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Caliciviruses (Caliciviridae) and astroviruses (Astroviridae) are among the leading cause of non-bacterial foodborne disease and gastroenteritis in human. These non-enveloped RNA viruses infect a wide range of vertebrate species including rodents. Rodents are among the most important hosts of infectious diseases globally and are responsible for over 80 zoonotic pathogens that affect humans. Therefore, screening pathogens in rodents will be is necessary to prevent cross-species transmission to prevent zoonotic outbreaks. In the present study, we screened caliciviruses and astroviruses in order to describe their diversity and whether they harbor strains that can infect humans. RNA was then extracted from intestine samples of 245 rodents and retrotranscribed in cDNA to screen caliciviruses and astroviruses by PCRs. All the samples tested negative for caliciviruses and while astroviruses were detected in 18 (7.3%) samples of Rattus rattus species. Phylogenetic analyses based on the RdRp gene showed that all the sequences belonged to Mamastrovirus genus in which they were genetically related to R. rattus related AstVs previously detected in Gabon or in Rattus spp. AstV from Kenya and Asia. These findings suggested that transportation such as land and railway, as well national and international trade, are likely to facilitate spread of AstVs by the dissemination of rodents., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Astroviruses (AstVs) are RNA viruses infecting a large diversity of avian and mammalian species, including bats, livestock, and humans. We investigated AstV infection in a free-tailed bat species, Mormopterus francoismoutoui , endemic to Reunion Island. A total of 380 guano samples were collected in a maternity colony during 38 different sampling sessions, from 21 June 2016 to 4 September 2018. Each sample was tested for the presence of the AstV RNA-dependent RNA-polymerase (RdRp) gene using a pan-AstV semi-nested polymerase chain reaction assay. In total, 27 guano samples (7.1%) tested positive, with high genetic diversity of the partial RdRp gene sequences among positive samples. Phylogenetic analysis further revealed that the detected viruses were genetically related to AstVs reported in rats, reptiles, dogs, and pigs, but did not cluster with AstVs commonly found in bats. Although more investigations need to be conducted to assess the prevalence of infected bats in the studied population, our findings show that Reunion free-tailed bats are exposed to AstVs, and suggest that cross-species transmission may occur with other hosts sharing the same habitat.

Astroviruses are non-enveloped, single-stranded RNA viruses that infect mammalian and avian species. In humans, astrovirus infections are one of the most common causes of gastroenteritis in children. Infection has also been linked to serious neurological complications, especially in immunocompromised individuals. More extensive disease has also been characterized in non-human mammalian and avian species. To date, astroviruses have been detected in over 80 different avian and mammalian hosts. As the number of hosts continues to rise, the need to understand how astroviruses transmit within a given species as well as to new host species becomes increasingly important. Here, we review the current understanding of astrovirus transmission, the factors that influence viral spread, and the potential for cross-species transmission. Additionally, we highlight the current gaps in knowledge and areas of future research that will be key to understanding astrovirus transmission and zoonotic potential.

We investigated an outbreak of acute gastroenteritis due to human astrovirus in a daycare center, describing the transmission mechanism, the most affected age groups, conditioning factors and the extent of the outbreak among household contacts of the daycare center attenders. Data were collected from persons exposed at the daycare center and their home contacts. Fecal samples from affected and non-affected daycare center attenders were analyzed for viruses causing acute gastroenteritis by RT-PCR. The percentage of households affected and the attack rates (AR) were calculated. The attack rates were compared using the rate ratio (RR) with 95% confidence intervals. Information was obtained from 245 people (76 attenders and 169 contacts) of whom 49 were clinical cases. Five HAstV-4, two HAstV-8 and three non-typable HAstV cases were identified (six from clinical cases and four from asymptomatic infected people). The global AR was 20% (41.2% in children aged < 2 years). Data were obtained from 67 households: 20 households of affected attenders and 47 of non-affected attendees. Household contacts of affected attenders had a higher AR (74.3%) than that of non-affected attendees (2.4%). We found asymptomatic infections amongst daycare attendees. The transmission of HAstV during the outbreak was not limited to the daycare center but extended to household contacts of both affected and non-affected attenders.

In 2019, a novel goose astrovirus (GoAstV) epidemiological investigation on geese was conducted in Shandong province, China. During the investigation, a high prevalence of novel GoAstV was observed in symptom-free breeding geese flocks. Moreover, the novel GoAstV-specific RNA was detected in either breeder birds or their progenies. To verify the hypothesis that the novel GoAstV could be transmitted vertically, a total of 42 WuLong breeder geese, aged 335 days, were equally divided into three groups for experimental infection. The SDPY isolate of novel GoAstV (A/goose/Shandong/SDPY/2018, SDPY), preserved in our laboratory, was injected intramuscularly to subjects of group A while orally inoculated to those of group B. After the inoculation, novel GoAstV RNA was detected in vitelline membrane, embryos, and allantoic fluid of goose embryos in novel GoAstV infected groups. Moreover, the ORF2 gene of novel GoAstV from vitelline membrane, embryo, allantoic fluid as well as gosling shared almost 100 % nucleotide homology to a novel GoAstV virus isolated from the goose ovary which produced the egg, suggesting that the novel GoAstV can be vertically transmitted in the goose. Taken together, the findings provide evidence of possible vertical transmission of novel GoAstV from breeding goose to goslings., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Astroviruses are small, non-enveloped, positive-sense, single-stranded RNA viruses that belong to the Astroviridae family. Astroviruses infect diverse hosts and are typically associated with gastrointestinal illness; although disease can range from asymptomatic to encephalitis depending on the host and viral genotype. Astroviruses have high genetic variability due to an error prone polymerase and frequent recombination events between strains. Once thought to be species specific, recent evidence suggests astroviruses can spread between different host species, although the frequency with which this occurs and the restrictions that regulate the process are unknown. Recombination events can lead to drastic evolutionary changes and contribute to cross-species transmission events. This work reviews the current state of research on astrovirus evolution and emergence, especially as it relates to cross-species transmission and recombination of astroviruses., (Copyright © 2019. Published by Elsevier B.V.)

The aim of this study was to investigate canine astrovirus (CaAstV) infection in southwest China. We collected 107 faecal samples from domestic dogs with obvious diarrhoea. Forty-two diarrhoeic samples (39.3 %) were positive for CaAstV by RT-PCR, and 41/42 samples showed co-infection with canine coronavirus (CCoV), canine parvovirus-2 (CPV-2) and canine distemper virus (CDV). Phylogenetic analysis based on 26 CaAstV partial ORF1a and ORF1b sequences revealed that most CaAstV strains showed unique evolutionary features. Interestingly, putative recombination events were observed among four of the five complete ORF2 sequences cloned in this study, and three of the five complete ORF2 sequences formed a single unique group, suggesting that these strains could be a novel genotype. We successfully sequenced the complete genome of one CaAstV strain (designated 2017/44/CHN), which was 6628 nt in length. The features of this genome include putative recombination events in the ORF1a, ORF1b and ORF2 genes, while the ORF2 gene had a continuous insertion of 7 aa in region II compared with the other complete ORF2 sequences available in GenBank. Phylogenetic analysis showed that 2017/44/CHN formed a single group based on genome sequences, suggesting that this strain might be a novel genotype. The results of this study revealed that CaAstV circulates widely in diarrhoeic dogs in southwest China and exhibits unique evolutionary events. To the best of our knowledge, this is the first report of recombination events in CaAstV, and it contributes to further understanding of the genetic evolution of CaAstV.

Astroviruses are a common cause of gastroenteritis in children worldwide and can also cause infection in a range of domestic and wild animal species. Canine astrovirus (formally named as Mamastrovirus 5, MAstV5) has been reported worldwide, and its role as an enteric pathogen is still controversial. Herein, we describe the genomic characterization of a MAstV5 (strain crab-eating fox/2016/BRA) identified in a wild canid (Cerdocyon thous) diagnosed with canine distemper virus (CDV) as causa mortis. The nearly complete genome comprised 6579 nt in length and displayed the archetypal organization of astroviruses. The present report is the first evidence of MAstV5 infection in an animal species other than the dog and highlights a possible natural astrovirus spillover between domestic and wild canids. Moreover, these results show the first evidence of extra-intestinal MAstV5, suggesting a virus systemic spread. This work is expected to contribute to a better understanding of the astroviruses biology and their interactions with the wildlife health., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We report the identification of a neurotropic astrovirus associated with encephalitis in a sheep. This virus is genetically almost identical to an astrovirus recently described in neurologically diseased cattle. The similarity indicates that astroviruses of the same genotype may cause encephalitis in different species.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. Our goal was to determine the duration of MuAstV infection, susceptibility of pups, and efficacy of soiled-bedding sentinels and environmental monitoring. Eight CD1 dams and their litters of 3-d-old pups and 8 CD1 dams and their litters of 13-d-old mice were inoculated orally with MuAstV. Neither dams nor offspring demonstrated any clinical signs, and MuAstV had little to no effect on weight gain in pups. MuAstV RNA was detected in feces from 15 of the 16 dams through postnatal day (PND) 21, and 9 dams were still shedding MuAstV at PND 42. MuAstV RNA was highest in intestines of mice. Low levels of MuAstV RNA were sporadically detected in the spleen, liver, and kidney. MuAstV was detected in 97% of feces from 3- to 9-wk-old mice born to infected dams. Several weanlings became pregnant, and intestines from their pups were MuAstV-negative at PND 0 through 5. Weekly swabs of cages housing MuAstV-infected mice were MuAstV-positive through PND 42. Swabs of the rear exhaust manifold of the ventilated rack were MuAstV-positive at 21 through 56 d after inoculation. In addition, 98% of sentinels that received soiled bedding from dams and their litters and 83% of sentinels that received soiled bedding from weaned mice were MuAstV-positive. Feces from most sentinels exposed to soiled bedding that had been stored for 1, 2 or 3 wk before addition of the sentinels were MuAstV-positive.

Astroviruses (AstVs) can infect a variety of hosts, including mammalian and avian species, and are commonly associated with enteric infections. Recently, mammalian AstVs have been linked to extra-intestinal manifestations, including neurologic disorders in humans, cattle and minks, demonstrating zoonotic potential. So far, five porcine AstV (PAstV) genotypes have been identified, with PAstV1, PAstV2, PAstV3 and PAstV5 implicated in cross-species transmission. Our knowledge about PAstV epidemiology in China is still limited. In this study, two duplex differential RT-PCR assays were developed to investigate the distribution and prevalence of PAstV1, PAstV2, PAstV4 and PAstV5. Two hundred eighteen samples were collected from 33 farms and pigs with known diarrhea status in nine regions of Hunan province in China. Specifically, 126 small intestines, 51 fecal swabs, 20 lungs, 19 spleens and two kidneys were obtained. PAstVs were detected in all nine regions and in 81.8% (27/33) of the pig farms investigated. The overall prevalence of PAstV was 46.3% (101/218), with PAstV5 as the predominant type, with a positive rate of 24.8% (54/218). The prevalence of PAstV4, PAstV1 and PAstV2 was 16.1% (35/218), 14.7% (32/218) and 10.1% (22/218), respectively. Besides being present in intestines and fecal swabs, PAstV RNA was also detected in lungs, spleens and kidneys. Sequencing revealed a high level of genetic divergence within each genotype, and a higher positive rate of PAstV5 was associated with pigs with diarrhea compared to pigs without diarrhea. This study revealed for the first time that PAstV4 is circulating in China, and that PAstV5 is the dominant genotype in pig herds in Hunan province in China.

Canine astrovirus RNA was detected in the stools of 17/63 (26.9%) samples, using either a broadly reactive consensus RT-PCR for astroviruses or random RT-PCR coupled with massive deep sequencing. The complete or nearly complete genome sequence of five canine astroviruses was reconstructed that allowed mapping the genome organization and to investigate the genetic diversity of these viruses. The genome was about 6.6kb in length and contained three open reading frames (ORFs) flanked by a 5' UTR, and a 3' UTR plus a poly-A tail. ORF1a and ORF1b overlapped by 43 nucleotides while the ORF2 overlapped by 8 nucleotides with the 3' end of ORF1b. Upon genome comparison, four strains (HUN/2012/2, HUN/2012/6, HUN/2012/115, and HUN/2012/135) were more related genetically to each other and to UK canine astroviruses (88-96% nt identity), whilst strain HUN/2012/126 was more divergent (75-76% nt identity). In the ORF1b and ORF2, strains HUN/2012/2, HUN/2012/6, and HUN/2012/135 were related genetically to other canine astroviruses identified formerly in Europe and China, whereas strain HUN/2012/126 was related genetically to a divergent canine astrovirus strain, ITA/2010/Zoid. For one canine astrovirus, HUN/2012/8, only a 3.2kb portion of the genome, at the 3' end, could be determined. Interestingly, this strain possessed unique genetic signatures (including a longer ORF1b/ORF2 overlap and a longer 3'UTR) and it was divergent in both ORF1b and ORF2 from all other canine astroviruses, with the highest nucleotide sequence identity (68% and 63%, respectively) to a mink astrovirus, thus suggesting a possible event of interspecies transmission. The genetic heterogeneity of canine astroviruses may pose a challenge for the diagnostics and for future prophylaxis strategies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bats belong to the order Chiroptera that represents the second largest order of mammals with more than 1200 species and an almost global distribution. Environmental changes and deforestation have severely influenced many ecosystems, intensifying the contact between wildlife and humans. In recent years, bats have been found to harbor a number of different viruses with zoonotic potential, as well as a great diversity of astroviruses, for which the question of zoonotic potential remains unanswered to date. Human astroviruses have been identified as the causative agent for diarrhea in children and immunocompromised patients. For a long time, astroviruses have been considered to be strictly species-specific. However, a great genetic diversity has recently been discovered among animal and human astroviruses that might indicate the potential of these viruses to cross species barriers. Furthermore, our knowledge about the tissue tropism of astroviruses has been expanded to some neurotropic strains that have recently been shown to be responsible for encephalitis in humans and livestock. This review gives an overview on what is known about astroviruses in bats, humans and livestock, especially bovines and pigs. Future research activities are suggested to unravel astrovirus infection dynamics in bat populations to further assess the zoonotic potential of these viruses.

The transmission routes of duck astrovirus CPH (DAstV/CPH) and its pathogenicity in duck embryos were investigated. Using a reverse transcription-polymerase chain reaction (RT-PCR) developed in this study, DAstV/CPH was detected in 23/50 fresh droppings of breeder ducks, 39/65 breeding eggs, 26/31 dead embryos, and 6/10 newly hatched ducklings, which were taken from a Pekin duck farm where DAstV/CPH had previously been identified. This finding, and the detection of DAstV/CPH in 36/130 dead-in-shell duck embryo samples collected from different hatcheries located in six provinces, suggests that the virus may be horizontally and vertically transmitted and associated with hatchability problems. Inoculation and repeated passages in embryonating duck eggs resulted in isolation of DAstV/CPH. The virus caused severe chorioallantoic membrane lesions as well as growth retardation and embryo mortality, indicating that DAstV/CPH is pathogenic for duck embryos. The effect of DAstV/CPH on hatching was confirmed by an embryo infection experiment in which 8/10 9-day-old duck embryos inoculated with the third passage of DAstV/CPH were unable to hatch, with most embryos succumbing in the final stage of incubation. The use of RT-PCR on the hatched ducklings provided evidence that the embryos could develop into infected ducklings.

A viral metagenomics approach was used to investigate fecal samples of Japanese calves with and without diarrhea. Of the different viral pathogens detected, read counts gave nearly complete astrovirus-related RNA sequences in 15 of the 146 fecal samples collected in three distinct areas (Hokkaido, Ishikawa, and Kagoshima Prefectures) between 2009 and 2015. Due to the lack of genetic information about bovine astroviruses (BoAstVs) in Japan, these sequences were analyzed in this study. Nine of the 15 Japanese BoAstVs were closely related to Chinese BoAstVs and clustered into a lineage (tentatively named lineage 1) in all phylogenetic trees. Three of 15 strains were phylogenetically separate from lineage 1, showing low sequence identities, and clustered instead with an American strain isolated from cattle with respiratory disease (tentatively named lineage 2). Interestingly, two of 15 strains clustered with lineage 1 in the open reading frame (ORF)1a and ORF1b regions, while they clustered with lineage 2 in the ORF2 region. Remarkably, one of 15 strains exhibited low amino acid sequence similarity to other BoAstVs and was clustered separately with porcine astrovirus type 5 in all trees, and ovine astrovirus in the ORF2 region, suggesting past interspecies transmission.

Although RNA viruses exhibit a high frequency of host jumps, major differences exist among the different virus families. Astroviruses infect a wide range of hosts, affecting both public health systems and economic production chains. Here we delineate the ecological and adaptive processes that drive the cross-species transmission of astroviruses. We observe that distinct transmission zones determine the prevailing astrovirus host and virus diversity, which in turn suggests that no single host group (e.g., bats) can be the natural reservoir, as illustrated through our phylogenetic analysis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Avian astroviruses, of the genus Avastrovirus, are recognized as being the cause of enteritis in different bird species worldwide. In particular, turkeys are very susceptible and can be severely affected by this viral agent. More recently, astroviruses were detected in diseased guinea fowl in Italy but whether or not they were the causative agents of the clinical disease was not established. Despite the distribution and relevance of Avastrovirus infection, very little information on pathogenesis or factors influencing the pathogenicity of astroviruses is available. To increase available data on the pathogenesis of these viruses and to test the hypothesis of possible interspecies transmission, experimental infections were carried out in turkeys and guinea fowl with two genetically distinct avian astroviruses, namely TK-6363 and GF-5497, originating respectively from diseased turkey poults and guinea fowl. Data obtained in our study show that both of the viruses selected were able to infect young birds of the species in which they were originally detected. Additionally, these viruses were able to infect young birds of different species causing clinical signs, thus providing experimental evidence for the infection of distinct avian astroviruses in different avian species.

Astroviruses can infect mammalian and avian species and are often responsible for gastroenteric disease symptoms. In this study, the complete open reading frame (ORF) 2, the 3' end of ORF1b and the corresponding intergenic region of astroviruses identified in farmed guinea fowl (Numida meleagris) were sequenced and genetically analysed. Overall, the genetic sequence of guinea fowl astroviruses was related to turkey astrovirus type 2 (TastV2), although a marked genetic distance was revealed based on ORF2, which might indicate the circulation of a distinct virus genotype and serotype in guinea fowl. Furthermore, the genetic data presented herein suggest that either recombination between different astroviruses infecting distinct hosts or adaptation of a given astrovirus to a new host had occurred. In either case, direct or indirect interspecies transmission of astroviruses is likely to have occurred between turkey and guinea fowl, indicating the ability of viruses belonging to the family Astroviridae to cross species barriers.

Astroviruses are small, non-enveloped, positive sense, single-stranded RNA viruses first identified in 1975 in children suffering from diarrhea and then described in a wide variety of animals. To date, the list of animal species susceptible to astrovirus infection has expanded to 22 animal species or families, including domestic, synantropic and wild animals, avian, and mammalian species in the terrestrial and aquatic environments. Astrovirus infections are considered among the most common cause of gastroenteritis in children, second only to rotavirus infections, but in animals their association with enteric diseases is not well documented, with the exception of turkey and mink astrovirus infection. Genetic variability has been described in almost all astrovirus species sufficiently examined infecting mammals and birds; however, antigenic variability has been demonstrated for human astroviruses but is far less investigated in animal viruses. Interestingly, there is an increasing evidence of recombination events occurring in astroviruses, which contributes to increase the genetic variability of this group of viruses. A wide variety of species infected, the evident virus genetic diversity and the occurrence of recombination events indicate or imply either cross-species transmission and subsequent virus adaptation to new hosts or the co-infection of the same host with different astroviruses. This can also favor the emergence of novel astroviruses infecting animals or with a zoonotic potential. After more than 30 years from their first description in humans, there are many exciting streams of research to be explored and intriguing questions that remain to be answered about the relatively under-studied Astroviridae family. In the present work, we will review the existing knowledge concerning astrovirus infections in humans and animals, with particular focus on the molecular biology, interspecies transmission and zoonotic potential of this group of viruses., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An epidemic outbreak of both norovirus (NV) and astrovirus (ASV) occurred on a research ship surveying Tokyo Bay, causing acute gastroenteritis in 26 of its 37 crew members. The presence of viral pathogens in fecal specimens was analyzed, and noroviruses were identified by reverse transcription-PCR in 18 (48.6%) of these specimens. In addition, astroviruses were identified in 14 (37.8%) of the fecal samples from the affected crew members, and multiple viral infections of both NV and ASV were observed in 6 cases. The genogrouping of the NV-positive samples was then examined by dot blot hybridization, and it was determined that all of the isolates were from genogroup II (GII). No bacterial pathogens were subsequently isolated from fecal specimens. Furthermore, a variety of NV strains were identified by sequencing and single-stranded conformational polymorphism (SSCP) analyses of PCR products from the fecal samples. One recombinant NV isolate, Minato/14, was identified as a recombinant NV strain of GII/6 and GII/1. The other NV isolates from this outbreak were classified into three NV genotypes (GII/1 [Minato/10], GII/4 [Minato/33], and GII/5 [Minato/6]). Furthermore, ASVs in positive samples were determined to belong to serotypes 1 and 2 by sequencing analysis. Our findings thus indicate that coinfections with NV and ASV, including a number of NV genotypes, persisted during an outbreak of gastroenteritis in a closed environment.

Purpose of Review: Public health concerns related to enteric viral agents, such as astroviruses and caliciviruses, include their ability to cause sporadic diarrhea, large outbreaks of gastroenteritis, and hospitalizations or deaths resulting from vomiting, diarrhea, and dehydration. Improved surveillance and application of sensitive molecular assays has increased awareness of these enteric pathogens and reduced the 'diagnostic gap' or unknown causes of non-bacterial gastroenteritis., Recent Findings: Molecular assays have been applied to further describe the epidemiology of human astroviruses from a variety of geographic areas. The burden of astrovirus infections compared with other enteric viral agents, including rotaviruses, caliciviruses, and enteric adenoviruses have been reported. New methods for detection of astroviruses such as reverse transcription-polymerase chain reaction and molecular typing methods have advanced the understanding of the epidemiology. Additional molecular studies have described the protein processing mechanisms of this single-stranded RNA virus., Summary: Astroviruses are increasingly recognized as significant gastrointestinal pathogens. The understanding of molecular epidemiology and molecular processing of the virus may lead to specific prevention strategies.

The persistence of human astroviruses dried on representative porous (paper) and nonporous (china) surfaces was investigated. Long-term astrovirus survival on fomites was monitored by an integrated cell culture-reverse transcription-PCR procedure. Viruses were applied to inanimate surfaces in the presence and absence of fecal material, and their survival was assayed at 4 and 20 degrees C with high relative humidity. Astroviruses exhibited a notable persistence when dried on porous and nonporous materials, particularly at low temperature. Short-term survival of astroviruses on fomites was compared to that of other enteric viruses significant for health, such as rotavirus, adenovirus, poliovirus, and hepatitis A virus. Overall, astroviruses persisted better than poliovirus and adenovirus, although they exhibited a shorter survival than rotavirus and hepatitis A virus. Astroviruses show a high level of persistence at the desiccation step, which is of major significance in determining the chance of subsequent virus survival dried on fomites. Astroviruses are able to survive on inert surfaces long enough to suggest that fomites may play a relevant role in the secondary transmission of astrovirus diarrhea.

Human astrovirus (HAstV) is a significant cause of acute diarrhea among children, resulting in outbreaks of diarrhea and occasionally in hospitalization. Improved detection methods for eight antigenic types of HAstV and studies assessing the frequency and severity of HAstV diarrhea have further defined the impact of HAstV infections in children. These studies have shown that HAstV infections are clinically milder (diarrhea, vomiting, fever) than rotavirus infections. However, frequent coinfection of HAstV with rotavirus and caliciviruses in childhood diarrhea complicates the epidemiology. Seroprevalence studies have provided evidence that the majority of children are infected by HAstV by 6 years of age. The route of transmission is probably fecal-oral from food or water sources. Recent and planned studies will help to define the epidemiology and in the future lead to prevention strategies, which could include vaccination.