Metagenome-Assembled Viral Genomes Analysis Reveals Diversity and Infectivity of the RNA Virome of Gerbillinae Species.

Rodents are a known reservoir for extensive zoonotic viruses, and also possess a propensity to roost in human habitation. Therefore, it is necessary to identify and catalogue the potentially emerging zoonotic viruses that are carried by rodents. Here, viral metagenomic sequencing was used for zoonotic virus detection and virome characterization on 32 Great gerbils of Rhombomys opimus, Meriones meridianus, and Meiiones Unguiculataus species in Xinjiang, Northwest China. In total, 1848 viral genomes that are potentially pathogenic to rodents and humans, as well as to other wildlife, were identified namely Retro-, Flavi-, Pneumo-, Picobirna-, Nairo-, Arena-, Hepe-, Phenui-, Rhabdo-, Calici-, Reo-, Corona-, Orthomyxo-, Peribunya-, and Picornaviridae families. In addition, a new genotype of rodent Hepacivirus was identified in heart and lung homogenates of seven viscera pools and phylogenetic analysis revealed the closest relationship to rodent Hepacivirus isolate RtMm-HCV/IM2014 that was previously reported to infect rodents from Inner Mongolia, China. Moreover, nine new genotype viral sequences that corresponded to Picobirnaviruses (PBVs), which have a bi-segmented genome and belong to the family Picobirnaviridae, comprising of three segment I and six segment II sequences, were identified in intestines and liver of seven viscera pools. In the two phylogenetic trees that were constructed using ORF1 and ORF2 of segment I, the three segment I sequences were clustered into distinct clades. Additionally, phylogenetic analysis showed that PBV sequences were distributed in the whole tree that was constructed using the RNA-dependent RNA polymerase (RdRp) gene of segment II with high diversity, sharing 68.42-82.67% nucleotide identities with other genogroup I and genogroup II PBV strains based on the partial RdRp gene. By RNA sequencing, we found a high degree of biodiversity of Retro-, Flavi-, Pneumo-, and Picobirnaridae families and other zoonotic viruses in gerbils, indicating that zoonotic viruses are a common presence in gerbils from Xinjiang, China. Therefore, further research is needed to determine the zoonotic potential of these viruses that are carried by other rodent species from different ecosystems and wildlife in general.

Global Gene Expression Analysis of the Brainstem in EV71- and CVA16-Infected Gerbils.

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two most important pathogens of hand, foot, and mouth disease (HFMD). However, the neuropathogenesis of EV71 and CVA16 has not been elucidated. In our previous study, we established gerbils as a useful model for both EV71 and CVA16 infection. In this work, we used RNA-seq technology to analyze the global gene expression of the brainstem of EV71- and CVA16-infected gerbils. We found that 3434 genes were upregulated while 916 genes were downregulated in EV71-infected gerbils. In CVA16-infected gerbils, 1039 genes were upregulated, and 299 genes were downregulated. We also found significant dysregulation of cytokines, such as IP-10 and CXCL9, in the brainstem of gerbils. The expression levels of 10 of the most upregulated genes were confirmed by real-time RT-PCR, and the upregulated tendency of most genes was in accordance with the differential gene expression (DGE) results. Our work provided global gene expression analysis of virus-infected gerbils and laid a solid foundation for elucidating the neuropathogenesis mechanisms of EV71 and CVA16.

Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis.

Hepatitis E virus (HEV) infection has been associated with extrahepatic manifestations, particularly neurological disorders. Although it has been reported that HEV infection induced hepatocyte apoptosis associated with mitochondria injury, activation of mitochondrial apoptotic pathway in the central nervous system during HEV infection was not clearly understood. In this study, the induction of mitochondrial apoptosis-associated proteins and pro-inflammatory cytokines were detected in HEV infected Mongolian gerbil model and primary human brain microvascular endothelial cells (HBMVECs). Mitochondrial exhibited fragments with loss of cristae and matrix in HEV infected brain tissue by transmission electron microscope (TEM). In vitro studies showed that expression of NADPH oxidase 4 (NOX4) was significantly increased in HEV infected HBMVECs (p < 0.05), while ATP5A1 was significantly decreased (p < 0.01). Expressions of pro-apoptotic proteins were further evaluated. Bax was significantly increased in both HEV infected brain tissues and HBMVECs (p < 0.01). In vivo studies showed that caspase-9 and caspase-3 were activated after HEV inoculation (p < 0.01), associated with PCNA overexpression as response to apoptosis. Cytokines were measured to evaluate tissue inflammatory levels. Results showed that the release of TNFα and IL-1ß were significantly increased after HEV infection (p < 0.01), which might be attributed to microglia activation characterized by high level of IBA1 expression (p < 0.01). Taken together, these data support that HEV infection induces high levels of pro-inflammatory cytokines, associated with mitochondria-mediated apoptosis. The results provide new insight into mechanisms of extra-hepatic injury of HEV infection, especially in the central nervous system.

Antigen detection and apoptosis in Mongolian gerbil's kidney experimentally intraperitoneally infected by swine hepatitis E virus.

We examined the effect of hepatitis E virus (HEV) on the renal tissue pathogenesis, morphological damages and related molecular mechanisms following swine HEV suspension intraperitoneally inoculation in Mongolian gerbils. The microscopic and ultramicroscopic analyses of kidney tissue structure were carried out at different points after inoculation of HEV. The immunohistochemistry, real-time PCR and Western blot were performed to explore the molecular mechanisms associated with HEV presence in the renal tissues. Real-time PCR revealed that the copies of HEV RNA in the kidney were detected at 7 dpi, and peaked at 14 dpi at a concentration was 7.18 logs g(-1), with detection of HEV ORF2 antigen by immunohistochemistry. Hematoxylin and eosin (HE) staining showed pathological lesions including glomerular atrophy, degeneration, edema and necrosis of renal tubular epithelial cells and Mallory and Sirius red staining indicated the presence of collagen fibers and fibrosis in kidney tissues of inoculated gerbils. Ultrastructural studies of basal membrane of renal tubules demonstrated the rough and uneven with mitochondria swelling and vacuolation in the tissues of HEV inoculated animals. Similarly, significantly higher number of (TUNEL)-positive cells were seen in renal tubule tissues compared to control group. Moreover, immuno histochemical results indicated that significant increase expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), FAS and Caspase-3 in HEV inoculated Mongolian gerbils at each time points. Relative mRNA expression by real-time PCR revealed a significantly higher (P<0.05) mRNA level of BAX, Bcl-2 and caspase-3 transcription in HEV inoculated Mongolian gerbils. Our results demonstrates that activation of mitochondria and Caspase-3 protease might be induced the apoptosis which subsequently cause the necrosis and cell death of renal epithelial cells during acute phase of HEV infection in HEV inoculated Mongolian gerbils.

Enterovirus 71 infection causes severe pulmonary lesions in gerbils, meriones unguiculatus, which can be prevented by passive immunization with specific antisera.

Neurogenic pulmonary edema caused by severe brainstem encephalitis is the leading cause of death in young children infected by Enterovirus 71 (EV71). However, no pulmonary lesions have been found in EV71-infected transgenic or non-transgenic mouse models. Development of a suitable animal model is important for studying EV71 pathogenesis and assessing effect of therapeutic approaches. We had found neurological disorders in EV71-induced young gerbils previously. Here, we report severe pulmonary lesions characterized with pulmonary congestion and hemorrhage in a gerbil model for EV71 infection. In the EV71-infected gerbils, six 21-day-old or younger gerbils presented with a sudden onset of symptoms and rapid illness progression after inoculation with 1×105.5 TCID50 of EV71 via intraperitoneal (IP) or intramuscular (IM) route. Respiratory symptoms were observed along with interstitial pneumonia, pulmonary congestion and extensive lung hemorrhage could be detected in the lung tissues by histopathological examination. EV71 viral titer was found to be peak at late stages of infection. EV71-induced pulmonary lesions, together with severe neurological disorders were also observed in gerbils, accurately mimicking the disease process in EV71-infected patients. Passive transfer with immune sera from EV71 infected adult gerbils with a neutralizing antibody (GMT=89) prevented severe pulmonary lesion formation after lethal EV71 challenge. These results establish this gerbil model as a useful platform for studying the pathogenesis of EV71-induced pulmonary lesions, immunotherapy and antiviral drugs.

[Taxonomic status of the Chim virus (CHIMV) (Bunyaviridae, Nairovirus, Qalyub group) isolated from the Ixodidae and Argasidae ticks collected in the great gerbil (Rhombomys opimus Lichtenstein, 1823) (Muridae, Gerbillinae) burrows in Uzbekistan and Kazakhstan].

Full-length genome of the Chim virus (CHIMV) (strain LEIV-858Uz) was sequenced using the next-generation sequencing approach (ID GenBank: KF801656). The CHIMV/LEIV-858Uz was isolated from the Ornithodoros tartakovskyi Olenev, 1931 ticks collected in the great gerbil (Rhombomys opimus Lichtenstein, 1823) burrow in Uzbekistan near Chim town (Kashkadarinsky region) in July of 1971. Later, four more CHIMV strains were isolated from the O. tartakovskyi, O. papillipes Birula, 1895, Rhipicephalus turanicus Pomerantsev, 1936 collected in the great gerbil burrows in Kashkadarinsky, Bukhara, and Syrdarya regions of Uzbekistan, and three strains--from the Hyalomma asiaticum Schulze et Schlottke, 1930 from the great gerbil burrows in Dzheskazgan region of Kazakhstan. The virus is a potential pathogen of humans and camels. The phylogenetic analysis revealed that the CHIMV is a novel member of the Nairovirus genus (Bunyaviridae) and closely related to the Qalyub virus (QYBV), which is prototype for the group of the same name. The amino acid homology between the CHIMV and QYBV is 87% for the RdRp catalytic center (L-segment) that is coincident with both QYBV and CHIMV associated with the Ornithodoros ticks and burrow of rodents as well. The CHIMV homologies with other nairoviruses are 30-40% for the amino acid sequences of precursor polyprotein GnGc (M-segment), whereas 50%--for the nucleocapsid N (S-segment). The data obtained permit to classify the CHIMV as a member of the QYBV group in the genus of Nairovirus (Bunyaviridae).

[Taxonomic status of the Artashat virus (ARTSV) (Bunyaviridae, Nairovirus) isolated from the ticks Ornithodoros alactagalis Issaakjan, 1936 and O. verrucosus Olenev, Sassuchin et Fenuk, 1934 (Argasidae Koch, 1844) collected in Transcaucasia].

The Artashat virus (ARTSV) was originally isolated fom the Ornithodoros alactagalis Issaakjan, 1936 (Argasidae Koch, 1844), which were collected in the burrow of small five-toed jerboa (Allactaga elater Lichtenstein, 1825) in Armenia in 1972. Later, the ARTSV was isolated from the O. verrucosus Olenev, Sassuchin et Fenuk, 1934 collected in the burrows of Persian gerbil (Meriones persicus Blanford, 1875) in Azerbaijan. Based on the virion morphology, the ARTSV was assigned to the Bunyaviridae viruses. In this work, the ARTSV genome was partially sequenced (GenBank ID: KF801650) and it was shown that the ARTSV is a new member of the Nairovirus genus. ARTSV has from 42% (Issyk-Kul virus) to 58% (Raza virus, Hughes group) similarity with the nairoviruses for nucleotide sequence of part of RNA-dependent RNA-polymerase (RdRp). The similarity on the amino acid level is 65-70%. Low level of homology and the equidistant position of the ARTSV on phylogenetic tree indicate that the ARTSV is a new prototype species of the Nairovirus genus (Bunyaviridae) forming a separate phylogenetic branch.

[Genetic characterization of the Geran virus (GERV, Bunyaviridae, Nairovirus, Qalyub group) isolated from the ticks Ornithodoros verrucosus Olenev, Zasukhin and Fenyuk, 1934 (Argasidae) collected in the burrow of Meriones erythrourus Grey, 1842 in Azerbaijan].

The full-length genome of the unclassified Geran virus (GERV, strain LEIV-10899Az) isolated from the ticks (Ornithodoros verrucosus Olenev, Zasukhin and Fenyuk, 1934 (Argasidae, Ornithodorinae)) collected in the burrow of the red-tailed gerbils (Meriones (Cricedidae) erythrourus Grey, 1842) near the Geran station (Azerbaijan) was sequenced using the next-generation approach (GenBank ID: KF801649). It was shown that the GERV is a new representative of the Nairovirus genus (family Bunyaviridae). The comparative analysis of the full-length genome sequences of the GERV with other nairoviruses showed that the highest level of homology (55.6% for N protein (S-segment) of 54.2% for the polyprotein Gn/Gc (M-segment) and 74.8% for the RNA-dependent RNA polymerase (L-segment)) GERV had with the Chim virus (CHIMV) that is also associated with the shelters biocenoses (rodent burrows) in Central Asia and was previously assigned to the Qalyub virus group (QYBV). Comparing the GERV with the QYBV sequences (partial sequence 413 n.o. of RdRp gene) revealed a high level of homology: 74.3 and 97.4% for the nucleotide and amino acid sequences, respectively. The data obtained in this work provided an opportunity to classify the GERV to the QYBV group; the Nairovirus genus, to the family Bunyaviridae.

Isolation and characterization of a new strain of lymphocytic choriomeningitis virus from rodents in southwestern France.

A total of 821 tissue samples from rodents trapped during field campaigns organized in Europe and Africa were screened for the presence of arenaviruses by molecular methods and cell culture inoculation when feasible. Two Mus musculus domesticus trapped in the southwestern part of France were infected with a potentially new strain of lymphocytic choriomeningitis virus (LCMV), here referred to as LCMV strain HP65-2009, which was isolated and genetically characterized by whole genome sequencing. Genetic and phylogenetic analyses comparing LCMV HP65-2009 with 26 other LCMV strains showed that it represents a novel highly-divergent strain within the group of Mus musculus-associated LCMV.

Hygienic monitoring of Mongolian gerbils: which mouse viruses should be included?

The Mongolian gerbil (Meriones unguiculatus) serves as an animal model for a wide range of diseases. A practical limitation in its use is the definition of the hygienic status, as not much is known about viruses that potentially infect gerbils and might be transmitted to other rodents. As successful re-derivation was recently described for gerbils, we now aimed at investigating which mouse viruses induce seroconversion in gerbils and might be transmitted to mice. Gerbils were inoculated with viral agents of mice and co-housed with mouse contact sentinels. Seroconversion in gerbils was observed after oronasal inoculation with Sendai virus (SeV), mammalian orthoreovirus serotype 3 (Reo-3) and rotavirus A (RV-A, EDIM), seroconversion to RV-A also in sentinel mice. Pneumonia virus of mice (PVM) was not detected by serology but by polymerase chain reaction in gerbils and respective sentinel mice. No seroconversion towards or transmission of murine hepatitis virus, murine norovirus, minute virus of mice or mouse cytomegalovirus was detected. Anti-gerbil IgG antibodies did not increase sensitivity of indirect immunofluorescence (IFA) compared with anti-mouse IgG. In conclusion, seroconversion to SeV, Reo-3 and RV-A as well as transmission of RV-A and PVM indicate that these agents should be included in health monitoring of gerbils. Furthermore, anti-mouse IgG is suitable as a secondary antibody for IFA with gerbil serum.

Betaherpesvirus sequences in eastern spiny mice and Wagner's dipodils.

Eastern spiny mice (Acomys dimidiatus; also known as Sinai spiny mice) have been extensively studied in terms of the influence of parasite load on population size and reproductive fitness. The physical isolation of these rodent populations makes them interesting models for disease interactions in a real-life population as opposed to a laboratory. We identify betaherpesvirus sequences in eastern spiny mice and Wagner's dipodils (Dipodillus dasyurus), species that inhabit dry montane wadis (dry creek valleys) of the Sinai, highlighting the need for a comprehensive analysis of the full pathogen repertoire of these rodents in long-term studies.

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils.

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.

Age- and host-dependent control of Borna disease virus spread in the developing brains of gerbils and rats.

Borna disease virus (BDV) is a non-cytolytic, neurotropic RNA virus that has a broad host range in warm-blooded animals, probably including humans. Recently, we have demonstrated that the neonatal gerbil is a unique model for analyzing BDV-induced acute neurological disease. In this report, to understand the effects of the brain development of gerbils in BDV-induced neuropathogenesis, as well as to investigate the host-dependent differences in BDV propagation and pathogenesis in the brains, we performed experimental infection of BDV using two different infant rodent models, gerbils and rats. We demonstrated here that most of the gerbils infected with BDV on postnatal days (PD) 14, but not on PD1 and PD7, could survive neurological disorders during the observation period of PD85. Interestingly, the levels of BDV RNA and antigen in surviving PD14 inoculated gerbil brains were extremely low, whereas diseased gerbils and both PD7 and PD14 inoculated rats contained significant amounts of BDV antigen in the central nervous system, suggesting that PD14 gerbils successfully controlled BDV spread in the brain. Furthermore, the viral distribution, as well as the expression levels of cytokine and CD8 mRNAs, in the brains was markedly different between the rodent models and between diseased and non-diseased statuses of the gerbils. These results demonstrated that developmentally regulated and host-specific factors could contribute to the prevention of BDV spread in developing animal brains. Studies using different animal systems would provide novel insights into the mechanisms of host defense responses to neurotropic virus infections.

Borna disease virus induces acute fatal neurological disorders in neonatal gerbils without virus- and immune-mediated cell destructions.

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that is known to cause neurological disturbances in various animal species. Our previous experiment demonstrated that neonate gerbils develop an acute fatal neurological disease following infection with BDV, Virology 282, 65-76). The study suggested that BDV directly causes functional damage of neuronal cells resulting in the lethal disorder in neonatal gerbils. To extend this finding, we examined whether BDV can induce neurological diseases in the absence of virus- and immune-mediated cell destruction, by using cyclosporine A (CsA)-treated neonatal gerbils. Although CsA completely suppressed specific antibody production and brain inflammation in the infected gerbil brains, the fatal neurological disorder was not inhibited by the treatment. Furthermore, we demonstrated that CsA treatment significantly decreased brain levels of cytokines, except interleukin (IL)-1 beta, in the infected gerbils. These results suggested that BDV replication, as well as brain cytokines, at least IL-1 beta, rapidly induces fatal disturbances in gerbil brain. We demonstrate here that BDV exhibits a unique neuropathogenesis in neonatal gerbil that may be pathologically and immunologically different from those in two other established rodent models, rats and mice. With this novel rodent model of virus infection it should be possible not only to examine acute neurological disturbances without severe neuroanatomical and immunopathological alterations but also to analyze molecular and cellular damage by virus replication in the central nervous system.

[Geographical divisions of rat transmissible diseases].

In order to prevent rat transmissible diseases, it is necessary to know the geographical distribution of rats, their external parasitic arthropod and rat-borne disease in human, and to make a graph of geographical division. Stratified sampling was conducted by county to capture rats, and to sort out flea for identification of their genus and species. A total of 55,064 rats sorting as 6 families, 17 genera and 21 species were captured, and a total of 34,733 flea sorting as 4 families, 25 genera and 53 species were picked. Three kinds of rat-borne diseases, namely plague, leptospirosis and epidemic hemorrhagic fever have been occurred and epidemic in Shanxi Province. Following the general principles for geographical division, Shanxi Province was divided into 4 sub-regions: (1) basin among mountains and prairie in Yanbei; (2) loess plateau, hill and prairie with shrub in the west; (3) hill, shrub and basin, prairie with forest in the middle part; (4) hill, basin with forest and prairie in the southeast. When bubonic plague was epidemic, its transmissible mode was probably Meriones meridianus--Xenopsylla conformis--Mus musculus, Rattus norvegicus--Xenopsylla cheoopis--human. Bubonic plague has been occurred within Xingxian and Linxian conxian counties of the second sub-region. Leptospirosis has been occurred in Xiangfen county of the third sub-region, the source of infection was from pigs instead of rats. Epidemic hemorrhagic fever has been occurred in the third and fourth sub-regions, the source of infection was mainly from Ruttus norvegicus.

High susceptibility of Mongolian gerbil (Meriones unguiculatus) to Borna disease virus.

Borna disease virus (BDV) is a neurotropic enveloped virus with a nonsegmented, single-, negative-stranded RNA genome. This virus induced encephalitis in experimentally infected adult rats, but in newborn rats BDV established a persistent, tolerant infection with no apparent clinical signs. Here, we report evidence that newborn Mongolian gerbils (Meriones unguiculatus) are more susceptible to experimental intracranial inoculation of horse-derived BDV in persistently infected MDCK cells, compared with similar inoculation in newborn rats. All inoculated newborn gerbils, but not rats, died 30 days after infection. Reverse transcriptase-polymerase chain reaction amplified BDV-specific sequences in several regions including the brain. Histopathological analysis revealed apparent inflammatory reactions in the brains of inoculated gerbils but not rats, although similar levels of BDV RNA were detected in both gerbil and rat brains. BDV-specific antigen and RNA were identified predominantly in neurons in the brains by immunohistochemistry with antibodies to BDV and in situ hybridization with BDV-specific riboprobes, respectively. BDV in the gerbil brain was easily rescued by co-cultivation of the brain homogenate with human oligodendroglioma cells. Thus, gerbils seem to be a useful animal model for studying BDV-induced pathogenesis in the brain.

La Crosse viremias in Mongolian gerbils (Meriones unguiculatis).

We examined the usefulness of mongolian gerbils (Meriones unguiculatus) as a new animal model for La Crosse virus (LACV) studies. Gerbils were exposed to LACV by either intramuscular (im) inoculation or exposure to transovarially infected Aedes triseriatus. Our studies indicate that gerbils may be a suitable animal model for LACV infection. Gerbils were susceptible to LACV, survived viral infection, and developed viremias and neutralizing antibody titers following exposure by im injection and by the bite of infected mosquitoes. Moreover, they are attractive to mosquito vectors. Gerbils have other advantages as laboratory vertebrate hosts for LACV; they are inexpensive, breed in captivity, and are usually mild-mannered and easy to handle. Thus, gerbils are a suitable model in the study of LACV pathogenesis as well as of transplacental and vector transmission.