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1.       Detection of Astrovirus

·         Transmission electron microscope (laborious, time consuming, subjective, involve personal experience)

·         Immune Electron Microscope (IEM)  or solid phase microscope (use antibody to enhance sensitivity of essay and enable confirmation of viral agent. Limitation: limited availability of antibody) – IEM and EIA is less time consuming

·         RT-PCR (offer threshold of detection as low as 10-100 genome copies per gram of stool. Sensitivity: depends on primer/probe, enzyme amplification reaction condition. Differentiation of classic and novel Human Astrovirus (HAstV)

·         Microarray (distinguish representative of eight serotype of classic HAstVs

·         Acid sequence-based amplification (NASBA)

·         RT-quantitative PCR (RT-qPCR) – high speed, sensitive, reproducibility, reduction of contamination risk in the HAstV. (quantification technique)

2.    From the above detection techniques, can you classify your virus into respective family?  Explain your answer with a phylogenetic tree.

First, use the above detection techniques to obtain the genome sequence to determine the relatedness between each genotype based on difference in complete capsid sequence which is the capsid protein level. Within genotype, strain can be grouped in serotype based on their antigenicity. Strain can also be classified based on their ORF2 variability.

(phylogenetic tree picture)

3.    Illustrate, in detail, the structure and the life cycle of the virus.

-       Main cellular receptor remains unknown.

-       Have dimeric surface spike that can recognize a polysaccharide molecule.

-       However, there are several substance that can prevent the infectivity which is dextran sulphate, heparin sulphate

-       The mechanism of entry is via clathrin-mediated endocytosis.

-       Entry depends on acidification and maturation of endosome,

-       Binding time of the virus to host cell surface is about 10 minutes while the uncoating process is 130 minutes.

-       Interaction of virus and host cell will activate extracellular signal-regulated kinase (ERK1/2) and phosphoinositide3-kinase (P13K pathway).

Translation and replication

-       VPg protein on viral genome plays a role in infectivity. The cleavage of VPg using proteolytic treatment will inactivate the viral genome infectivity.

-       VPg protein has a role in recruitment cellular translation initiation factors.

-       When the genome is translated, proteins formed are nsP1a1b and nsP1a polyprotein. The polyprotein will be cleaved into individual nonstructural proteins via serine proteases.

-       The cleavage process of viral nonstructural polyprotein is unknown.

-       Proteolytic processing of polyprotein nsP1a1b produces nsP1b protein and nsP1a protein which is cleaved to yield matured products.

-       Endoplasmic reticulum derived intracellular membrane will be the cellular site for genome replication.

-       However, the genome replication process remains unknown.

-       The negative-sense genomic RNA will be synthesized and use as template to produce positive-sense genomic RNA and positive-sense subgenomic RNA.

Expression of structural proteins, virion assembly and cell release

-       VP90 structural polyprotein initially assembles in immature virion in association with intracellular membrane.

-       VP90 polyprotein is further cleaved into VP70 polyprotein.

-       VP70 containing viral particles would be released from the cell and proteolytically processed by trypsin to enhance infectivity.

-       The process was carry out without cell lysis.

(Life cycle picture)

4.    4. Describe how the virus transmitted from a patient to another. Does it also involve age factor?

The virus is transmitted via fecal-oral route, contamination of food and contamination of water. The transmission via fecal-oral route often happen at nursery, daycare center, hospitals, schools and military facilities. Transmission of virus via contamination of water occurs from freshwater, drinking water and marine water. The waste water treatment practices could not completely remove the virus. While the transmission of food occurs due to the production of food using contaminated water or animals that have consumed the virus. Also, it can be due to poor hygiene during post-harvest and cooking utensils.

This virus targets children, elderly and immunocompromised individual.

5.

- control of transmission route (include virus detection and virus inactivation in food and  disinfection of contaminated fomites.)

- prevention and control of the disease at host level.

- anti-viral using synthetic flavonoids.

- insertion of fluid to prevent dehydration

- treatment with IgG to treat immunocompromised host.

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Component in Infographic and Montage Video.

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Component in Infographic and Montage Video.

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-introduction astrovirus (general, gmbar astrovirus, characteristic, family)
-transmission
-life cycle (incubation period, ocurrence)
-individual infected
-symptom, detection, treatment

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1. Write anything about our group project Virtual Microbe (anything and any form (e.g. microsoft word, mindmap, video)
2. Attach the reflection to link below.

Link to Blendspace:
https://www.tes.com/lessons/Hp4hf2Wujjq17Q/collab/tbFZRJEPQCNrHtaanNGAxpGMZiHBPJdC/register

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1. What would you do to detect the presence of the virus? Polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) or other techniques? 2. From the above detection techniques, can you classify your virus into respective family? Explain your answer with a phylogenetic tree.
3. Illustrate, in detail, the structure and the life cycle of the virus.
4. Describe how the virus transmitted from a patient to another.Does it also involve age factor?
5. Suggest a possible treatment.

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Targeted individuals, signs and symptoms, incubation period and infection occurence. 

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Targeted individuals, signs and symptoms, incubation period and infection occurence. 

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(1)

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http://www.sciencedirect.com/science/article/pii/S0166093403002647

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Acknowledgement: the picture of Astrovirus was sourced from Enteric Virus Laboratory website of University of Barcelona and is reproduced under Creative Commons' license

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Acknowledgement: the picture of Astrovirus was sourced from BioQuick News and is reproduced under Creative Commons' license

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http://www.bioquicknews.com/node/3176

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Maria da Penha Trindade Pinheiro Xavier,

Filipe Aníbal Carvalho Costa,

Mônica Simões Rocha,

Juliana da Silva Ribeiro de Andrade,

Fernanda Kreischer Bandeira Diniz,

Thais Ramos de Andrade,

Marize Pereira Miagostovich,

José Paulo Gagliardi Leite,

Eduardo de Mello Volotão

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Abstract

Human astrovirus (HAstV) represents the third most common virus associated with acute diarrhea (AD). This study aimed to estimate the prevalence of HAstV infection in Brazilian children under 5 years of age with AD, investigate the presence of recently described HAstV strains, through extensive laboratory-based surveillance of enteric viral agents in three Brazilian coastal regions between 2005 and 2011. Using reverse transcription-polymerase chain reaction (RT-PCR), the overall HAstV detection rate reached 7.1% (207/2.913) with percentage varying according to the geographic region: 3.9% (36/921) in the northeast, 7.9% in the south (71/903) and 9.2% in the southeast (100/1.089) ( p < 0.001). HAstV were detected in cases of all age groups. Detection rates were slightly higher during the spring. Nucleotide sequence analysis of a 320-bp ORF2 fragment revealed that HAstV-1 was the predominant genotype throughout the seven years of the study. The novel AstV-MLB1 was detected in two children with AD from a subset of 200 samples tested, demonstrating the circulation of this virus both the in northeastern and southeastern regions of Brazil. These results provide additional epidemiological and molecular data on HAstV circulation in three Brazilian coastal regions, highlighting its potential to cause infantile AD.

Citation: Xavier MdPTP, Carvalho Costa FA, Rocha MS, Andrade JdSRd, Diniz FKB, Andrade TRd, et al. (2015) Surveillance of Human Astrovirus Infection in Brazil: The First Report of MLB1 Astrovirus. PLoS ONE 10(8): e0135687. https://doi.org/10.1371/journal.pone.0135687

Editor: Charles Y. Chiu, University of California, San Francisco, UNITED STATES

Received: December 30, 2014; Accepted: July 24, 2015; Published: August 14, 2015

Copyright: © 2015 Xavier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper. The nucleotide sequence data reported in this study is freely available in GenBank under the accession numbers: KM269039-KM269070, KM408170-KM408171 and KC294576-KC294577.

Funding: The authors thank the Excellence Program of Research (PROEP-CNPq/IOC) and the General Coordination of Laboratories/Secretary of Health Surveillance, Ministry of Health for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Human astrovirus belongs to the Astroviridae family and contains single-stranded, positive-sense, polyadenylated RNA 6.2-7.8 kilobases (kb) in length without an envelope, encased within an icosahedral capsid. The genome contains three open reading frames (ORFs) designated ORF1a, ORF1b and ORF2. The two first ORFs encode non-structural proteins, including viral proteinase and RNA polymerase and ORF2 encodes the capsid protein precursor [ 2, 3].

The Astroviridae family contains two distinguished genera: Mamastrovirus and Avastrovirus. These viruses were originally classified into genera and species based only on the host of origin. Recently, the Astroviruses Study Group, 9 th Report ICTV (International Committee on Taxonomy of Viruses), 2011 [ 6], proposed a classification based on the amino acid sequence, which encodes the capsid polyprotein and represents the most variable region of the genome [ 2, 3]. In this classification, Mamastrovirus genera associated to human disease is subdivided into four divergent species: MAstV 1 (the classical human astrovirus 1-8), MAstV 6 (AstV MLB1-3), MAstV 8 (AstV VA1 and VA3, also known as HMO-C and HMO-B, respectively) and MAstV 9 (AstV VA2, also Known as HMO-A, and VA4) [ 2].

In general, HAstV-1 is the most common type found in children, but the predominant genotype can vary with time and location [ 3, 7, 8]. The recent identification of novel AstV in humans using highly sensitive methods [ 9- 13], highlights the need to analyze the prevalence of these viruses to recognize their actual impact in public health, since they were found to be genetically related to animal viruses and some were isolated from patients with more severe diseases, such as encephalitis [ 14- 16].

This study aimed to associate the HAstV infection to Brazilian children under 5 years old affected with AD in three Brazilian coastal regions in a seven years period (2005-2011), providing epidemiological and molecular characteristics of HAstV genotypes. Recently described strains of HAstV were also studied.

Materials and Methods

Ethical statement

This study, including consent procedures, was approved by the Ethics Committee of Oswaldo Cruz Foundation (CEP 311/06) and is part of an ongoing official Brazilian Ministry of Health surveillance of enteric pathogens to investigate the viral etiology of AD. In this context, stool samples from AD cases were obtained by request of in and out patients attending health centers, including hospitals and public central laboratories, following outbreaks or sporadic cases of AD. For this study, consent was obtained verbally from the parents or relatives guardians on behalf of the children who were enrolled in this study. Data are maintained anonymously.

Clinical samples and studied areas

For this study, stool samples of 2.913 patients under 5 years old with negative diagnosis for RVA and NoV were obtained between January 2005 and December 2011 from three different Brazilian coastal regions (northeast, southeast and south) with distinct demographic and environmental scenarios and were selected for HAstV investigation. The population of the southern and southeastern regions is of a higher income compared to that of the northeastern region, where much of the population do not have access to sanitation, and where AD-related mortality is higher [ 17]. Acute diarrhea was defined when children presented three or more liquid or semi-liquid evacuations in a 24-h period.

Screening of novel HAstVs from stool samples

To search for novel and recently described HAstVs, 200 stool samples obtained between January and December 2011 from patients under two years old were randomly selected from three different Brazilian coastal regions (northeast, southeast and south). All samples were tested previously and were negative for RVA, NoV and HAstV1-8.

Statistical analysis

Rates of HAstV positivity and association between the frequency of symptoms and the different age groups were compared using the chi-squared test. Statistical significance was established at p < 0.05.

Nucleic acid extraction and detection

Fecal suspension was prepared in a 10% (w/v) Tris/HCL/Ca ²+ (0,01M, pH 7.2). RNA Extraction was performed using QIAamp Viral RNA extraction kit (Qiagen) methodology according to the manufacturer's instructions. Detection of classic HAstV and complementary DNA (cDNA) was synthesized using the Applied Biosystems High Capacity cDNA Reverse Transcription kit (Foster City, Ca, USA) with a previous denaturation step with dimethylsulfoxide for 7 min at 97°C. For classic HAstV detection, a PCR protocol was performed using a set of specific primers that targeted the ORF2 region capsid (Mon 269/270 [0.4 μM of each primer]) in a final reaction mixture of 50 μl in volume, according to the PCR conditions described previously [ 18].

For novel AstV detection, screening was carried out by amplifying a 409 bp segment of the ORF1b (RNA polymerase) region of the AstV genome with consensus primers SF0073/SF0076 designed with a bias for detection of a highly divergent AstV (Classic HAstV 1-8, MLB1-3 and VA1-3) [ 13]. Positive samples were sequenced and submitted to BLAST (software) [ 19] searching and those that indicated MLB1 homology were then subjected to amplification and sequencing of the 402 bp segment of the MLB1 ORF2 specific genome region using primers SF0053 and SF0061 for characterization [ 13]. All amplifications were performed using SuperScript III One-Step RT-PCR System (Invitrogen), according to the manufacturer´s instructions.

For all PCR reactions, final primers concentrations were of 0.2 μM for both forward and reverse primers, in a final reaction volume of 25 uL. The products were analyzed in a 2% agarose gels and visualized by ethidium bromide staining.

Molecular sequencing

For the molecular characterization of HAstV strains, PCR products were sequenced in both directions using the ABI Prism 3100 Genetic Analyzer and Big Dye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, Foster City, CA) with the same primers used for the amplification reactions [ 13, 18]. Centri-Sep columns (Princeton Separations, Foster City, CA) were used to purify the sequencing reaction products, according to the manufacturer's instructions.

Phylogenetic analysis

The chromatograms obtained from sequencing reactions were carried out using BioEdit software version 7.1.3.0 [ 20] for sequence editing. Sequence similarity searches were performed with sequences deposited in GenBank using the basic local alignment search tool (BLAST) software [ 19]. For phylogenetic analysis, reference sequences available for the same genomic region (ORF2) and size were selected taking into account the standards of each genetic type of human and/or environmental AstV and other similar sequences representing different geographical regions and time periods were also used. Sequences analyzes were performed using MEGA software version 5.1 [ 21] and multiple sequence alignments were carried out using the ClustalW program. Phylogenetic trees based on nucleotide (nt) sequences were obtained using the neighbor-joining method with Kimura two-parameter model with the bootstrap probabilities of each node calculated using 2,000 replicates. Values above 70 were considered significant and are represented in the trees. The nucleotide sequence data reported in this study is available in GenBank under the accession numbers: KM269039-KM269070, KM408170-KM408171 and KC294576-KC294577.

Results

Diagnostic and clinical aspects

The overall HAstV positivity rate among RVA and NoV negative AD cases was of 7.1% (207/2.913). Considering the distinct Brazilian coastal regions, HAstV was detected more frequently in the southeastern (9.2% [100/1.089]) and southern (7.9% [71/903]) regions, compared to the northeastern region (3.9% [36/921]); p < 0.001 ( Table 1).

Human astrovirus was detected in all age groups analyzed: 82/1.224 (6.7%) in infants aged 1-11 months, 64/1.031 (6.2%) in children aged 12-24 months, 48/549 (8.7%) in subjects aged 2-48 months and 13/109 (11.9%) among patients aged 49-60 months. The prevalence rate tended to be higher among children aged 49-60 months ( p = 0.054; chi-squared test for linear trend), although there was no statistical significance.

Differences in the frequency of some symptoms were observed between HAstV-positive and HAstV-negative patients ( Table 2). The presence of mucus in feces was significantly more frequent in HAstV-positive children, particularly in the 1-11-month age group ( p = 0.012).

Positivity for HAstV was detected throughout the whole year and seasonally was of 30/513 (5.8%) from January to March, 63/857 (7.4%) from April to June, 46/767 (6.0%) from July to September and 68/776 (8.8%) from October to December ( Fig 1).

Laboratory-based HAstV in three Brazilian regions, between 2005 and 2011. The rate of HAstV detection by month and number of HAstV-positive samples. Each month represents cumulative observations.

Molecular Characterization

Phylogenetic analysis of a 320-bp nucleotide sequence (ORF2) revealed that almost all HAstV genotypes (except HAstV-5 and HAstV-7) circulated in these three Brazilian coastal regions during the studied period. Considering HAstV-positive sequenced strains, HAstV-1 was the most frequent genotype identified, characterized in 77.7% strains.

In all regions studied, HAstV-1 was detected and represented 25% of the strains observed in the northeastern, 66% in the southeastern and 93% in the southern of Brazil. Other genotypes were also observed, including HAstV-3, which represented 45% of the strains detected in the northeastern, followed by HAstV-6 (25%) and HAstV-2 (8%). In the southeastern, the HAstV genotype-2 (23%) and HAstV-8 (6%) and HAstV-4 (5%) were recorded. In the southern region, besides HAstV-1 (93%), HAstV-8 was the only genotype detected (7%) ( Fig 2).

Brazilian HAstV-1 strains grouped into two different genetic clusters ( Fig 3A). Cluster 1 strains were closely related to each other (98.4-100% identity at the nt level) and were detected in all studied periods in different geographic regions of Brazil and displayed the highest identity (97.8-100%) to worldwide 1999-2011 strains. Cluster 2 grouped Brazilian strains detected from 2004-2005 and some Asiatic 2008-2011 strains were restricted to the southeastern region of Brazil. Brazilian HAstV-2 strains also grouped into two different clusters ( Fig 3B). Cluster 1 strains were closely related to each other (96.8-100% identity at the nt level) and were detected in 2006-2007 in different geographic regions (south and northeast), and displayed the highest identity (97.8-100%) to worldwide strains. The second HAstV cluster grouped Brazilian strains detected in 2005 and 2007 together with other Brazilian strains, as well as with Italian and Honduran strains, restricted to the southeastern Brazilian region. The Brazilian HAstV-3 strains showed the highest nucleotide identity (>97%) to HAstV-3 strains reported in Japan, Italy, Pakistan and Egypt. Notably, the unique HAstV-4 strain characterized in this study showed the lowest genetic relationship (93.1-93.7%) with Brazilian samples previously described and the highest nucleotide identity observed with worldwide samples (>96%).

(A) Phylogenetic analyzes were based on the 320-bp capsid gene between HAstV-1 strains detected in this study and HAstV-1 worldwide strains. (B) Phylogenetic analyzes between strains HAstV-2-HAstV-8 detected in this study and HAstV worldwide strains. The Brazilian strains described in this study are denoted by black circles. The scale bar indicates nucleotide substitutions per site. Bootstrap values (2.000 pseudo-replicates) above 70 are shown. The HAstV reference strains included in this analysis were obtained from GenBank.

Screening of novel HAstVs

Concerning novel HAstV screening, two samples were positive and characterized as AstV-MLB1 ( Fig 4). AstV- VA1 or other unusual HAstV rather than MLB1 were not detected. The two positive MLB1 samples were collected from a one year old child admitted in casualty due to AD in February and November 2011, in two different Brazilian regions: Maranhão State (northeast) and Rio de Janeiro State (southeast).

The Brazilian strains described in this study are denoted by black circles. The scale bar indicates nucleotide substitutions per site. Bootstrap values (2.000 pseudo-replicates) above 70 are shown. Reference sequences accession number in GenBank Database are cited in order of appearance: NC_011400; FJ402983; NC_014320; JN871245; KC294576; KC294577; HQ674649; HQ674647; HQ674648; HQ6746450; JQ086552; NC_016155; GQ502193; NC_002469; NC_001943; L13745; GU732187; DQ070852; DQ028633; GQ901902; AF260508; HM450382; to Turkey AstV (NC_002470).

Discussion

This study reports an overview of the distribution of HAstV genotypes in three coast regions of Brazil. It was the first time that the recently novel HAstV was investigated, increasing knowledge about its characteristics and genotypes circulation. Regarding HAstV infection prevalence in Brazil, initial studies conducted in different regions yielded positivity rates of 3-5% [ 22- 24], lower than those reported in the present survey. Nevertheless, our data showed similar rates to those in other studies carried out in Brazil [ 8, 25- 27] probably due to the evolution of molecular detection methods with increased sensitivity improving diagnosis. High HAstV positivity rates is normally described in AD outbreaks, such as in a native Brazilian population and in southeastern Mexico, where the prevalence of HAstV infection reached 56% and 28%, respectively [ 28, 29].

As expected, HAstV were commonly detected in all age group analyzed. The prevalence rate observed tended to be higher among children aged 40-60 months, although there was no statistical significance between age group and HAstV infection. HAstVs affect predominantly the pediatric population and the age of children infected with classic HAstV varies, ranging from newborns to over 5 years old [ 2, 8, 30].

Concerning seasonality, it has been proposed that the peak of HAstV detection in temperate climate countries occurs in the colder months and in tropical areas, the maximum incidence of HAstV infections tends to occur in the rainy season (2). The current study was performed in a seven years' time span, and a slight trend towards higher detection rates during the rainy season was observed. Similar to our data, the detection of HAstV in tropical climate countries was more frequent in the rainy season [ 29, 32]. Previous studies carried out in Brazil showed no seasonality or a higher detection frequency in warmer months [ 8, 24, 25], but the seasonal HAstV pattern is controversial and depends on the climate, geographical region analyzed and the year of study.

The recent discovery from humans of novel AstV strains that show genetic similarities to animal strains has aroused interest and the perception that these potentially zoonotic agents might have a greater impact on public health and on the etiological profile of AD [ 9- 12, 41]. In Brazil, the diagnosis of novel and classic HAstV are restricted to research center, not available in laboratories routine and their importance as etiological agents of AD in both sporadic cases and outbreaks remains poorly understood.

AstV-MLB1 is not yet a well-established AD agent and its role in human health remains unknown. A recent control case study conducted in India could not determine the association between AstV-MLB1 and AD [ 43]. Another important point is the fact that a large number of samples remain without an etiologic agent, drawing attention to other pathogens that may be involved. Other enteric viruses such as Adenovirus or Sapovirus, as well as bacteria and parasites may also be responsible. This study reinforces the role of distinct HAstV genotypes in the etiological AD profile in different Brazilian coastal regions, describing the detection of AstV-MLB1 in Brazil for the first time and suggesting potential changes in the etiological AD profile considering new pathogens agents as well as novel AstV.

Acknowledgments

We thank the Central Laboratories of the Brazilian Regions States staff (LACEN) for sending the clinical specimens, the Excellence Program of Research (PROEP-CNPq/IOC) and the General Coordination of Laboratories/Secretary of Health Surveillance, Ministry of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Conceived and designed the experiments: MPTPX EMV FACC JPGL MPM. Performed the experiments: MPTPX MSR JSRA FKBD TRA. Analyzed the data: MPTPX EMV FACC MPM. Contributed reagents/materials/analysis tools: JPGL MPM FACC EMV. Wrote the paper: MPTPX EMV FACC.

# data[].attributes.attachment_link.title
Surveillance of Human Astrovirus Infection in Brazil: The First Report of MLB1 Astrovirus

# data[].attributes.attachment_link.description
Human astrovirus (HAstV) represents the third most common virus associated with acute diarrhea (AD). This study aimed to estimate the prevalence of HAstV infection in Brazilian children under 5 years of age with AD, investigate the presence of recently described HAstV strains, through extensive laboratory-based surveillance of enteric viral agents in three Brazilian coastal regions between 2005 and 2011.

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· 28–35 nm diameter, icosahedral viruses that have a characteristic five- or sixpointed star-like surface structure when viewed by electron microscopy.

·Along with the Picornaviridae and the Caliciviridae, the Astroviridae comprise a third family of nonenveloped viruses whose genome is composed of plus-sense, single-stranded RNA.

·Astrovirus has a non-segmented, single stranded, positive sense RNA genome within a non-enveloped icosahedral capsid.