Characterization of a mouse-adapted strain of bat severe acute respiratory syndrome-related coronavirus.

Bats carry genetically diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). Some of them utilize human angiotensin-converting enzyme 2 (hACE2) as a receptor and cannot efficiently replicate in wild-type mice. Our previous study demonstrated that the bat SARSr-CoV rRsSHC014S induces respiratory infection and lung damage in hACE2 transgenic mice but not wild-type mice. In this study, we generated a mouse-adapted strain of rRsSHC014S, which we named SMA1901, by serial passaging of wild-type virus in BALB/c mice. SMA1901 showed increased infectivity in mouse lungs and induced interstitial lung pneumonia in both young and aged mice after intranasal inoculation. Genome sequencing revealed mutations in not only the spike protein but the whole genome, which may be responsible for the enhanced pathogenicity of SMA1901 in wild-type BALB/c mice. SMA1901 induced age-related mortality similar to that observed in SARS and COVID-19. Drug testing using antibodies and antiviral molecules indicated that this mouse-adapted virus strain can be used to test prophylactic and therapeutic drug candidates against SARSr-CoVs. IMPORTANCE The genetic diversity of SARSr-CoVs in wildlife and their potential risk of cross-species infection highlights the importance of developing a powerful animal model to evaluate the antibodies and antiviral drugs. We acquired the mouse-adapted strain of a bat-origin coronavirus named SMA1901 by natural serial passaging of rRsSHC014S in BALB/c mice. The SMA1901 infection caused interstitial pneumonia and inflammatory immune responses in both young and aged BALB/c mice after intranasal inoculation. Our model exhibited age-related mortality similar to SARS and COVID-19. Therefore, our model will be of high value for investigating the pathogenesis of bat SARSr-CoVs and could serve as a prospective test platform for prophylactic and therapeutic candidates.

Lethal Swine Acute Diarrhea Syndrome Coronavirus Infection in Suckling Mice.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a recently emerging bat-borne coronavirus responsible for high mortality rates in piglets. In vitro studies have indicated that SADS-CoV has a wide tissue tropism in different hosts, including humans. However, whether this virus potentially threatens other animals remains unclear. Here, we report the experimental infection of wild-type BALB/c and C57BL/6J suckling mice with SADS-CoV. We found that mice less than 7 days old are susceptible to the virus, which caused notable multitissue infections and damage. The mortality rate was the highest in 2-day-old mice and decreased in older mice. Moreover, a preliminary neuroinflammatory response was observed in 7-day-old SADS-CoV-infected mice. Thus, our results indicate that SADS-CoV has potential pathogenicity in young hosts. IMPORTANCE SADS-CoV, which likely has originated from bat coronaviruses, is highly pathogenic to piglets and poses a threat to the swine industry. Little is known about its potential to disseminate to other animals. No efficient treatment is available, and the quarantine strategy is the only preventive measure. In this study, we demonstrated that SADS-CoV can efficiently replicate in suckling mice younger than 7 days. In contrast to infected piglets, in which intestinal tropism is shown, SADS-CoV caused infection and damage in all murine tissues evaluated in this study. In addition, neuroinflammatory responses were detected in some of the infected mice. Our work provides a preliminary cost-effective model for the screening of antiviral drugs against SADS-CoV infection.

The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model.

The basis for rotavirus (RV) host range restriction (HRR) is not fully understood but is likely multigenic. RV genes encoding VP3, VP4, NSP1, NSP2, NSP3, and NSP4 have been associated with HRR in various studies. With the exception of NSP1, little is known about the relative contribution of the other RV genes to HRR. VP4 has been linked to HRR because it functions as the RV cell attachment protein, but its actual role in HRR has not been fully assessed. We generated a collection of recombinant RVs (rRVs) in an isogenic murine-like RV genetic background, harboring either heterologous or homologous VP4 genes from simian, bovine, porcine, human, and murine RV strains, and characterized these rRVs in vitro and in vivo. We found that a murine-like rRV encoding a simian VP4 was shed, spread to uninoculated littermates, and induced diarrhea comparably to rRV harboring a murine VP4. However, rRVs carrying VP4s from both bovine and porcine RVs had reduced diarrhea, but no change in fecal shedding was observed. Both diarrhea and shedding were reduced when VP4 originated from a human RV strain. rRVs harboring VP4s from human or bovine RVs did not transmit to uninoculated littermates. We also generated two rRVs harboring reciprocal chimeric murine or bovine VP4. Both chimeras replicated and caused disease as efficiently as the parental strain with a fully murine VP4. These data suggest that the genetic origin of VP4 partially modulates HRR in the suckling mouse and that both the VP8* and VP5* domains independently contribute to pathogenesis and transmission. IMPORTANCE Human group A rotaviruses (RVs) remain the most important cause of severe acute gastroenteritis among infants and young children worldwide despite the introduction of several safe and effective live attenuated vaccines. The lack of knowledge regarding fundamental aspects of RV biology, such as the genetic basis of host range restriction (HRR), has made it difficult to predictively and efficiently design improved, next-generation live attenuated rotavirus vaccines. Here, we engineered a collection of VP4 monoreassortant RVs to systematically explore the role of VP4 in replication, pathogenicity, and spread, as measures of HRR, in a suckling mouse model. The genetic and mechanistic bases of HRR have substantial clinical relevance given that this restriction forms the basis of attenuation for several replication-competent human RV vaccines. In addition, a better understanding of RV pathogenesis and the determinants of RV spread is likely to enhance our ability to improve antiviral drug and therapy development.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry.

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.

Duplication and divergence of the retrovirus restriction gene Fv1 in Mus caroli allows protection from multiple retroviruses.

Viruses and their hosts are locked in an evolutionary race where resistance to infection is acquired by the hosts while viruses develop strategies to circumvent these host defenses. Forming one arm of the host defense armory are cell autonomous restriction factors like Fv1. Originally described as protecting laboratory mice from infection by murine leukemia virus (MLV), Fv1s from some wild mice have also been found to restrict non-MLV retroviruses, suggesting an important role in the protection against viruses in nature. We surveyed the Fv1 genes of wild mice trapped in Thailand and characterized their restriction activities against a panel of retroviruses. An extra copy of the Fv1 gene, named Fv7, was found on chromosome 6 of three closely related Asian species of mice: Mus caroli, M. cervicolor, and M. cookii. The presence of flanking repeats suggested it arose by LINE-mediated retroduplication within their most recent common ancestor. A high degree of natural variation was observed in both Fv1 and Fv7 and, on top of positive selection at certain residues, insertions and deletions were present that changed the length of the reading frames. These genes exhibited a range of restriction phenotypes, with activities directed against gamma-, spuma-, and lentiviruses. It seems likely, at least in the case of M. caroli, that the observed gene duplication may expand the breadth of restriction beyond the capacity of Fv1 alone and that one or more such viruses have recently driven or continue to drive the evolution of the Fv1 and Fv7 genes.

Long-Term Rodent Surveillance after Outbreak of Hantavirus Infection, Yosemite National Park, California, USA, 2012.

In 2012, a total of 9 cases of hantavirus infection occurred in overnight visitors to Yosemite Valley, Yosemite National Park, California, USA. In the 6 years after the initial outbreak investigation, the California Department of Public Health conducted 11 rodent trapping events in developed areas of Yosemite Valley and 6 in Tuolumne Meadows to monitor the relative abundance of deer mice (Peromyscus maniculatus) and seroprevalence of Sin Nombre orthohantavirus, the causative agent of hantavirus pulmonary syndrome. Deer mouse trap success in Yosemite Valley remained lower than that observed during the 2012 outbreak investigation. Seroprevalence of Sin Nombre orthohantavirus in deer mice during 2013-2018 was also lower than during the outbreak, but the difference was not statistically significant (p = 0.02). The decreased relative abundance of Peromyscus spp. mice in developed areas of Yosemite Valley after the outbreak is probably associated with increased rodent exclusion efforts and decreased peridomestic habitat.

Human Exposure to Hantaviruses Associated with Rodents of the Murinae Subfamily, Madagascar.

We conducted a national human serologic study of a hantavirus detected in Madagascar rodents using a commercial kit and a new ELISA targeting the virus. Our results suggest a conservative estimate of 2.7% (46/1,680) IgG seroprevalence. A second single-district study using the new ELISA revealed a higher prevalence (7.2%; 10/139).

In Vivo Dynamics of Reporter Flaviviridae Viruses.

Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

Lassa Virus in Pygmy Mice, Benin, 2016-2017.

Lassa virus has been identified in 3 pygmy mice, Mus baoulei, in central Benin. The glycoprotein and nucleoprotein sequences cluster with the Togo strain. These mice may be a new reservoir for Lassa virus in Ghana, Togo, and Benin.

Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice.

On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus.

Lymphocytic choriomeningitis virus meningoencephalitis in a renal transplant recipient following exposure to mice.

Solid organ transplant recipients (SOTR) are at increased risk for a wide variety of typical and atypical infections as a consequence of impaired cell mediated and humoral immunity. We report a case of meningoencephalitis in a renal transplant recipient caused by lymphocytic choriomeningitis virus (LCMV) acquired by exposure to mice excreta. The clinical course was complicated by the development of hydrocephalus, requiring a ventriculoperitoneal shunt. To our knowledge, this is the first reported case of LCMV infection in a SOTR that was not organ donor derived.

Sequence Diversity, Intersubgroup Relationships, and Origins of the Mouse Leukemia Gammaretroviruses of Laboratory and Wild Mice.

UNLABELLED: Mouse leukemia viruses (MLVs) are found in the common inbred strains of laboratory mice and in the house mouse subspecies ofMus musculus Receptor usage and envelope (env) sequence variation define three MLV host range subgroups in laboratory mice: ecotropic, polytropic, and xenotropic MLVs (E-, P-, and X-MLVs, respectively). These exogenous MLVs derive from endogenous retroviruses (ERVs) that were acquired by the wild mouse progenitors of laboratory mice about 1 million years ago. We analyzed the genomes of seven MLVs isolated from Eurasian and American wild mice and three previously sequenced MLVs to describe their relationships and identify their possible ERV progenitors. The phylogenetic tree based on the receptor-determining regions ofenvproduced expected host range clusters, but these clusters are not maintained in trees generated from other virus regions. Colinear alignments of the viral genomes identified segmental homologies to ERVs of different host range subgroups. Six MLVs show close relationships to a small xenotropic ERV subgroup largely confined to the inbred mouse Y chromosome.envvariations define three E-MLV subtypes, one of which carries duplications of various sizes, sequences, and locations in the proline-rich region ofenv Outside theenvregion, all E-MLVs are related to different nonecotropic MLVs. These results document the diversity in gammaretroviruses isolated from globally distributedMussubspecies, provide insight into their origins and relationships, and indicate that recombination has had an important role in the evolution of these mutagenic and pathogenic agents. IMPORTANCE: Laboratory mice carry mouse leukemia viruses (MLVs) of three host range groups which were acquired from their wild mouse progenitors. We sequenced the complete genomes of seven infectious MLVs isolated from geographically separated Eurasian and American wild mice and compared them with endogenous germ line retroviruses (ERVs) acquired early in house mouse evolution. We did this because the laboratory mouse viruses derive directly from specific ERVs or arise by recombination between different ERVs. The six distinctively different wild mouse viruses appear to be recombinants, often involving different host range subgroups, and most are related to a distinctive, largely Y-chromosome-linked MLV ERV subtype. MLVs with ecotropic host ranges show the greatest variability with extensive inter- and intrasubtype envelope differences and with homologies to other host range subgroups outside the envelope. The sequence diversity among these wild mouse isolates helps define their relationships and origins and emphasizes the importance of recombination in their evolution.

Detection of the Emerging Picornavirus Senecavirus A in Pigs, Mice, and Houseflies.

Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.

EBV Infection of Mice with Reconstituted Human Immune System Components.

Epstein-Barr virus (EBV) was discovered 50 years ago as the first candidate human tumor virus. Since then, we have realized that this human γ-herpesvirus establishes persistent infection in the majority of adult humans, but fortunately causes EBV-associated diseases only in few individuals. This is an incredible success story of the human immune system, which controls EBV infection and its transforming capacity for decades. A better understanding of this immune control would not only benefit patients with EBV-associated malignancies, but could also provide clues how to establish such a potent, mostly cell-mediated immune control against other pathogens and tumors. However, the functional relevance of EBV-specific immune responses can only be addressed in vivo, and mice with reconstituted human immune system components (huMice) constitute a small animal model to interrogate the protective value of immune compartments during EBV infection, but also might provide a platform to test EBV-specific vaccines. This chapter will summarize the insights into EBV immunobiology that have already been gained in these models and provide an outlook into promising future avenues to develop this in vivo model of EBV infection and human immune responses further.

Pathogenicity of reassortant H9 influenza viruses with different NA genes in mice and chickens.

To better understand the influence of different NA genes on pathogenicity of H9 viruses, three reassortant H9 viruses (rH9N1, H9N2 and rH9N3) were generated and characterized. All three viruses replicated efficiently in eggs and MDCK cells, whereas the rH9N1 and rH9N3 replicated more efficiently than H9N2 in A549 cells. The rH9N3 replicated more efficiently than rH9N1 and H9N2 viruses in mice, however, rH9N3 replicated and shed less efficiently than the H9N2 virus in chickens. Further studies indicate that N3 had higher NA activity and released virus from erythrocytes faster, which may improve the adaptation of H9 influenza virus to mammals.

Virulence of a novel reassortant canine H3N2 influenza virus in ferret, dog and mouse models.

An outbreak of a canine influenza virus (CIV) H3N2 reassortant derived from pandemic (pdm) H1N1 and CIV H3N2 in companion animals has underscored the urgent need to monitor CIV infections for potential zoonotic transmission of influenza viruses to humans. In this study, we assessed the virulence of a novel CIV H3N2 reassortant, VC378, which was obtained from a dog that was coinfected with pdm H1N1 and CIV H3N2, in ferrets, dogs, and mice. Significantly enhanced virulence of VC378 was demonstrated in mice, although the transmissibility and pathogenicity of VC378 were similar to those of classical H3N2 in ferrets and dogs. This is notable because mice inoculated with an equivalent dose of classical CIV H3N2 showed no clinical signs and no lethality. We found that the PA and NS gene segments of VC378 were introduced from pdmH1N1, and these genes included the amino acid substitutions PA-P224S and NS-I123V, which were previously found to be associated with increased virulence in mice. Thus, we speculate that the natural reassortment between pdm H1N1 and CIV H3N2 can confer virulence and that continuous surveillance is needed to monitor the evolution of CIV in companion animals.

Molecular determinants of influenza virus pathogenesis in mice.

Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account.

Experimental infection of mice with bovine viral diarrhea virus.

The objective of this study was to test the ability of bovine viral diarrhea virus (BVDV) to infect mice. Two mice each were either mock infected or inoculated with one of three BVDV strains by the intraperitoneal (IP) (n = 8) or intranasal (IN) (n = 8) route. All mice were euthanized at day 7 postinfection (p.i.). None of the infected mice exhibited any clinical signs of illness; however, the tissues harvested after BVDV challenge showed significant histopathological changes. Blood samples from five mice that were injected IP and one mouse that was inoculated IN were positive for BVDV by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry (IHC) was used to assess the presence of viral antigen in the organs of mice infected with three BVDV strains. In IP-injected mice, BVDV antigen was detected in the spleen (5/6), mesenteric lymph nodes (4/6), lymphatic tissue of the lung (3/6), lung (1/6), and stomach (1/6) of the infected mice; however, it was not detected in the liver (0/6) or kidney (0/6). In IN-inoculated mice, BVDV antigen was detected in the lung and mesenteric lymph nodes of one BVDV-infected mouse but was not detected in other tissues. The results of this study suggest that the spleen is the most reliable tissue for BVDV antigen detection using IHC in the IP-injected group. Our study demonstrates that mice can be infected by BVDV. This is the first report of BVDV infection in mice.

Cell-associated transmission of HIV type 1 and other lentiviruses in small-animal models.

Small-animal models of lentivirus transmission have repeatedly demonstrated transmission by cell-associated virus via vaginal, rectal, and oral routes. The earliest experiments were in the cat/feline immunodeficiency virus model, followed a decade later by successful vaginal transmission of cell-associated human immunodeficiency virus (HIV) in mice bearing transplanted human immune cells. After early unsuccessful attempts at cell-associated transmission in nonhuman primates, renewed investigation in diverse primate models has now confirmed the findings from the cat and humanized mouse models. Improvements in humanized mouse models have made them the preferred small-animal models to study HIV mucosal transmission. They provide complementary systems to nonhuman primate models to aid in the elucidation of the many remaining questions on the mechanism of and means to prevent both cell-associated and cell-free HIV transmission across mucosal barriers.

Characterization of Mus musculus papillomavirus 1 infection in situ reveals an unusual pattern of late gene expression and capsid protein localization.

Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism.