Flies (Insecta, Diptera) collected in the environment of dairy farms as carriers of Rotavirus A and betacoronavirus.

AIMS: We aimed to investigate the prevalence of rotavirus and coronavirus in dipterans that commonly inhabit the environment of dairy farms. METHODS AND RESULTS: We collected 217 insect specimens from nine dairy farms, which were examined through hemi-nested RT-PCR followed by Sanger sequencing in search of VP1 and N genes for rotavirus and bovine coronavirus-BCoV, respectively. With a predominance of Muscidae (152/217 = 70%) 11 families of Diptera were identified. Rotavirus A (RVA) and betacoronavirus (BCoV) were detected in 14.7% (32/217) and 4.6% (10/217) of the dipterans, respectively. Sequencing of the amplicons was possible for 11.5% (25/217) of RVA and 0.5% (1/217) of BCoV, confirming the presence of these pathogens. CONCLUSIONS: Our findings highlight the role of dipterans as carriers of RVA and BCoV of great relevance for public and animal health.

Australian Rotavirus Surveillance Program: Annual Report, 2021.

Abstract: This report from the Australian Rotavirus Surveillance Program describes the circulating rotavirus genotypes identified in children and adults during the period 1 January to 31 December 2021. During this period, 521 faecal specimens had been referred for rotavirus G- and P- genotype analysis, of which 474 were confirmed as rotavirus positive. Of these, 336/474 were wildtype rotavirus strains and 138/474 were identified as vaccine-like. Of the 336 wildtype samples, 87.5% (n = 294/336) were identified as G8P[8], and were detected in five of the six jurisdictions that provided samples for the reporting period. Two rotavirus outbreaks, located in the Northern Territory and Western Australia, were also attributed to G8P[8]. As with the 2020 reporting period, a low number of stool samples were received for this reporting period as a result of the COVID-19 pandemic. However, an unexpectedly high proportion of samples with unusual genotypes were identified which were potentially zoonotic in nature, including feline G3, P[9], bovine-like G8, P[14], and porcine-like G4, G6, P[1], and P[6]. Ongoing rotavirus surveillance is crucial to identify changes in genotypic patterns and to provide diagnostic laboratories with quality assurance by reporting incidences of wildtype, vaccine-like, or false positive rotavirus results.

A comprehensive molecular survey of viral pathogens associated with canine gastroenteritis.

Viral pathogens are the primary cause of canine gastroenteritis. However, few structured comprehensive studies on the viral etiology of canine gastroenteritis have been conducted. In this study, 475 rectal swabs collected over three years (2018-2021) from clinical canine gastroenteritis cases were screened for the presence of six major enteric viruses - canine parvovirus 2 (CPV-2), canine distemper virus (CDV), canine adenovirus 2 (CAdV-2), canine coronavirus (CCoV), canine astrovirus (CaAstV), and canine rotavirus (CRV) - by real-time PCR. The most frequently detected virus was CPV-2, which was present in 64.8% of the samples (subtype 2a, 21.1%; 2b, 77.4%; 2c, 1.5%), followed by CDV (8%), CaAstV (7.2%), CCoV (5.9%), and CAdV-2 (4.6%). Two to four of these viruses in different combinations were found in 16.8% of the samples, and CRV was not detected. The complete genome sequences of Indian isolates of CDV, CCoV, and CaAstV were determined for the first time, and phylogenetic analysis was performed. This study highlights the need for routine prophylactic vaccination with the appropriate vaccines. Notably, 70.3% of animals vaccinated with DHPPiL were found to be positive for at least one virus. Hence, regular molecular analysis of the prevalent viruses is crucial for addressing vaccination failures.

One-Year Surveillance of SARS-CoV-2 and Rotavirus in Water Matrices from a Hot Spring Area.

The pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is still impacting not only on human health but also all economic activities, especially in those related to tourism. In this study, in order to characterize the presence of SARS-CoV-2 in a hot spring park in Uruguay, swimming pools water, wastewater, and surface water from this area were analyzed by quantitative PCR. Wastewater from Salto city located next to the hydrothermal spring area was also evaluated as well as the presence of Rotavirus (RV). Overall, SARS-CoV-2 was detected in 13% (13/102) of the analyzed samples. Moreover, this virus was not detected in any of the samples from the swimming pools water and was present in 18% (3/17) of wastewater samples from the hotels area showing the same trend between the titer of SARS-CoV-2 and the number of infected people in Salto city. SARS-CoV-2 was also detected in wastewater samples (32% (11/34)) from Salto city, detecting the first positive sample when 105 persons were positive for SARS-CoV-2. Rotavirus was detected only in 10% (2/24) of the wastewater samples analyzed in months when partial lockdown measures were taken, however, this virus was detected in nearly all wastewater samples analyzed when social distancing measures and partial lockdown were relaxed. Wastewater results confirmed the advantages of using the detection and quantification of viruses in this matrix in order to evaluate the presence of these viruses in the population, highlighting the usefulness of this approach to define and apply social distancing. This study suggests that waters from swimming pools are not a source of infection for SARS-CoV-2, although more studies are needed including infectivity assays in order to confirm this statement.

Rotavirus Spike Protein VP4 Mediates Viroplasm Assembly by Association to Actin Filaments.

Rotavirus (RV) viroplasms are cytosolic inclusions where both virus genome replication and primary steps of virus progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, that contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wild-type (wt) VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in coexpression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCE The spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens.

Rotavirus meningitis in an adult with transient aphasia.

We identified an additional case of documented Rotavirus meningitis in an adult with full medical history. A previously healthy 37-year-old patient presented herself for transient aphasia associated with fever and headaches at the end of a one-week history of gastroenteritis. Cerebrospinal fluid (CSF) analysis revealed lymphocytic meningitis, and treatment with aciclovir was initiated. Rotavirus A reverse transcription-polymerase chain reaction (RT-PCR) was positive in CSF and the patient's stools in favor of Rotavirus meningitis. Testing for other viruses was negative. Magnetic resonance imaging (MRI) showed no signs of encephalitis. Aphasia was resolutive in less than 12 hours, and no neurological symptoms relapsed. All symptoms evolved favorably despite aciclovir discontinuation. Viral sequencing methods have recently identified unexpected viruses as potential causative agents in meningitis, including Rotavirus. We confirm the detectability of Rotavirus in the analysis of CSF in the context of Rotavirus gastroenteritis in an adult. This case suggests postviral headache and neurological deficits with cerebrospinal fluid lymphocytosis (HaNDL) syndrome may be linked to previously undetected direct viral infection of the central nervous system. Therefore, clinicians should consider Rotavirus meningitis in diagnosing meningitis associated with gastroenteritis in adults.

A TaqMan Probe-Based Multiplex Real-Time PCR for Simultaneous Detection of Porcine Epidemic Diarrhea Virus Subtypes G1 and G2, and Porcine Rotavirus Groups A and C.

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/µL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/µL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.

Egyptian cobra (Naja haje haje) venom phospholipase A2: a promising antiviral agent with potent virucidal activity against simian rotavirus and bovine coronavirus.

Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC50 of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log10 TCID50 and 2.5 log10 TCID50, respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro.

Rotavirus and Norovirus Infections in Children Under 5 Years Old with Acute Gastroenteritis in Southwestern China, 2018-2020.

OBJECTIVE: Rotaviruses and noroviruses are important causes of acute gastroenteritis in children. While previous studies in China have mainly focused on rotavirus, we investigated the incidence of norovirus in addition to rotavirus in Southwestern China. METHODS: From January 2018 to December 2020, cases of rotavirus or norovirus infections among children under five ages with acute gastroenteritis were evaluated retrospectively. RESULTS: The detection rate of rotavirus was 24.5% (27,237/111,070) and norovirus was 26.1% (4649/17,797). Among 17,113 cases submitted for dual testing of both rotavirus and norovirus, mixed rotavirus/norovirus infections were detected in 5.0% (859/17,113) of cases. While there was no difference in norovirus incidence in outpatient compared to hospitalized cases, rotavirus was detected two times more in outpatients compared to hospitalized cases (26.6% vs.13.6%; P < 0.001). Both rotavirus and norovirus infections peaked in children aged 12-18 months seeking medical care with acute gastroenteritis (35.6% rotavirus cases; 8439/23,728 and 32.5% norovirus cases; 1660/5107). Rotavirus infections were frequent between December and March of each year while norovirus was detected earlier from October to December. Our results showed significant correlation between virus detection and environmental factors such as average monthly temperature but not relative humidity. In addition, we observed a reduction in the detection rates of rotavirus and norovirus at the beginning of the SARS-CoV-2 pandemic in 2020. CONCLUSION: Our results indicate that rotavirus and norovirus are still important viral agents in pediatric acute gastroenteritis in Southwestern China.

Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes.

Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

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.

Should countries switch to using five- or ten-dose rotavirus vaccines now that they are available?

INTRODUCTION: Single-dose rotavirus vaccines, which are used by a majority of countries, are some of the largest-sized vaccines in immunization programs, and have been shown to constrain supply chains and cause bottlenecks. Efforts have been made to reduce the size of the single-dose vaccines; however, with two-dose, five-dose and ten-dose options available, the question then is whether using multi-dose instead of single-dose rotavirus vaccines will improve vaccine availability. METHODS: We used HERMES-generated simulation models of the vaccine supply chains of the Republic of Benin, Mozambique, and Bihar, a state in India, to evaluate the operational and economic impact of implementing each of the nine different rotavirus vaccine presentations. RESULTS: Among single-dose rotavirus vaccines, using Rotarix RV1 MMP (multi-monodose presentation) led to the highest rotavirus vaccine availability (49-80%) and total vaccine availability (56-79%), and decreased total costs per dose administered ($0.02-$0.10) compared to using any other single-dose rotavirus vaccine. Using two-dose ROTASIIL decreased rotavirus vaccine availability by 3-6% across each supply chain compared to Rotarix RV1 MMP, the smallest single-dose vaccine. Using a five-dose rotavirus vaccine improved rotavirus vaccine availability (52-92%) and total vaccine availability (60-85%) compared to single-dose and two-dose vaccines. Further, using the ten-dose vaccine led to the highest rotavirus vaccine availability compared to all other rotavirus vaccines in both Benin and Bihar. CONCLUSION: Our results show that countries that implement five-dose or ten-dose rotavirus vaccines consistently reduce cold chain constraints and achieve higher rotavirus and total vaccine availability compared to using either single-dose or two-dose rotavirus vaccines.

The effect of Cryptosporidiumparvum, rotavirus, and coronavirus infection on the health and performance of male dairy calves.

The objective of this prospective cohort study was to investigate the effect of bovine coronavirus (BCoV), bovine rotavirus (BRoV), and Cryptosporidiumparvum on dairy calf health and performance and to determine the prevalence of these pathogens. A total of 198 male dairy calves housed at a grain-fed veal facility were examined from June 11, 2018, to October 9, 2018. Calves were fed milk replacer twice daily and housed individually until weaning at 56 d. Once weaned, calves were moved into groups of 5 until they were moved to a finishing facility at 77 d. At the grain-fed veal facility, calves were scored for fecal consistency for the first 28 d and had fecal samples taken on arrival and at 7 and 14 d. Fecal samples were frozen and submitted to a commercial laboratory, where they were tested for BCoV, C.parvum, and 2 groups of BRoV: group A (BRoV A) and group B (BRoV B). Calves were weighed on arrival and at 14, 49, 56, and 77 d using a digital body scale. Treatments for disease and mortalities that occurred over the 77 d were also recorded. Statistical models, including Cox proportional hazards and repeated measures models, were built to determine the effect of infection with 1 of the pathogens. Over the 3 sampling points, 151 (85.8%), 178 (94.2%), 3 (1.5%), and 97 (57.4%) calves tested positive at least once for BCoV, BRoV A, BRoV B, and C.parvum, respectively. The source of the calves and the level of serum total protein measured on arrival were associated with testing positive for a pathogen. Calves that tested positive for C.parvum had an increased proportion of days with diarrhea and severe diarrhea; calves that tested positive for BCoV and BRoV A had an increased proportion of days with severe diarrhea. In addition, calves that tested positive for C.parvum had a higher hazard of being treated for respiratory disease. With respect to body weight, calves that had diarrhea or severe diarrhea had lower body weight at 49, 56, and 77 d. Specifically, calves that had an increased proportion of days with diarrhea showed a reduction in weight gain of up to 15 kg compared to calves without diarrhea. Calves that tested positive for C.parvum had a lower body weight at 49, 56, and 77 d; calves that tested positive for BCoV had a lower body weight at 56 and 77 d. This study demonstrates that the prevalence of BCoV, BRoV A, and C.parvum infection is high in this population of calves and has significant effects on the occurrence of diarrhea and body weight gain. Future studies should evaluate approaches for minimizing the effect of infection with these pathogens to improve the welfare, health, and productivity of dairy calves.