。

Genetic characterization of G12P[6] and G12P[8] rotavirus strains collected in six African countries between 2010 and 2014

BackgroundG12 rotaviruses were first observed in sub-Saharan Africa in 2004 and since then have continued to emerge and spread across the continent and are reported as a significant human rotavirus genotype in several African countries, both prior to and after rotavirus vaccine introduction. This study investigated the genetic variability of 15 G12 rotavirus strains associated with either P[6] or P[8] identified between 2010 and 2014 from Ethiopia, Kenya, Rwanda, Tanzania, Togo and Zambia.MethodsThe investigation was carried out by comparing partial VP7 and partial VP4 sequences of the African G12P[6] and G12P[8] strains with the available GenBank sequences and exploring the recognized neutralization epitopes of these strains. Additionally, Bayesian evolutionary analysis was carried out using Markov Chain Monte Carlo (MCMC) implemented in BEAST to estimate the time to the most recent ancestor and evolutionary rate for these G12 rotavirus strains.ResultsThe findings suggested that the VP7 and VP4 nucleotide and amino acid sequences of the G12 strains circulating in African countries are closely related, irrespective of country of origin and year of detection, with the exception of the Ethiopian strains that clustered distinctly. Neutralization epitope analysis revealed that rotavirus VP4 P[8] genes associated with G12 had amino acid sequences similar to those reported globally including the vaccine strains in RotaTeq and Rotarix. The estimated evolutionary rate of the G12 strains was 1.016 × 10− 3 substitutions/site/year and was comparable to what has been previously reported. Three sub-clusters formed within the current circulating lineage III shows the diversification of G12 from three independent ancestries within a similar time frame in the late 1990s.ConclusionsAt present it appears to be unlikely that widespread vaccine use has driven the molecular evolution and sustainability of G12 strains in Africa. Continuous monitoring of rotavirus genotypes is recommended to assess the long-term impact of rotavirus vaccination on the dynamic nature of rotavirus evolution on the continent.

Molecular survey of parvovirus, astrovirus, coronavirus, and calicivirus in symptomatic dogs.

Gastrointestinal disorders caused by enteric viruses are frequently reported in dogs worldwide, with significant mortality rates in unvaccinated individuals. This study reports the identification and molecular characterization of Canine parvovirus (CPV-2), Canine coronavirus (CcoV), Canine astrovirus (AstV), and Canine calicivirus (CcaV) in a panel of dogs showing severe enteric clinical signs sampled in a typical Mediterranean environment (Sardinia, Italy). At least one of these viral species was detected in 92.3% samples. CPV-2 was the most frequently detected virus (87.2%), followed by AsTv (20.5%), CCoV-IIa (18%), and CCoV-I (10.3%). CCoV-IIb and CaCV were not detected in any sample. Single infection was detected in 24 samples (66.7%), mainly related to CPV-2 (91.7%). Coinfections were present in 33.3% samples with constant detection of CPV-2. Canine coronavirus was present only in coinfected animals. The VP2 sequence analysis of CPV-2 positive samples confirmed the presence of all variants, with CPV-2b most frequently detected. Phylogeny based on the CcoV-IIa spike protein (S) gene allowed to identify 2 different clades among Sardinian isolates but failed to distinguish enteric from pantropic viruses. Study on presence and prevalence of enteroviruses in dogs increase our knowledge about the circulation of these pathogens in the Mediterranean area and highlight the need for dedicated routine vaccine prophylaxis. Molecular analyses of enteric viruses are fundamental to avoid failure of vaccines caused by frequent mutations observed in these enteroviruses.

Molecular characterization of VP2 and VP3 proteins of Chicken Anemia Virus isolates in Turkey

Chicken anemia virus (CAV) has a circular 2.3 kb DNA genome encoding VP1, VP2, and VP3 proteins. VP1 protein is assembled into viral capsid. VP2 is a nonstructural protein required for the correct assembly of VP1. Also, there is an interaction between VP2 and VP3 protein, known as apoptin. Mostly, VP1 protein of CAV isolates have been investigated in previous studies but the information about VP2 and VP3 proteins is insufficient. The aim of this study is molecular characterization of VP2 and VP3 proteins from 20 CAV isolates previously collected in different regions of Turkey using in silico tools. The open reading frames (ORFs) for VP2 and VP3 were determined to be 651 and 366 base pairs (bp) encoding 216 and 121 amino acids, respectively. Nucleotide substitutions were identified at 21 and 14 positions for VP2 and VP3, respectively. Three isolates had one apiece amino acid substitutions in VP2 sequences. The MW and pI values for VP2 were predicted as 24.08?24.138 kDa and 6.96?8.35, respectively. Moreover, seven isolates had one to three amino acid substitutions for VP3 protein. The MW and pI values for VP3 were calculated as 13.188-13.372 kDa and 9.44-9.95, respectively. The secondary structures were predicted and phylogenetic trees were constructed for VP2 and VP3 proteins. As a conclusion, molecular characterization of VP2 and VP3 proteins from 20 CAV isolates contributes to the current knowledge on these proteins having diverse functions and immunological potentials.

Molecular characterization and the first full sequencing genome of chicken infectious anemia virus (CIAV) in Iran.

The chicken infectious anemia virus (CIAV) is an important pathogen that causes severe immunosuppression in young chickens. The study aims to characterize the genotype and full-length sequencing of CIAV strains in Iran. First, the collected thymus samples were investigated by conventional PCR for CIAV detection. Second, one of the CIAV positive samples (UT-Zahraee) was chosen for full genome sequencing. Throughout 2017, we detected 13 CIAVs isolated from 40 broiler flocks of different provinces of Iran. A comparison of the complete sequences of the genome and homologies of the nucleotides revealed that UT-Zahraee had a high similarity with American and Egyptian CIAV isolates. Moreover, VP1 sequence analysis showed that UT-Zahraee shared high homology with previously reported Iranian CIAV strains, Chinese, and Egyptian isolates. This study is the first report of full genome sequencing of CIAV strain from Iran. It will be beneficial to understand better the epidemiology and molecular characteristics of CIAV circulating in Iran.

Detection and molecular characteristics of bovine rotavirus A in dairy calves in China.

BackgroundBovine group A rotavirus (BoRVA) is a major cause of severe gastroenteritis in newborn dairy calves. Only one study has investigated the G and P genotypes among dairy calves in a few regions of China, which were G6 and P[5]. Therefore, data on the prevalence and molecular characteristics of BoRVA in dairy calves in China remains limited.ObjectivesThe purpose of this study was to investigate the prevalence and molecular characteristics of BoRVA in dairy calves in China.Methods269 dairy calves diarrheic samples from 23 farms in six provinces in China were collected to detect BoRVA using reverse transcription polymerase chain reaction.Results71% of samples were determined to be BoRVA-positive. Two G genotypes (G6, G10) and two P genotypes (P[1], P[5]) were identified, and G6P[1] BoRVA was the predominant strain. Moreover, the VP7 and VP4 gene sequences of these dairy calf BoRVA strains revealed abundant genetic diversity. Interestingly, eight out of 17 complete G6 VP7 sequences were clustered into G6 lineage VI and analysis showed the strains were closely related to Chinese yak BoRVA strains.ConclusionsThe results of this study show that BoRVA circulates widely among dairy calves in China, and the dominant genotype in circulation is G6P[1], first report on molecular characteristics of complete P[5] VP4 genes in chinese dairy calves. These results will help us to further understand the prevalence and genetic evolution of BoRVA among dairy calves in China and, thus, prevent the disease more effectively.

VP2 Gene-Based Molecular Evolutionary Patterns of Major Circulating Bluetongue Virus Serotypes Isolated during 2014–2018 from Telangana and Andhra Pradesh States of India

Introduction: Bluetongue disease is an economically important viral disease of livestock caused by bluetongue virus (BTV) having multiple serotypes. It belongs to the genus Orbivirus of family Reoviridae and subfamily Sedoreovirinae. The genome of BTV is 10 segmented dsRNA that codes for 7 structural and 4 nonstructural proteins, of which VP2 was reported to be serotype-specific and a major antigenic determinant. Objective: It is important to know the circulating serotypes in a particular geographical location for effective control of the disease. The present study unravels the molecular evolution of the circulating BTV serotypes during 2014–2018 in Telangana and Andhra Pradesh states of India. Methods: Multiple sequence alignment with available BTV serotypes in GenBank and phylogenetic analysis were performed for the partial VP2 sequences of major circulating BTV serotypes during the study period. Results: The multiple sequence alignment of circulating serotypes with respective reference isolates revealed variations in antigenic VP2. The phylogenetic analysis revealed that the major circulating serotypes were grouped into eastern topotypes (BTV-1, BTV-2, BTV-4, and BTV-16) and Western topotypes (BTV-5, BTV-12, and BTV-24). Conclusion: Our study strengthens the need for development of an effective vaccine, which can induce the immune response for a range of serotypes within and in between topotypes.

N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles.

Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on the sequence of the major capsid protein VP1, further enhance this public threat. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells were characterized in detail using a set of biophysical and structural tools. We used native mass spectrometry, gas-phase electrophoretic mobility molecular analysis, and proteomics to get clear insights into particle size, structure, and composition, as well as stability. Generally, noroviruses have been known to form mainly T = 3 particles. Importantly, we identified a major truncation in the capsid proteins as a likely cause for the formation of T = 1 particles. For vaccine development, particle production needs to be a reproducible, reliable process. Understanding the underlying processes in capsid size variation will help to produce particles of a defined capsid size presenting antigens consistent with intact virions. Next to vaccine production itself, this would be immensely beneficial for bio-/nano-technological approaches using viral particles as carriers or triggers for immunological reactions.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens

ObjectiveTo develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. MethodsA panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 per μL and copies per μL, and the newly established methods were tested in clinical specimens collected in recent years. ResultsThe sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 per μL and 10 virus copies per μL, and for the complete VP1 gene was 1 CCID50 per μL and 100 virus copies per μL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons. ConclusionThis RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Full-length genome sequencing of a very virulent infectious bursal disease virus isolated in Tunisia

Infectious bursal disease (IBD), an acute, highly contagious, and immunosuppressive avian disease, is caused by infectious bursal disease virus (IBDV) and constitutes one of the main threats to the poultry industry, worldwide. This study was performed to isolate and characterize IBDV isolates circulating in Tunisia. Eleven collected bird samples were identified using an SYBR Green–based one-step real-time reverse transcriptase polymerase chain reaction. The full-length genome sequencing of 7 of the 11 IBDV isolates has been realized. VP2 gene data showed limited sequence variations for all the 7 tested samples. The few nucleotide changes were silent and the deduced amino acid sequences were identical with the exception of a unique and characteristic nonsilent mutation (C1203) detected for the TN37/19 isolate, with a change of amino acid (L) to (F) at position 401. In addition, the serine-rich heptapeptide SWSASGS, characteristic of virulent IBDV, as well the amino acid residues, conserved in most very virulent IBDV (vvIBDV) strains, were detected in all the Tunisian tested isolates. Nucleotide sequences of VP5 gene revealed the presence of 5 substitutions leading to changes in the amino acid sequences of the virus. Two of these mutations were unique and characteristic of the Tunisian isolates. Besides, the alternative AUG start codon, characteristic of vvIBDV, was observed in all obtained VP5 gene sequences. The Tunisian protein sequences of VP1 showed E242 and the TDN triplet at positions 145, 146, and 147, a motif specific of vvIBDV. Phylogenetic analyses of the 5 genes confirmed the sequence alignment results and showed that the Tunisian strains are closely related to the very virulent Algerian IBDV strains.

Natural infection of free-ranging mandrills (Mandrillus sphinx) by enteroviruses and astroviruses in southern Gabon

Enteroviruses (Picornaviridae) and astroviruses (Astroviridae) cause various diseases in humans and animals, including in non-human primates (NHPs). Some enteroviruses and astroviruses detected in NHPs are genetically related to those infecting humans, indicating the occurrence of interspecies transmissions. In this study, we screened 200 fecal samples of 56 free-ranging mandrills (Mandrillus sphinx) by nested reverse transcription-PCR with primers targeting the VP1 and RdRp genes, to evaluate the diversity of enterovirus and astrovirus infection, respectively, and the associated zoonotic risk. Overall, ten samples from six mandrills were enterovirus-positive (5%), and three samples from three mandrills were astrovirus-positive (1.5%). This is the first evidence of astrovirus infection in mandrills. Phylogenetic analyses based on the VP1 sequences revealed that all ten enterovirus sequences were part of the species Enterovirus J, suggesting low zoonotic risk. Phylogenetic analysis of the three astrovirus sequences showed that they all belonged to the Mamastrovirus genus. Two astrovirus sequences were highly divergent from all human astrovirus sequences (63.4–73% nucleotide identity), while one sequence (AstV-5) suggested cross-species transmission from humans to mandrills. Additional studies are needed to better characterize the identified astroviruses and to confirm whether mandrills are host of astroviruses than can be transmitted to humans.

Multiple Introductions and Predominance of Rotavirus Group A Genotype G3P[8] in Kilifi, Coastal Kenya, 4 Years after Nationwide Vaccine Introduction.

Globally, rotavirus group A (RVA) remains a major cause of severe childhood diarrhea, despite the use of vaccines in more than 100 countries. RVA sequencing for local outbreaks facilitates investigation into strain composition, origins, spread, and vaccine failure. In 2018, we collected 248 stool samples from children aged less than 13 years admitted with diarrheal illness to Kilifi County Hospital, coastal Kenya. Antigen screening detected RVA in 55 samples (22.2%). Of these, VP7 (G) and VP4 (P) segments were successfully sequenced in 48 (87.3%) and phylogenetic analysis based on the VP7 sequences identified seven genetic clusters with six different GP combinations: G3P[8], G1P[8], G2P[4], G2P[8], G9P[8] and G12P[8]. The G3P[8] strains predominated the season (n = 37, 67.2%) and comprised three distinct G3 genetic clusters that fell within Lineage I and IX (the latter also known as equine-like G3 Lineage). Both the two G3 lineages have been recently detected in several countries. Our study is the first to document African children infected with G3 Lineage IX. These data highlight the global nature of RVA transmission and the importance of increasing global rotavirus vaccine coverage.