Synanthropic rodents as virus reservoirs and transmitters

Abstract This review focuses on reports of hepatitis E virus, hantavirus, rotavirus, coronavirus, and arenavirus in synanthropic rodents (Rattus rattus, Rattus norvegicus, and Mus musculus) within urban environments. Despite their potential impact on human health, relatively few studies have addressed the monitoring of these viruses in rodents. Comprehensive control and preventive activities should include actions such as the elimination or reduction of rat and mouse populations, sanitary education, reduction of shelters for the animals, and restriction of the access of rodents to residences, water, and food supplies.


INTRODUCTION
Rodents (Order: Rodentia) are distributed on all continents except for Antarctica 1 . Their heterogeneous and cosmopolitan distribution expands as their interaction with humans increases 2 . Some species are better able to adapt to urban environments (synanthropism).
In a study conducted in Buenos Aires (Argentina), black rats (Rattus rattus) were found in residential and industrial areas, while house mice (Mus musculus) and brown rats (Rattus norvegicus) were captured in green areas and shantytowns 3 , where their presence was favored by easy access, availability of shelter, and large food supply 4 .
These animals are natural reservoirs of infectious diseases 5,6 , and are involved in the emergence and dissemination of viruses, bacteria, and protozoa. The transmission of these agents can occur through both direct (bite, contact) and indirect (urine, feces) means, through vectors (ticks, fleas, and mites) that infest rodents, or when they are predated by other species 1,7,8 .
Although little information is currently available on the size of the population of synanthropic rats, some indicators allow the subjective estimation of their presence, such as the presence of excrement, marks on walls, trails on the ground, food sources, or the visual observation of rodents and/or the damage caused by them [9][10][11] .
To evaluate the size of rodent populations, the concept of the 'minimum known number' is used 12 , where individuals have to be captured and recaptured to estimate the infestation rate. This statistical formula can be simplified by multiplying the number of animals caught in a trap in a single catch by 100 11,13 .
A survey carried out in 1529 dwellings in a low-income region of São Paulo (Brazil) showed an initial synanthropic rodent infestation rate of 40%, which was reduced to 14.4% after the implementation of sanitary education and pest control 13 . Similarly, in Pau de Lima (Bahia, Brazil), 62% of households (137/221) presented signs of active rodent infestations 14 .
Besides synanthropic species, other rodents can cocirculate in rural environments and wild and urban interface areas, where they contact other animals and people 10,15 . Wild rodents are reservoirs for hantavirus, vaccinia virus, and Lassa virus, among others [16][17][18][19] .  20 investigated rural settlements in Goiás (Brazil), and found 2.57% (n=12) positive rate for Orthohantavirus, equally distributed between women and men (n=6). In contrast, similar studies showed higher frequency in middle-aged men due to their risk behaviors 21,22 .
Refugee camps accommodate large number of people and consequently there is accumulation of food and residues, attracting rodents (synanthropic and wild). In Africa, studies have revealed the circulation of Mastomys natalensis infected by Lassa virus in Thus, hepatitis E, hantavirus, rotavirus, coronavirus, and arenavirus are the focus of this review, as they are neglected diseases transmitted by rodents.

HEPATITIS E (HEV)
The hepatitis E virus (HEV) has a single-stranded RNA genome of approximately 7 kb length 28 . As a member of the Hepeviridae family, the genus Orthohepevirus has four species (from A to D) 29 , among which Orthohepevirus A and C have already been described in rodents 30 .
Infection occurs via the fecal-oral route through the consumption of water contaminated with excrement or the consumption of raw/undercooked meat and the viscera of infected animals 31 . The prevalence rate in humans reaches 40% in industrialized countries, and the virus has been detected in blood banks 32 . Socially vulnerable people may be an important epidemiological group at risk, along with patients who depend on blood transfusions 33,34 .
The virus shows tropism to digestive system and is eliminated in stool after 4 to 23 days of infection 35 , remaining in this state for additional 5 weeks 36,37 .
The symptoms are mainly nonspecific, and include fever, headaches, abdominal pain, but these infections may also be asymptomatic (depending on the HEV dose to which the patient was exposed), hindering the detection, and potentiating the agent's spread 38-40 .
Mortality rates are higher among infected people presenting previous liver disease, immunocompromised patients, and pregnant women, as they present higher chances of renal failure leading to death 33,41,42 .
The role of black rats (R. rattus) and brown rats (R. norvegicus) as reservoirs and transmitters of this viral agent and its prevalence rate remains unknown 30 .
Orthohepevirus A has seven different genotypes (HEV1-7) defined by the concatenated amino acid distance between the open reading frames of ORF1 (nonstructural proteins) and ORF2 (capsid proteins) 43 . HEV-1 and HEV-2 occur only in humans; HEV-3 has been isolated from humans and several animal species; HEV-4 has been isolated from humans and pigs; HEV-5 and HEV-6 have been identified only in wild boars; and HEV-7 has only been found in camels 44 .
The presence of anti-HEV IgG 30,45 , and the detection of viral particles in the feces of these rodents (with or without seroconversion) have been demonstrated 37,46,47 . However, only a single study has shown similarities between rodent (R. norvegicus) strains with regard to the HEV-3 genotype, which is most closely related to the genotypes found in rabbits 37 .
On the other hand, rat HEV (genotype C1), belonging to the Orthohepevirus C group, has been reported in R. norvegicus and R. rattus, although its potential to cause disease in humans is still questioned 37,47 .
In Vietnam, animals captured at bus stations and hospitals have tested positive for rat HEV 48 . These findings are supported by serological evidence from domestic animals and rodents in other studies 45,49,50 .
Detection methods for HEV include serological (specific IgG), histopathological, and molecular (RT-PCR) techniques 41 . Commercially, there are no specific prophylatic measures available on the market, although Chinese researchers have developed the HEV p239 vaccine from the HEV1 genotype 51 . Its efficacy is considered high (> 90%), requiring three doses (0,.1 and 6 months), and it may be used even in pregnant women 52 .
More than 50 species of hantaviruses have been reported worldwide 55 ; however, some of them do not cause diseases, including the Prospect Hill virus 56 . Rodents, bats, and moles are reservoirs of these agents 57 ; transmission occurs through bites (saliva), and especially via the inhalation of viral particles from the feces and urine of these animals 58 . Despite a report of transmission between humans, this form is rare 59 .
The Orthohantavirus genus includes the greatest number of pathogenic species of public health importance 60 . Its presence is associated with the geographic distribution of rodents (Murinae, Avicolinae, and Sigmodontinae families), which can harbor distinct forms of the disease.
The earliest reports of hemorrhagic fever with renal syndrome (HFRS), caused by viruses known as Old World hantaviruses (Europe and Asia), come from Chinese writings dating from 960 BC. Later, in the Korean War (1951)(1952)(1953)(1954), these diseases caused the death of over 3,000 soldiers 61 . The Hantaan virus (HTNV) species was related to this outbreak. HTNV was isolated for the first time in 1978 62 and was linked to the rodent Apodemus agrarius; the virus was detected in blood, urine, feces, and respiratory tract samples. In humans, this species causes a severe form of HFRS, which has thus far been restricted to rural areas of China, Korea, and Russia 63,64 .
The most commonly detected viral agent of this group in Western Europe, Puumala virus (PUUV), is disseminated by Myodes glareolus, whose proliferation is favored by the underbrush vegetation of this region, and the spread of the virus is further affected by virus-host coevolution 68 . In humans, it causes moderate nephropathy, and can lead to subclinical infections 69 .
Seoul virus usually causes mild infections and without medical care the mortality rates reach 1%. The host of this virus (R. norvegicus) is found in urban areas, leading to a cosmopolitan distribution of the disease, in contrast to those caused by other Old World hantaviruses 60,70 . The incubation period varies from 2-3 weeks, and the endothelial cell tropism of the virus 71 produces nonspecific symptomatology (fever, headaches, muscle pain, nausea, vomiting), in addition to respiratory problems, dizziness, and diarrhea. Thrombocytopenia entails the development of petechias, and decreased blood pressure affects kidney function, causing renal failure followed by disseminated intravascular coagulation 72 .
Hantavirus cardiopulmonary syndrome (HCPS) is another pathology associated with agents of the genus New World hantaviruses (Americas), and appears to be related to climatic phenomena such as El Niño 58 .
The first report of hantavirus in Brazil dates from 1993, when Juquitiba virus, transmitted by Oligoryzomys nigripes, was detected by Silva et al. (1997) 73  The incubation period of HCPS ranges between 16-24 days, with initial nonspecific HFRS-like symptomatology differentiated by pulmonary edema and lymphoid organ impairment 74 , which may cause cardiovascular shock and death 72 . Studies indicate that this pulmonary phase lasts approximately 1 week, but long-lasting sequelae have been reported, such as dyspnea and weakness 61 . According to available data from the Brazilian Ministry of Health, 2061 people had been infected in the country as of 2017, with a lethality rate of 40.1% 75 .
In rodents, this virus causes a chronic infection with mild symptomatology such as decreased growth 76 and renal problems 77 , although it is usually asymptomatic. This can be explained by the coevolution of rodents with the virus over millions of years 78 . Hantavirus can be diagnosed by associating the patient's history with the presence of wild or synanthropic rodents. Tests for hantavirus include specific serological detection using ELISA (IgM or IgG) or viral detection using RT-PCR or real-time PCR 79 .
The occurrence of HFRS has not yet been reported in Brazil, despite serologically positive human and rodent samples 80 [83][84][85] . Additionally, studies have demonstrated that the use of antiviral ribavirin increases the survival rate in hantavirus-infected rats [86][87][88] .
Myriad mechanisms of viral variability occur in RVs, such as point mutations, rearrangements, reassortments, and intragenic recombination, conferring great genetic diversity 91,92 . This genus is divided into nine different groups (RVA-RVI) based on the antigenic properties and nucleotide sequences of the VP6 protein 89,93,94 , and there is a potential candidate tenth group, RVJ 95 .
Although rotaviruses are considered species-specific, heterologous infections may occur 100 . Wa-like and DS1-like (RVA)

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strains primarily cause disease in humans, and infections caused by genotypes and serotypes common to animals have also been documented 100,101 .
Synanthropic rodents are usually not associated with RVs, however, their efficiency in disease transmission and their close contact with other animals and people highlight their importance to the epidemiology of these viruses 101,102 .
Transmission initially occurs through the fecal-oral route, via particles present in the soil and water, causing diarrhea due to the loss of absorptive capacity of injured intestinal cells during viral replication 103 . Diarrhea is a leading cause of infant mortality worldwide, and rotavirus infections are responsible for more than 35% of these cases 104 .
RVs are widely distributed in Brazil, and have been described in animals (both young and adults) such as cattle 105 , birds 106 , and pigs 107,108 . In rodents, there is a single report of RVA associated with swine production 109 .
A metagenomic analysis of R. norvegicus in Germany characterized a sample of RVA, revealing close identity between the identified strain and other animal and human strains, namely, genotypes G3, P [3], and N2 110 .
As the associated symptomatology is mainly nonspecific, the diagnosis can easily be misleading 111 . Commercial ELISA kits, RT-PCR (single or multiplex), and qPCR assays 99,102,110 are available for the detection of these infections.

CORONAVIRUS (COV)
Coronaviruses are enveloped, nonsegmented, positive, single-stranded RNA viruses associated with the structural N-phosphoprotein in a nucleocapsid with helical symmetry 112,113 . They are found in a wide variety of animals causing respiratory, enteric, hepatic, and neurological diseases of varying severity 114 . According to the International Committee on Taxonomy of Viruses, two subfamilies belong to the Coronaviridae family; Letovirinae, which has one subgenus, Milecovirus, found only in frogs and a sea hare thus far 115 , and Orthocoronavirinae, which is found in birds and mammals, and is divided into four genera due to the antigenic and genetic characteristics of the viruses 53, 116 .
Despite the many uncertainties about the epidemiology and reservoirs of severe acute respiratory syndrome (SARS) and Middle Eastern respiratory syndrome (MERS), bats have been identified as the most likely reservoirs, while palm civets (Paguma larvata) 128 and dromedary camels (Camelus dromedarius) 129,130 act as intermediary hosts before dissemination to humans 120,131,132 . Both diseases have caused worldwide health problems, affecting 27 countries and causing hundreds of deaths in 2002 (SARS) and 2012 (MERS), aggravated by nosocomial transmission or transmission by family members 126 .
In general, the virion contains at least three proteins: the spike (S), membrane (M), and envelope (E) proteins. In addition, some coronaviruses include hemagglutinin esterase (HE) 133 . Proteins M and E are related to viral assembly 134 , while the S protein show hemagglutinating activity and is the main target for neutralizing antibodies 135 .
The S protein, which shows great variability, is responsible for host specificity because its S1 and S2 subunits are used for binding the virus to host cell receptors, and are associated with the antigenicity and pathogenicity of the virus 112,136 .
Many similarities exist between the CoVs of rats and bats 119,120,140,141 , suggesting that rodents could act as important reservoirs 142 . A survey conducted on 330 intestinal content samples from rodents (Apodemus sp., Myodes glareolus, Arvicola terrestris, and Microtus sp.), collected between 2014 and 2016 in different regions of France 121 , revealed positivity of 6.3% (21 samples), all belonging to Alphacoronavirus groups. This study also revealed Alpha and Betacoronavirus in bats, rabbits, and hedgehogs from the same area 121 .
Besides field investigations, rodent CoV also has an important role as Murine hepatitis virus (MHV) (137) , and has been used for experimental infections, mostly for the identification of potential viruses showing interspecies transmission 119 . Although there is little information about the prevalence and diversity CoV in rodents 138,139 , many new species of Alpha and Betacoronaviruses (LRNV, LAMV, LRLV, and HKU24) have been identified in rodents in China and Europe 119,120,140,141 .
Some vaccine candidates are being developed only for MERS-CoV, including whole-virus, vectored virus, DNA, and proteinbased vaccines, however, lack of investment is delaying their development 143 .

ARENAVIRUS (AV)
Arenaviruses (genus Arenavirus, family Arenaviridae) are enveloped viruses with an RNA genome segmented in two ambisense single stranded molecules: small (S) and large (L) (53) . The S portion encodes nucleocapsid protein and envelope glycoproteins, while L segment contains RNA dependent RNA polymerase (RdRp) and zincbinding protein genes. Both S and L intergenic regions may potentially form one or more hairpins, which regulate mRNA transcription 53, [144][145][146] .
This viral family has four genera, based on phylogenetic analysis involving pairwise sequence comparisons (PASC) of complete genomes 53, 147 . Antennavirus genus includes viruses that infect frogfish 148 , Hartmanivirus and Reptarenavirus infect snakes, and Mammarenavirus, which have been reported in bats, ticks, rodents, and primates, including humans 146,147 .
Mammarenaviruses are correlated to the geographical location where their hosts are found 147 ; currently classified as: (a) Old World (OW) (Lassa virus, Lujo virus, among others), and found mainly in Africa in rodents of the Murinae family as natural hosts 149 . Although Lymphocytic choriomeningitis virus (LCMV) belongs to this category, it circulates globally 146 . Approximately 5% of the human population have been exposed to LCMV, due to the ubiquity of the virus host, M. musculus 150 ; (b) New World (NW), found in the American continent, are divided into four clades (A-D) 151 155 . Sigmodontinae rodents are the main hosts of this class, even though Tacaribe virus has already been described in bats 156 and Amblyomma americanum ticks 157 .
Members of both OW and NW arenaviruses can cause hemorrhagic fever and severe human diseases affecting the central nervous system 158 . Zoonotic transmission is through contact with rodents' urine or feces, and human-to-human transmission is possible 146 . Because of their impact on human health and rapid spread, they are potential bioterrorism agents 159 .
In Colombia, a study conducted with M. musculus, collected from residential areas, detected 10% (8/80) of positives in serological analysis for LCMV. When brain samples of the same animals were submitted to RT-PCR, serologically negative individuals showed positive results in this second analysis, highlighting the importance of parallel diagnosis 160 . It can be justified by the vertical transmission among rodents that may deactivate cytotoxic T-lymphocytes, generating immune-complexes that may lead to misdiagnosis of ELISA reactions 160 .
A research conducted in French Guiana sampled 37 animals (M. musculus) of which two were positive for LCMV by heminested PCR (from lung and kidney samples) 161 ; another inquiry in Argentina reported that 9.4% of the mice collected were positive for arenavirus, and the serological rate was 4.6% and 2.6% for men and women, respectively 162 . In Baltimore (USA), 9% of the mice were seropositive for LCMV 163 , and 4.7% of the people were analyzed 164 . In Brazil, to our knowledge, there are no serological records on the prevalence of LCMV in rodents or humans.
Lassa virus (OW) is endemic in African countries, with seroprevalence reaching about 50% of the human population; this disease causes about 5,000 deaths every year 165 . Mastomys natalensis is considered the main reservoir of the virus 166 , but it can also be found in Hylomyscus pamfi and Mastomys erythroleucus 167 .
Surveys conducted in Nigeria reported positive animals for Lassa virus in M. natalensis and M. erythroleucus, R. rattus 168 , and M. musculus 169 . In China, RT-PCRs performed in organs of R. rattus and R. norvegicus showed positive rates of 75% and 17%, respectively, and a new viral species, the Wenzhou virus, was isolated 145 .
Junin virus is considered endemic in Argentina 170 and sporadic outbreaks have been reported 171 . There are several promising vaccinal prototypes being developed for this virus, most are in the preclinical stage 172 and one, based on plasmidial DNA, has already reached human test phase 173 .
In Brazil, the most remarkable arenavirus is Sabiá virus, reported in São Paulo (Brazil) in 1994. Initial symptoms were described as flu-like (fever, sickness, headaches, and lethargy), quickly leading to hemorrhage and death (within 3 days) 154,174 . There have been two reports of this virus, caused by occupational exposure in a laboratory environment, one in Pará (Brazil) and the other in Connecticut (USA), both with non-fatal courses 175,176 . The 4th case was described in São Paulo (Brazil) in 1999 177 and the 5th, on January 2020 in Sorocaba (São Paulo, Brazil) 178 as a natural infection with lethal outcome.
Arenaviruses can be diagnosed using: (a) RT-PCR (fluids, feces, and tissues) followed by viral RNA sequencing for differentiation; (b) serology, through detection of specific IgG and IgM employing immunofluorescence and/or ELISA tests; (c) viral isolation in cell culture.
Recommended treatment is support therapy that can be combined with the antiviral ribavirin, which should be administered during the first 7-10 days after infection. Despite its efficacy, there are significant side effects, such as hemolytic anemia, progressive weight loss, respiratory difficulty, insomnia, and dermatitis, among others [180][181][182] . Alternative drugs with less side effects have been tested, such as favipiravir 183 and triarylmethane clotrimazole 184 . Cocktails using multiple antiviral drugs that target different steps of the viral life cycle appear to be the best strategy to limit viral multiplication with lower risk of drug resistance 185 .
In literature we find description of viral rodent-infections, usually from within the context of biological models or experimentation. In this review, we focus on rodents within an urban environment, especially R. rattus, R. norvegicus, and M. musculus, although with the advancement of human populations, the interaction with 6/11 wild rodents increases, and different viruses can emerge. There are relatively few studies addressing the monitoring of viruses in these hosts, favoring the occurrence of outbreaks.
Control and preventive activities should go beyond the elimination or reduction of the populations of these hosts and involve sanitary education to aid the human population in the reduction of shelters for the hosts, the restriction of rodent access to residences, and the reduction of their water and food supply. Basic sanitation actions are a generic but effective measure in the reduction of rodents and, consequently, the propagation of diseases.

AUTHORS' CONTRIBUTION
MLG and FG: revision design, participated in data analysis, discussion, writing draft and review; CMB and RMS: participated in data analysis, discussion, writing draft and review.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.