Outbreak of human astroviruses 1 and Melbourne 2 in acute gastroenteritis pediatric patients in Japan during the COVID-19 pandemic, 2021.

BACKGROUND: Human astrovirus (HAstV) infection is one of the leading causes of acute gastroenteritis in young children. The present study reports the outbreak of HAstV in children with acute gastroenteritis in Kyoto, Japan, during the COVID-19 pandemic, 2021. METHODS: A total of 61 stool samples were collected from children with acute gastroenteritis who visited a pediatric outpatient clinic in Maizuru city, Kyoto, Japan from July to October, 2021. HAstV was screened by RT-PCR, and the genotypes were identified by nucleotide sequence analysis. RESULTS: Of 61 cases of acute gastroenteritis, 20 were mono-infected with HAstV alone. In addition, mixed infection of HAstV and NoV, and HAstV and RVA were also detected in 15 and 1 cases, respectively. Of 36 HAstV strains detected in this outbreak, 29 and 7 were HAstV1 and MLB2 genotypes, respectively. All HAstV1 strains were closely related to the HAstV1 reported from Thailand and Japan in 2021 and all of them belonged to subgenotype HAstV1a. Among MLB2, they were most closely related to the MLB2 strains reported from China in 2016 and 2018. CONCLUSIONS: After the kindergartens and schools were re-opened at the middle of 2021 in Japan, an outbreak of HAstV was reported. Control measures against the COVID-19 pandemics might affect the spread of diarrheal virus infection. Here we report the outbreak of HAstV1 and MLB2 in Kyoto, Japan, during COVID-19 pandemic in 2021.

Coinfection in the cases of feline panleukopenia: a literature review

Coinfection caused by bacteria, parasites, or viruses complicates almost all feline panleukopenia virus (FPV) infections. Pathogens that colonize the gastrointestinal tract, Clostridium perfingens, Clostridium piliforme, Cryptosporidium spp, Giardia spp, Tritrichomonas fetus, canine parvovirus type 2,Salmonella sp., feline coronavirus, feline bocavirus, and feline astrovirus were isolated in the presence of FPV infection. Complex mechanisms between viruses, bacteria, protozoa, and hosts contribute to the pathogenesis and severity of coinfection. Prompt and accurate diagnosis, vaccination precautions, and appropriate treatment play important roles in reducing morbidity and mortality. This article outlines the etiology, pathogenesis, diagnosis, prevention, and treatment that can help veterinarians and pet owners improve their knowledge of managing the diseases.

Viral enteritis in cattle: To well known viruses and beyond

Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Epidemiological investigation of bovine viral diarrhea virus infection in swinery

From 2017 to 2020, 1 078 piglet diarrhea samples were collected from 6 pig farms in different districts of Shanghai. Multiple RT-PCR method was used for detection and analysis to study the infection status of bovine viral diarrhea virus (BVDV) in swinery in Shanghai. The results showed that the overall detection rate of BVDV in swinery in Shanghai was 7.14% (77/1 078), and showed an increasing trend year by year. The mixed infection rate of BVDV and other diarrhea pathogens was high, with the highest dual infection rate (65%, 26/40), mainly BVDV/PASTV (61.54%, 16/26). On this basis, the triple infection rate was 25% (10/40), mainly BVDV/PAStV/PKoV (40%, 4/10) infection mode;The quadruple infection rate was 10% (4/40), which was dominated by BVDV/PAStV/PEDV/PSV (50%, 2/4) infection. The BVDV prevalence in swinery was seasonal, and the prevalence in spring (10.36%) and autumn (13.59%) was higher than that in summer (6.8%) and winter (2.66%). The positive rate of BVDV in different pig farms was significantly different by 0-24.07%. In view of the detection rate of diarrhea virus dominated by PEDV in pig farm 2 had been high in recent years, this study further monitored the infection of BVDV in this pig farm, and found that the detection rate of BVDV in this pig farm was increasing year by year from 2017 to 2019, with the highest detection rate in 2019 (8.61%, 42/488);The mixed infection of BVDV and other diarrhea pathogens was also serious, with the dual infection rate of 57.58% (19/32), triple infection rate of 21.21% (7/32), quadruple infection rate of 21.21% (7/32), respectively. This study enriched the epidemic data of BVDV in swinery in Shanghai, and could provide reference for the prevention and control of pig epidemics.

Synchronicity of viral shedding in molossid bat maternity colonies.

Infection dynamics in vertebrates are driven by biological and ecological processes. For bats, population structure and reproductive cycles have major effects on RNA virus transmission. On Reunion Island, previous studies have shown that parturition of pregnant females and aggregation of juvenile Reunion free-tailed bats (Mormopterus francoismoutoui) are associated with major increase in the prevalence of bats shedding RNA viruses. The synchronicity of such shedding pulses, however, is yet to be assessed between viruses but also maternity colonies. Based on 3422 fresh faeces collected every 2-5 weeks during four consecutive birthing seasons, we report the prevalence of bats shedding astroviruses (AstVs), coronaviruses (CoVs) and paramyxoviruses (PMVs) in two maternity colonies on Reunion Island. We found that the proportion of bats shedding viruses is highly influenced by sampling collection periods, and therefore by the evolution of the population age structure. We highlight that virus shedding patterns are consistent among years and colonies for CoVs and to a lesser extent for PMVs, but not for AstVs. We also report that 1% of bats harbour co-infections, with two but not three of the viruses, and most co-infections were due to CoVs and PMVs.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

Discovery of novel Mamastroviruses in Bactrian camels and dromedaries reveals complex recombination history.

Virus emergence may occur through interspecies transmission and recombination of viruses coinfecting a host, with potential to pair novel and adaptive gene combinations. Camels are known to harbor diverse ribonucleic acid viruses with zoonotic and epizootic potential. Among them, astroviruses are of particular interest due to their cross-species transmission potential and endemicity in diverse host species, including humans. We conducted a molecular epidemiological survey of astroviruses in dromedaries from Saudi Arabia and Bactrian camels from Inner Mongolia, China. Herein, we deployed a hybrid sequencing approach coupling deep sequencing with rapid amplification of complementary deoxyribonucleic acid ends to characterize two novel Bactrian and eight dromedary camel astroviruses, including both partial and complete genomes. Our reported sequences expand the known diversity of dromedary camel astroviruses, highlighting potential recombination events among the astroviruses of camelids and other host species. In Bactrian camels, we detected partially conserved gene regions bearing resemblance to human astrovirus types 1, 4, and 8 although we were unable to recover complete reading frames from these samples. Continued surveillance of astroviruses in camelids, particularly Bactrian species and associated livestock, is highly recommended to identify patterns of cross-species transmission and to determine any epizootic threats and zoonotic risks posed to humans. Phylogenomic approaches are needed to investigate complex patterns of recombination among the astroviruses and to infer their evolutionary history across diverse host species.

A Novel Coronavirus and a Broad Range of Viruses in Kenyan Cave Bats.

BACKGROUND AND METHODS: To investigate virus diversity in hot zones of probable pathogen spillover, 54 oral-fecal swabs were processed from five bat species collected from three cave systems in Kenya, using metagenome sequencing. RESULTS: Viruses belonging to the Astroviridae, Circoviridae, Coronaviridae, Dicistroviridae, Herpesviridae and Retroviridae were detected, with unclassified viruses. Retroviral sequences were prevalent; 74.1% of all samples were positive, with distinct correlations between virus, site and host bat species. Detected retroviruses comprised Myotis myotis, Myotis ricketti, Myotis daubentonii and Galidia endogenous retroviruses, murine leukemia virus-related virus and Rhinolophus ferrumequinum retrovirus (RFRV). A near-complete genome of a local RFRV strain with identical genome organization and 2.8% nucleotide divergence from the prototype isolate was characterized. Bat coronavirus sequences were detected with a prevalence of 24.1%, where analyses on the ORF1ab region revealed a novel alphacoronavirus lineage. Astrovirus sequences were detected in 25.9%of all samples, with considerable diversity. In 9.2% of the samples, other viruses including Actinidia yellowing virus 2, bat betaherpesvirus, Bole tick virus 4, Cyclovirus and Rhopalosiphum padi virus were identified. CONCLUSIONS: Further monitoring of bats across Kenya is essential to facilitate early recognition of possibly emergent zoonotic viruses.

Pathogenic and metagenomic evaluations reveal the correlations of porcine epidemic diarrhea virus, porcine kobuvirus and porcine astroviruses with neonatal piglet diarrhea.

Porcine epidemic diarrhea virus (PEDV) frequently causes diarrhea outbreaks. However, whether newly discovered enteric viruses such as porcine kobuvirus (PKV) and porcine astroviruses (PAstVs) are also correlated with diarrhea is still unclear. Diarrhea outbreaks were reported in a PEDV-vaccinated pig farm in Xinjiang Uygur Autonomous Region of China from 2019 to 2020. PEDV was a common pathogen detected in fecal samples by routine RT-PCR assays. The PEDV positive fecal sample was used for pathogenic analysis due to the failure isolation of PEDV. The challenged neonatal piglets appeared watery diarrhea within one day post infection (dpi) and all died within 6 dpi. Histopathological and immunohistochemical examinations supported that PEDV is a major pathogen causing intestinal lesions. To further explore enteric viruses associated with neonatal piglet diarrhea, metagenomics sequencing was performed for the diarrheic piglets. Remarkably, PKV was the most abundant virus (58.33%) followed by PEDV (34.45%) and PAstVs (7.22%), which were also confirmed by real-time RT-PCR assays. Significant in vivo replications of PEDV and PKV could only be observed in challenged piglets whilst PAstVs maintained similar virus loads in both challenged and mock infected piglets. Overall, this study provides first pathogenic and metagenomic evidence that significant proliferations of PEDV and PKV are closely associated with severe diarrhea in neonatal piglets, while PAstVs likely play limited roles in neonatal piglet diarrhea.

Establishment and application of triple PCR method for FAstV, FPV and FECoV

Feline Astrovirus (FAstV), Feline Parvovirus(FPV) and Feline Enteric Coronavirus (FECoV) are important pathogens causing diarrhea in cats.In order to establish a molecular detection method which can differentiate the three pathogens in the same PCR system, an FAstV/FPV/FECoV triple PCR method was established with optimized primer concentrations and annealing temperature, and specificity, sensitivity and repeatability were tested. The results showed that the PCR method could only identify FAstV (320 bp), FPV (468 bp) and FECoV (664 bp) genes, while not other canine and feline related pathogens. The detection limits of FAstV, FPV and FECoV were 2x10~7 copy/L (7.1 pg/L),4.7x10~6 copy/L (2.4 pg/L) and 7x10~6 copy/L (5.1 pg/L) respectively. The established triple PCR method was used to detect 207 cat fecal samples collected in Chengdu from 2019 to 2020, including 141 diarrhea samples and 66 clinical health samples. The detection rates of FAstV, FPV and FECoV were 24.15% (50/207), 37.20% (77/207) and 15.46% (32/207) respectively, and the co-infection rates of FAstV/FPV, FPV/FECoV and FAstv/FECoV were 9.18%,6.28% and 6.28% respectively. In conclusion, the triple PCR method of FAstV/FPV/FECoV was successfully established, and could be applied for virus detection and epidemiological investigation.

Impact of the SARS-CoV-2 Pandemic on the Prevalence and Incidence of Gastrointestinal Viruses in Children up to Five Years Old: a Retrospective Cohort Study.

The aim is determining the impact of non-pharmaceutical measures (NPIs) against SARS-CoV-2 in the incidence and prevalence of gastrointestinal viruses (GV) in children. Demographic, analytical, and clinical data of children from which samples were received at the Hospital Universitario La Paz (Madrid, Spain) and that had a gastrointestinal infection with a positive sample through multiplex-PCR for GV were collected. The time periods included were prepandemic (P1): March 14, 2019 to March 14, 2020 and pandemic (P2): March 15, 2020 to March 15, 2021. The global prevalence, relative incidence (RI, per 1,000 admissions) and absolute incidence (AI, per 100,000 population) of GV were compared for both time periods. The prevalence of GV versus SARS-CoV-2 was determined for P2. Seven-hundred and 50 out of 2,547 children analyzed in P1 and 106 out of 1,368 in P2 were positive by PCR for GV (46.3% decrease in P2). Prevalence and RI of GV declined in P2, except for the RI of rotavirus. Adenovirus showed the largest decreased of prevalence and RI (100%), followed by sapovirus. Astrovirus reduction was less pronounced (3.1% versus 0.4%). Norovirus was the most frequent virus in both time periods and its prevalence and RI also decreased in P2 (15.2% versus 4.7% and 3.40 versus 1.74, respectively). Rotavirus had the smallest decrease in prevalence (2.6% versus 2.5%), and its RI increased during P2 from 0.7 to 0.93. After removing the rotavirus vaccine strains from the analysis, the prevalence and RI decreased during P2 (2.1% to 0.7% and 0.5 to 0.3, respectively). The AI decreased during P2 in all GV, and the prevalence of SARS-CoV-2 and GV was inversely proportional over time. Prevalence and incidence of GV have decreased during the pandemic, probably due to the implementation of NPIs against this virus and the reduction of health care attention to infections other than COVID-19. The differences in the decrease of prevalence and incidence for each virus may be explained by differences in the transmission and the resistance in the environment. Prevalence and RI of rotavirus might be biased since the PCR used detects both the infecting and the vaccine strains. IMPORTANCE Our original article contains an analysis of the impact of the measures applied against SARS-CoV-2 on the prevalence and incidence of GV in children. The small number of studies published to date that analyze the impact of these measures individually on each of the GV makes our study of great interest at this time.

Development of a Multiplex RT-PCR Assay for Simultaneous Detection of Four Potential Zoonotic Swine RNA Viruses.

Swine viruses like porcine sapovirus (SaV), porcine encephalomyocarditis virus (EMCV), porcine rotavirus A (RVA) and porcine astroviruses (AstV) are potentially zoonotic viruses or suspected of potential zoonosis. These viruses have been detected in pigs with or without clinical signs and often occur as coinfections. Despite the potential public health risks, no assay for detecting them all at once has been developed. Hence, in this study, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of SaV, EMCV, RVA and AstV from swine fecal samples. The PCR parameters were optimized using specific primers for each target virus. The assay's sensitivity, specificity, reproducibility, and application to field samples have been evaluated. Using a pool of plasmids containing the respective viral target fragments as a template, the developed mRT-PCR successfully detected 2.5 × 103 copies of each target virus. The assay's specificity was tested using six other swine viruses as a template and did not show any cross-reactivity. A total of 280 field samples were tested with the developed mRT-PCR assay. Positive rates for SaV, EMCV, RVA, and AstV were found to be 24.6% (69/280), 5% (14/280), 4.3% (12/280), and 17.5% (49/280), respectively. Compared to performing separate assays for each virus, this mRT-PCR assay is a simple, rapid, and cost-effective method for detecting mixed or single infections of SaV, EMCV, RVA, and AstV.

Where have the enteric viruses gone? - Differential effects on frequent causes of infectious diarrhoea by SARS-CoV-2 pandemic lockdown measures.

BACKGROUND: Measures of distancing, wearing face/medical masks and lockdown introduced in many countries to meet the challenges of the SARS-CoV-2 pandemic have led to gross changes in the epidemiology of important infections. The observation of decline of positive norovirus tests after introduction of lockdown in Germany led us to investigate changes in the detection of major causes of diarrhoea by comparing pre-pandemic quarters (PPQ: 1Q/17 through 1Q/20) since 2017 and pandemic quarters (PQ: 2Q/20 through 1Q/21). METHODS AND SETTING: Bioscientia Laboratory Ingelheim is a large regional clinical pathology laboratory serving > 50 hospitals and > 5000 general practitioners and specialist outpatient practices located in the federal states Hesse, Rhineland-Palatinate and North Rhine-Westphalia, Germany. Antigen detection assays were used for detection of astrovirus, adenovirus, rotavirus, and Campylobacter antigen and Clostridium difficile Toxin A/B, while norovirus was detected by qualitative RT-PCR. FINDINGS: The mean positivity-ratios of norovirus, adenovirus and astrovirus assays were 3-20 fold lower in periods PQ (2Q/20 through 1Q/21) compared to PPQ (1Q/17 through 1Q/20) (p<.01). The mean positivity-ratio was lower in PQ compared to PPQ for rotavirus (p=.31), but failed to reach statistical significance, while for campylobacter antigen (p=.91) and C. difficile Toxin A/B (p=.17) the mean positivity-ratio was even higher in PQ compared to PPQ. CONCLUSIONS: Apparently, hygienic measures used to contain the SARS-CoV-2 pandemic have differential effects on incidence of diarrhoea viruses as compared to bacterial gastrointestinal agents, particularly C. difficile, which may lead to re-evaluate measures implemented against this important cause of nosocomial diarrhoea.

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.

Human Astrovirus 1-8 Seroprevalence Evaluation in a United States Adult Population.

Human astroviruses are an important cause of viral gastroenteritis globally, yet few studies have investigated the serostatus of adults to establish rates of previous infection. Here, we applied biolayer interferometry immunosorbent assay (BLI-ISA), a recently developed serosurveillance technique, to measure the presence of blood plasma IgG antibodies directed towards the human astrovirus capsid spikes from serotypes 1-8 in a cross-sectional sample of a United States adult population. The seroprevalence rates of IgG antibodies were 73% for human astrovirus serotype 1, 62% for serotype 3, 52% for serotype 4, 29% for serotype 5, 27% for serotype 8, 22% for serotype 2, 8% for serotype 6, and 8% for serotype 7. Notably, seroprevalence rates for capsid spike antigens correlate with neutralizing antibody rates determined previously. This work is the first seroprevalence study evaluating all eight classical human astrovirus serotypes.

Development of SYBR Green I-based real-time reverse transcription polymerase chain reaction for the detection of feline astrovirus.

In this study, a SYBR Green I-based real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for the clinical diagnosis of feline astroviruses (FeAstVs). Specific primers were designed based on the conserved region of the FeAstV ORF1b gene. Experiments for specificity, sensitivity, and repeatability of the assay were carried out. In addition, the assay was evaluated using clinical samples. Specificity analysis indicated that the assay showed negative results with samples of Feline Parvovirus, Feline Herpesvirus, Feline Calicivirus, Feline Bocavirus, and Feline Coronavirus, indicating good specificity of the assay. Sensitivity analysis showed that the SYBR Green I-based real-time RT-PCR method could detect as low as 3.72 × 101 copies/µL of template, which is 100-fold more sensitive compared to the conventional RT-PCR. Both intra-assay and inter-assay variability were lower than 1 %, indicating good reproducibility. Furthermore, an analysis of 150 fecal samples showed that the positive detection rate of SYBR Green I-based real-time RT-PCR was higher than that of the conventional RT-PCR, indicating the high reliability of the method. The assay is cheap and effective. Therefore, it could provide support for the detection of FeAstV in large-scale clinical testing and epidemiological investigation.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.