Viral Protein Gene Research Articles

Prevalence of Human Parvovirus B19 and Associated Risk Factors in Women with Bad Obstetric History in Babylon Province, Iraq

Abstract Background: Parvovirus infection is an important cause of fetal loss during all three trimesters of pregnancy. Objectives: This study was conducted to investigate the prevalence, susceptibility rate, and risk factors of B19-V in women aged 15 to 45 in the Al-Hamza region of Babil, Iraq, considering the negative effects of the infection. Materials and Methods: Between January 2022 and March 2023, blood samples were taken from 116 women, including those with and those without a history of bad obstetric history (BOH). Enzyme-linked immunosorbent assay (ELISA) was used to test sera taken from the study participants for B19 IgM and immunoglobulin G (IgG) antibodies. Those samples tested positive for IgG B19 were further subjected to polymerase chain reaction amplification and sequencing, targeting partial gene sequences of the nonstructural viral protein 1 (VP1) gene. Results: For human parvovirus IgG and IgM, the seropositivity was 44% and 16.5%, respectively. The following variables were associated in this study with greater seroprevalence rates of IgG and IgM, respectively: age 36–45 years (72.7%, 36.4%); abortion (80%, 32.5%); education (82.2%, 33.3%); unemployment (61%, 22.2%); and overcrowding (73.3%, 31.1%). In this case–control study, positive amplicons (700 bp) were detected from 27 samples out of 40 women of childbearing age who tested positive for IgG. Sequence analysis of the partial sequence of the VP1 gene in all positive amplicons was 100% identical with each other and with human parvovirus B19 (NC: 00088.3). According to the current study, about 66% of women are serologically negative and at risk of contracting parvovirus B19. Conclusion: Our findings conclude that pregnant women in Iraq, particularly those with BOH, must undergo frequent serological screening for B19-V during all three trimesters and be given the necessary advice to avoid B19-V infection to improve their pregnancy outcomes.

Epidemiological and molecular characterization of Coxsackievirus A6 causing hand, foot, and mouth disease in the Philippines, 2012-2017.

Coxsackievirus A6 (CVA6) is emerging as the dominant serotype among enteroviruses (EVs) responsible for hand, foot, and mouth disease (HFMD) outbreaks in multiple countries. However, details regarding this serotype in the Philippines are limited. In this study, we investigated the epidemiological and molecular characteristics of laboratory-confirmed CVA6 HFMD cases in the Philippines between 2012 and 2017. Data collected from case report forms submitted to the National Reference Laboratory for Poliovirus and other Enteroviruses were used to determine the distribution and clinical findings of laboratory-confirmed CVA6 HFMD. Phylogenetic analyses of the complete viral protein 1 (VP1) and partial 3D polymerase (3Dpol) gene sequences were performed to determine the genotype and recombinant (RF) form of the selected samples. An increase in the detection rate of CVA6 among enterovirus-positive HFMD cases was observed from 61.9% (140/226) in 2012 to 88.1% (482/587) in 2017, with most cases coming from the Luzon island group. Among the detected cases, the majority were children, with a median age of 2 years old (interquartile range: 1.17-3.40). Respiratory-related morbidities were the commonly reported complications (7.9%; 72/907). Based on the VP1 and 3Dpol gene sequence analysis, the CVA6 strains in this study were classified as genotype D3b and RF-A group, respectively. This study elucidated that CVA6 was the most prevalent enterovirus serotype causing HFMD in the Philippines in 2012-2017, with genotype D3b/RF-A circulating within this period. This study highlights the importance of viral surveillance and molecular epidemiological analysis to broaden our understanding of HFMD in the Philippines.

Detection of human neurotropic JCPyV DNA sequence in pediatric anaplastic xanthoastrocytoma

Due to its peculiar histopathological findings, pleomorphic xanthoastrocytoma (PXA), a rare cerebral tumor of young adults with a slow growth and a good prognosis, resembles to the lytic phase of progressive multifocal leukoencephalopathy, a fatal neurodegenerative disease caused by JC polyomavirus (JCPyV). Therefore, the presence of JCPyV DNA was examined in an 11-year-old child with xanthoastrocytoma, WHO grade 3, by quantitative PCR (qPCR) and nested PCR (nPCR) using primers amplifying sequences encoding the N- and C-terminal region of large T antigen (LTAg), the non-coding control region (NCCR), and viral protein 1 (VP1) DNA. The expression of transcripts from LTAg and VP1 genes was also evaluated. In addition, viral microRNAs’ (miRNAs) expression was investigated. Cellular p53 was also searched at both DNA and RNA level. qPCR revealed the presence of JCPyV DNA with a mean value of 6.0 × 104 gEq/mL. nPCR gave a positive result for the 5ʹ region of the LTAg gene and the NCCR, whereas 3ʹ end LTAg and VP1 DNA sequences were not amplifiable. Only LTAg transcripts of 5ʹ end were found whereas VP1 gene transcript was undetectable. Although in most cases, either Mad-1 or Mad-4 NCCRs have been identified in association with JCPyV-positive human brain neoplasms, the archetype NCCR structure was observed in the patient’s sample. Neither viral miRNA miR-J1-5p nor p53 DNA and RNA were detected. Although the expression of LTAg supports the possible role of JCPyV in PXA, further studies are warranted to better understand whether the genesis of xanthoastrocytoma could depend on the transformation capacity of LTAg by Rb sequestration.

Reduction of some viral protein's gene expression of recombinant influenza A/H1N1-PR8 virus upon treatment with Punica granatum crude extract

BackgroundIt was we known that Flu infections have a place with the class Orthomyxoviridae furthermore, three distinct sorts (A, B and C sort infections) are known. Among these, influenza viral infections display the broadest host range (birds, people, and different well evolved creatures) also, can develop into profoundly pathogenic strains. So, the scientists tried to find a way to restrict this invasion which can cause certain death to human raise. Subjects & methodsDifferent concentrations of Punica granatum extract (2.5, 5, 10 and 20 μg/ml) prepared from the stock solution were used against human influenza A/Puerto Rico/8/34 H1N1 (IAV/PR8) to test its ability to prevent the virus to grow on MDCKII and A549 cells to check the effect of this plant extract on viral replication titter using plaque assay, gene expression of some viral proteins like non-structural protein NS1, nucleoprotein NP and Protein basic PB1 using reverse transcriptase quantitative real time polymerase chain reaction. Also, localization of NP protein was checked using immune-florescent staining technique. ResultsOur finding showed that there was significant decrease in IAV/PR8 ability to grow and replicate in MDCKII cells at multiplicity of infection (MOI) in both of 0.1 and 0.01 MOI. this finding was confirmed upon checking of expression of NS1, PB1 and NP proteins of IAV/PR8 that were reduced significantly at 10 and 20 μg/ml. Interestingly the localization of viral NP protein affected negatively upon the treatment of cells with elected concentration of plant extract by the weak localization outside the nucleus. ConclusionMethanolic extract of Punica granatum produced a strong anti-viral activity against IAV/PR8 replication ability and that was due to the severe effect on viral proteins expression especially on PB1 and NP which responsible for viral protein synthesis and NS1 protein which is a strong IFN antagonistic protein thus; weakening its replication ability in different cell lines.

Construction of Genomic and Sub-Genomic Dengue Infectious Replicons.

Dengue replicons are powerful tools used in studying virus biology as well as in high-throughput screening of drug candidates. Replicon constructs are developed as genomic (consists of all the viral protein genes) or sub-genomic (consists of only nonstructural protein genes) and are used to study various aspects of the virus life cycle such as genome replication and virus assembly. In addition, a replicon usually includes a reporter gene used in monitoring virus replication. In this chapter, we provide methods to develop both genomic and sub-genomic dengue replicons with a luciferase reporter and describe different assays to utilize these systems.

Molecular epidemiology of coxsackievirus A16 circulating in children in Beijing, China from 2010 to 2019

BackgroundCoxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot and mouth disease (HFMD). This study aimed to investigate the molecular epidemiology and evolutionary characteristics of CVA16.MethodsThroat swabs were collected from children with HFMD and suspected HFMD during 2010–2019. Enteroviruses (EVs) were detected and typed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR. The genotype, evolutionary rate, the most recent common ancestor, population dynamics and selection pressure of CVA16 were analyzed based on viral protein gene (VP1) by bioinformatics software.ResultsA total of 4709 throat swabs were screened. EVs were detected in 3180 samples and 814 were CVA16 positive. More than 81% of CVA16-positive children were under 5 years old. The prevalence of CVA16 showed obvious periodic fluctuations with a high level during 2010–2012 followed by an apparent decline during 2013–2017. However, the activities of CVA16 increased gradually during 2018–2019. All the Beijing CVA16 strains belonged to sub-genotype B1, and B1b was the dominant strain. One B1c strain was detected in Beijing for the first time in 2016. The estimated mean evolutionary rate of VP1 gene was 4.49 × 10–3 substitution/site/year. Methionine gradually fixed at site-23 of VP1 since 2012. Two sites were detected under episodic positive selection, one of which (site-223) located in neutralizing linear epitope PEP71.ConclusionsThe dominant strains of CVA16 belonged to clade B1b and evolved in a fast evolutionary rate during 2010–2019 in Beijing. To provide more favorable data for HFMD prevention and control, it is necessary to keep attention on molecular epidemiological and evolutionary characteristics of CVA16.

Genotypes of rhinovirus detected among children in two communities of South-West Nigeria.

Rhinoviruses (RVs) are the most common etiological agent implicated in respiratory infections among infants and children. There are currently no approved antivirals and vaccine for use against the virus; hence, the need for information on the genotypes of rhinovirus from developing countries of the world with high burden of the infection. This study determined the genotypes of rhinovirus circulating among children in selected cities in Nigeria. Nasopharyngeal and oropharyngeal samples were carefully collected from children showing signs of respiratory infection in two communities in South-west Nigeria. Polymerase Chain Reaction was used to amplify the hypervariable part of the 5'- non-coding region, the entire viral protein gene 4 and the 5' terminus of the VP2 gene of RV. Nucleotide BLAST and phylogenetic analyses were used to genotype the isolates. Of the samples analysed, 12.7% showed rhinovirus positivity. All the three genotypes of rhinovirus were detected with genotype C (71.4%), being the predominant. Multiple strains of rhinovirus were found circulating. We showed for the first time the genotypes and strains of rhinovirus circulating in Nigeria. Further studies are required to highlight transmission patterns and disease severity among rhinovirus species in Nigeria.

Navigating the Diagnostics of COVID-19

Introduction/ObjectiveThe recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in Hubei province, China in December 2019 and has spread worldwide at an alarming rate. With millions of individuals infected and over two hundred thousand deaths, the necessity to develop fast and efficient diagnostic methods is of high importance. Diagnostic modalities rely on a combination of epidemiology, clinical presentation, laboratory examination, and appropriate imaging to diagnose and distinguish SARS-CoV-2 from other pulmonary infections. The purpose of this paper is to report on currently available diagnostic screening methods for patients infected with SARS-CoV-2 to guide frontline healthcare workers involved with COVID-19 patient care.MethodsAn electronic literature search was performed for peer-reviewed articles published from January 1, 2020, until April 26, 2020. Articles were then reviewed and included based on the applicability to the topic.ResultsThe preferred diagnostic approach is reverse transcription of the virus’ RNA followed by PCR amplification (RT-PCR). This method recognizes the gene-specific primers to target various viral protein genes, such as the envelope protein gene or the nucleocapsid protein gene, which enables this test to be both sensitive and specific toward SARS-CoV-2. However, this method has been proven to be time-consuming taking hours-to-days for the results. In order to improve the speed and efficiency of diagnostics, newer rapid diagnostic serological tests are being developed for testing SARS-CoV-2, each with its own unique advantages and disadvantages. They could potentially be used as triage tests to rapidly identify patients who are very likely to have COVID-19 in combination with other accurate diagnostic methods.ConclusionTherefore, a combination of diagnostic testing used in a timely manner may be beneficial for the rapid and accurate detection of SARS-CoV-2. This was evident in cases where despite initial negative RT-PCR tests for various patients, who later demonstrate chest CT scans with various degrees of consolidation and ground-glass opacity. Thus establishing the need for radiology diagnosis to be complementary to RT-PCR for COVID-19 patients. Hopefully, the continuous development and use of rapid diagnostic tests and the implementation of public health measures will help control the spread of the disease.

Conserved motifs in the invertebrate iridescent virus 6 (IIV6) genome regulate virus transcription

Invertebrate iridescent virus 6 (IIV6) is the type species of the Iridovirus genus in the Betairidovirinae subfamily of the Iridoviridae family. Transcription of the 215 predicted IIV6 genes is temporally regulated, dividing the genes into three kinetic classes: immediate-early (IE), delayed-early (DE), and late (L). So far, the transcriptional class has been determined for a selection of virion protein genes and only for three genes the potential promoter regions have been analyzed in detail. In this study, we investigated the transcriptional class of all IIV6 genes that had not been classified until now. RT-PCR analysis of total RNA isolated from virus-infected insect cells in the presence or absence of protein and DNA synthesis inhibitors, placed 113, 23 and 22 of the newly analyzed viral ORFs into the IE, DE and L gene classes, respectively. Afterwards, in silico analysis was performed to the upstream regions (200 bp) of all viral ORFs using the MEME Suite Software. The AA(A/T)(T/A)TG(A/G)A and (T/A/C)(T/G/C)T(T/A)ATGG motifs were identified in the upstream region of IE and DE genes, respectively. These motifs were validated by luciferase reporter assays as crucial sequences for promoter activity. For the L genes two conserved motifs were identified for all analyzed genes: (T/G)(C/T)(A/C)A(T/G/C)(T/C)T(T/C) and (C/G/T)(G/A/C)(T/A)(T/G) (G/T)(T/C). However, the presence of these two motifs did not influence promoter activity. Conversely, the presence of these two sequences upstream of the reporter decreased its expression. Single nucleotide mutations in the highly conserved nucleotides at the end of the second motif (TTGT) showed that this motif acted as a repressor sequence for late genes in the IIV6 genome. Next, upstream sequences of IIV6 L genes from which we removed this second motif in silico, were re-analyzed for the presence of potential conserved promoter sequences. Two additional motifs were identified in this way for L genes: (T/A)(A/T)(A/T/G)(A/T)(T/C)(A/G)(A/C)(A/C) and (C/G)(T/C)(T/A/C)C(A/T)(A/T)T(T/G) (T/G)(T/G/A). Independent mutations in either motif caused a severe decrease in luciferase expression. Information on temporal classes and upstream regulatory sequences will contribute to our understanding of the transcriptional mechanisms in IIV6.

Structural Analysis of Merkel Cell Polyomavirus (MCPyV) Viral Capsid Protein 1 (VP1) in HIV-1 Infected Individuals.

Merkel cell polyomavirus (MCPyV) viral protein 1 (VP1) is the capsid protein that mediates virus attachment to host cell receptors and is the major immune target. Given the limited data on MCPyV VP1 mutations, the VP1 genetic variability was examined in 100 plasma and 100 urine samples from 100 HIV+ individuals. Sequencing of VP1 DNA in 17 urine and 17 plasma specimens, simultaneously MCPyV DNA positive, revealed that 27 samples displayed sequences identical to VP1 of MCC350 strain. VP1 from two urine specimens had either Thr47Ser or Ile115Phe substitution, whereas VP1 of one plasma contained Asp69Val and Ser251Phe substitutions plus deletion (∆) of Tyr79. VP1 DNA in the remaining samples had mutations encoding truncated protein. Three-dimensional prediction models revealed that Asp69Val, Ser251Phe, and Ile115Phe caused neutral effects while Thr47Ser and Tyr79∆ produced a deleterious effect reducing VP1 stability. A549 cells infected with urine or plasma samples containing full-length VP1 variants with substitutions, sustained viral DNA replication and VP1 expression. Moreover, medium harvested from these cells was able to infect new A549 cells. In cells infected by samples with truncated VP1, MCPyV replication was hampered. In conclusion, MCPyV strains with unique mutations in the VP1 gene are circulating in HIV+ patients. These strains display altered replication efficiency compared to the MCC350 prototype strain in A549 cells.

Molecular characterization of the chicken anemia virus by using the VP1 gene of CAV/ SK/2017 strains, Iraq

Chicken infectious anemia virus (CIAV) is one amongst the numerous pathogens in the poultry industry. CIAV infection can cause restraint of the immune system, the excitement of co-infections, vaccination failures and mortality. chicks. Maternal antibodies not prevent infection but to prevent these symptoms, immunological disorder, or transmission of the virus. Genetic characterization for five sequences of chicken infectious anemia virus (CIAV) from different flocks of broiler industrial chickens were a consideration for the primary time in Iraq. Phylogenetic analysis of the viral protein 1 (VP1) gene, as well as the hypervariable region of the CIAV genome, indicated that Iraqi CIAV strains have belonged into genotype II. Amino acid comparison exhibit that the diversity of VP1 is indicated that the new strains were extremely pathogenic viruses. Our epidemiological study provided new insights into the prevalence of CIAV strain in recent years in Sulaimani province/Iraq

Analysis of codon usage pattern in the viral proteins of chicken anaemia virus and its possible biological relevance

Chicken anaemia virus (CAV) is an important poultry pathogen. CAV infection can lead to severe immunosuppression and mortality in birds. The viral proteins (VP1, VP2 and VP3) are major protective immunogens that determine the pathotype of CAV strains. The factors influencing the synonymous codon usage bias and the nucleotide composition of the viral protein genes of CAV have not been studied. In the present study, we have analysed the synonymous codon usage pattern in VP1, VP2 and VP3 genes of CAV. Our results showed that all the genes have a low codon usage bias; however, this bias was slightly higher in the VP2 gene as compared to VP1 and VP3. The major contributing factor for this pattern of codon usage bias in CAV is mutational pressure followed by the host factors. The conclusion was drawn based on codon usage, correspondence analysis, ENC-GC3 plot and correlation analyses among different indices. The study will help us to understand the codon usage bias of CAV and related single-stranded DNA viruses which could further be used to explore their biology.

Genomic sequence and pathogenicity of the first avian metapneumovirus subtype B isolated from chicken in China

Avian metapneumovirus (aMPV), which has been reported in many countries, causes an acute upper respiratory tract disease in chickens and turkeys. Although aMPV was first detected in China in 1999, there has been no further effort to isolate and characterize the aMPV subtype B (aMPV/B) from field outbreaks. In the present study, we used Vero cells to culture a viral strain, LN16, isolated from chickens with swollen head syndrome. The results of RT-PCR, indirect immunofluorescent antibody, and G gene sequence analyses confirmed that strain LN16 corresponds to aMPV/B. We amplified and sequenced the complete genome of strain LN16 and found it to be 13,513 nucleotides in length. Nine viral protein genes of the strain were between 93.2% and 98.4% identical to those of the pathogenic field isolate VCO3/60616. However, insertions and deletions were detected in the intergenic regions. Animal experiments showed that 72.7% of chickens infected with strain LN16 had excess mucus, nasal discharge, and inflammation in the lungs and turbinate. In addition, 27.2% of chickens infected with LN16 shed progeny virions. Viral tissue distribution analysis showed that aMPV could be detected in the turbinate and occasionally in immune organs. This is the first report of the isolation of aMPV/B in China and the first complete genome sequence of aMPV/B from chicken. These findings enrich the epidemiological data on aMPV and may contribute to the development of effective measures to prevent its further spread in China.

Molecular epidemiology of coxsackievirus A6 derived from hand, foot, and mouth disease in Fukuoka between 2013 and 2017.

Coxsackievirus (CV)-A6 has been the primary causative agent of hand, foot, and mouth disease (HFMD) in Japan since 2011. In Fukuoka, CV-A6-associated HFMD caused epidemics in 2013, 2015, and 2017. This paper reports the genetic characteristics of the CV-A6 entire viral protein 1 (VP1) derived from patients with HFMD in Fukuoka between 2013 and 2017. CV-A6 was detected in 105 of 280 clinical specimens, and the entire VP1 sequences could be analyzed for 90 of the 105 specimens. Phylogenetic analysis revealed that the CV-A6 strains were classified into clade A and subgrouped into subclade A3 or subclade A4. Each subclade strain carried amino acid substitutions in the presumed DE and GH loops of the VP1, and no amino acid substitutions were identified as deleterious to the protein function. No significant difference was found in the clinical symptoms between the genetic subclades using statistical analyses. In conclusion, this study clarified the genetic diversity of CV-A6 in Fukuoka from 2013 to 2017. The emergence of the CV-A6 strains was classified into derived new subclades based on phylogenetic analysis of the VP1 gene that may cause CV-A6-associated HFMD epidemics approximately every 2 years.

Different Strains of BK Polyomavirus: VP1 Sequences in a Group of Iranian Prostate Cancer Patients.

BK virus (BKV) has a worldwide seroprevalence in humans. Based on sequences of the major capsid proteins, i.e. viral protein 1 (VP1), there are four BKV genotypes. Each genotype has its own subtypes, and wasshown to be circulating independently in the human population. The aim of this study was to determine BKVgenotypes and subtypes among Iranian patients with prostatic cancer, benign prostatic hyperplasia, and kidney transplantation. BKV DNA was extracted from prostatic cancers and benign prostatic hyperplasia blocks and also urine of kidney transplantation patients. BKV (VP1) gene was amplified partially (327nt) by homemade polymerase chain reactions and subjected for sequencing and phylogenetic analysis. Bioedit version 7.0 and Mega version 5.0 were used for sequence analysis and for comparing the results with world-driven BKV sequences. All of BKV VP1 genes which were derived from Iranian patients were classified with subtype 1b2 strains from Germany and Turkey. Predicted amino acid sequences from the studied region of VP1 showed that all of these nucleotide diversities could change amino acid sequence numbers 60, 68, 72, 73 and 82 among VP1. The interesting point was that genetic analysis of derived sequences showed a different feature of genetic diversity among Iranian sequences. This feature has not been reported yet. This characteristic feature of Iranian BKV VP1 gene provides a unique cluster of sequences in phylogenetic tree.

Molecular analysis of norovirus in specimens from children enrolled in a 1982-1986 study in Belém, Brazil: A community-based longitudinal study

Fecal specimens were collected during a longitudinal, community-based study in the city of Belém, North Brazil, that was conducted over 3 years (October 1982 to March 1986), in which 20 children were included from birth to 3 years of age. A total of 229 fecal samples were screened by real time RT-PCR targeting the junction region (ORF 1/2) of the norovirus (NoV) genome. NoV-positive samples were subjected to PCR and sequencing of the viral polymerase (ORF1) and viral protein 1 (VP1) genes (ORF2). The junction region was also sequenced to assess for recombination when ORF1 and ORF2 genotyping results were dissimilar. Samples classified as GII.P4/GII.4 were further characterized by sequencing the P2 subdomain of the viral capsid to determine possible alterations. An overall positivity of 16.1% (37/229) was observed, including GI (16.2%-6/37) and GII (83.8%-31/37) genogroups. Cases of NoV reinfection in at least 2-month intervals were observed, and 12 children developed at least one case of asymptomatic NoV infection. In total, 48.6% (18/37) NoV-positive samples were subjected to nucleotide sequencing analysis targeting the following polymerase genes: GI.P3 (n = 1), GII.Pa (n = 1), GII.Pc (n = 1), GII.P4 (n = 5), GII.P6 (n = 5), GII.P7 (n = 3), GII.P12 (n = 1), and GII.P22 (n = 1). For the VP1 gene, characterization was performed in 14 (77.8%) samples: GI.3 (n = 1), GII.2 (n = 1), GII.4 (n = 4), GII.6 (n = 4), GII.7 (n = 1), GII.12 (n = 1), GII.14 (n = 1), and GII.23 (n = 1). Recombination events were confirmed in three cases (GII.P12/GII.2, GII.P7/GII.14, and GII.Pa/GII.12), and four samples genotyped as GII.P4/GII.4 were analyzed to identify variants. None had contemporary counterparts. Three children developed consecutive NoV infections by different genotypes. The present report documents the importance of NoV as a cause of childhood infection during a longitudinal study conducted more than 30 years ago.

A multiplex PCR for detection of six viruses in ducks

In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 102 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks.

Hairpin structures with conserved sequence motifs determine the 3′ ends of non-polyadenylated invertebrate iridovirus transcripts

Previously, we observed that the transcripts of Invertebrate iridescent virus 6 (IIV6) are not polyadenylated, in line with the absence of canonical poly(A) motifs (AATAAA) downstream of the open reading frames (ORFs) in the genome. Here, we determined the 3′ ends of the transcripts of fifty-four IIV6 virion protein genes in infected Drosophila Schneider 2 (S2) cells. By using ligation-based amplification of cDNA ends (LACE) it was shown that the IIV6 mRNAs often ended with a CAUUA motif. In silico analysis showed that the 3′-untranslated regions of IIV6 genes have the ability to form hairpin structures (22–56 nt in length) and that for about half of all IIV6 genes these 3′ sequences contained complementary TAATG and CATTA motifs. We also show that a hairpin in the 3′ flanking region with conserved sequence motifs is a conserved feature in invertebrate-infecting iridoviruses (genus Iridovirus and Chloriridovirus).

Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae).

BackgroundComparative studies of chloroplast genomes (plastomes) across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae), one from Hydrodictyon reticulatum and the other from Pediastrum duplex.MethodsGenomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs) ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed.ResultsThe plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae.DiscussionThe complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique orfs indicate recent, independent invasions into each plastome, a hypothesis testable with an expanded plastome investigation within the Hydrodictyaceae.

Molecular analysis of norovirus in specimens from children enrolled in a 1982-1986 study in Belém, Brazil: A community-based longitudinal study.

Fecal specimens were collected during a longitudinal, community-based study in the city of Belém, North Brazil, that was conducted over 3 years (October 1982 to March 1986), in which 20 children were included from birth to 3 years of age. A total of 229 fecal samples were screened by real time RT-PCR targeting the junction region (ORF 1/2) of the norovirus (NoV) genome. NoV-positive samples were subjected to PCR and sequencing of the viral polymerase (ORF1) and viral protein 1 (VP1) genes (ORF2). The junction region was also sequenced to assess for recombination when ORF1 and ORF2 genotyping results were dissimilar. Samples classified as GII.P4/GII.4 were further characterized by sequencing the P2 subdomain of the viral capsid to determine possible alterations. An overall positivity of 16.1% (37/229) was observed, including GI (16.2%-6/37) and GII (83.8%-31/37) genogroups. Cases of NoV reinfection in at least 2-month intervals were observed, and 12 children developed at least one case of asymptomatic NoV infection. In total, 48.6% (18/37) NoV-positive samples were subjected to nucleotide sequencing analysis targeting the following polymerase genes: GI.P3 (n = 1), GII.Pa (n = 1), GII.Pc (n = 1), GII.P4 (n = 5), GII.P6 (n = 5), GII.P7 (n = 3), GII.P12 (n = 1), and GII.P22 (n = 1). For the VP1 gene, characterization was performed in 14 (77.8%) samples: GI.3 (n = 1), GII.2 (n = 1), GII.4 (n = 4), GII.6 (n = 4), GII.7 (n = 1), GII.12 (n = 1), GII.14 (n = 1), and GII.23 (n = 1). Recombination events were confirmed in three cases (GII.P12/GII.2, GII.P7/GII.14, and GII.Pa/GII.12), and four samples genotyped as GII.P4/GII.4 were analyzed to identify variants. None had contemporary counterparts. Three children developed consecutive NoV infections by different genotypes. The present report documents the importance of NoV as a cause of childhood infection during a longitudinal study conducted more than 30 years ago.